Dr. S. M. Bhatt smbhatt@amity.edu , smbhatt_bhu@rediffmail.

com phone 9313993840 Amity University Uttar Pradesh ENZYMOLOGY AND ENZYME TECHNOLOGY Course Code: BTBBT 30602

Course Objective: The course aims to provide an understanding of the principles and application of proteins, secondary metabolites and enzyme biochemistry in therapeutic applications and clinical diagnosis. The theoretical understanding of biochemical systems would certainly help to interpret the results of laboratory experiments. Course Contents: Module I Enzymes: Introduction and scope, Nomenclature, Mechanism of Catalysis. Module II Specificity of enzyme action, monomeric and oligomeric enzymes, Enzyme inhibition. Module III Enzyme Kinetics: Single substrate steady state kinetics; Michaelis Menten equation, Linear plots, King-Altman’s method; Inhibitors and activators; Multisubstrate systems; ping-pong mechanism, Alberty equation, Sigmoidal kinetics and Allosteric enzymes Module IV Extraction & purification of enzymes. Module V Immobilization of Enzymes; Advantages, Carriers, adsorption, covalent coupling, cross-linking and entrapment methods, Micro-environmental effects. Module VI Biotechnological applications of enzymes: Large scale production and purification of enzymes, enzyme utilization in industry, enzymes and recombinant DNA technology Examination Scheme: Component Codes Weight age (%) H/Q 10 S 10 CT2 20 EE 60

Text & References: Text • Biotechnological Innovations in Chemical Synthesis, R.C.B. Currell, V.D. Mieras, Biotol Partners Staff, Butterworth Heinemann. • Enzyme Technology, M.F. Chaplin and C. Bucke, Cambridge University Press. • Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis, R.A. Copeland, John Wiley and Sons Inc. References • Enzymes Biochemistry, Biotechnology, Clinical Chemistry, Trevor Palner Dr. S. M. Bhatt smbhatt@amity.edu , smbhatt_bhu@rediffmail.com phone 9313993840 Amity University Uttar Pradesh • • Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems, I.H. Segel, Wiley-Interscience

Industrial Enzymes & their applications, H. Uhlig, John Wiley and Sons Inc.

ENZYMOLOGY AND ENZYME TECHNOLOGY LAB Course Code: BTBPL 30622

Course Objective: The laboratory will help the students to isolate enzymes from different sources, enzyme assays and studying their kinetic parameters which have immense importance in industrial processes. Course Contents: Module I Isolation of enzymes from plant and microbial sources. Module II Enzyme assay; activity and specific activity – determination of amylase, nitrate reductase, cellulase, protease Module III Purification of Enzyme by ammonium sulphate fractionation Module IV Enzyme Kinetics: Effect of varying substrate concentration on enzyme activity, determination of Michaelis-Menten constant (Km) and Maximum Velocity (Vmax.) using Lineweaver-Burk plot. Module V Effect of Temperature and pH on enzyme activity Module VI Enzyme immobilization Examination Scheme: Major Experiments: Minor Experiments: Spotting: Viva: Records: Total: 40 20 10 100

10 20

Note: Minor variation could be there depending on the examiner. Text: Practical Biochemistry, Sawhney and Singh

Chapter- 1

Enzymes an introduction 9

Definition Introduction

Historical aspects in enzyme discovery Classification of Enzymes. application of enzymes Chapter-2 Types of Enzymes 16 A. Simple enzymes B. Complex enzymes Complex enzymes Role of Coenzymes A. Catalysts B. Biocatalyst or Enzymes How enzymes speed up the reaction without taking part Unit of activity Chapter 3 Enzyme Specificity 20 ENZYME MECHANISM MODELS A. Lock and Key Model for Enzyme Activity B. Induced Fit (Hand and Glove) Model of Enzyme Activity Mechanisms of Enzyme Catalysis Chapter 4 Enzyme-Substrate Interactions 24 a. Catalysis by Bond Strain b. Catalysis by Proximity and Orientation c. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases) Concerted acid-base catalysis d. Covalent Catalysis: Covalent Intermediates e) Metal Ion Catalysis Catalytic Power a) Proximity b) Orientation f. Catalysis in organic solvents Some advantages to using enzymes in non-aqueous solvents include (Dordick, 1989): Importance and relevance of whole cell enzymes in biotechnology There may be some drawbacks to using whole cells in catalysis. These include Abzymes Exercises Chapter-5 ENZYME KINETICS & INHIBITION 46 Chemical Reactions and Rates Chemical Reaction Order Michaelis-Menton constant a. Pre-steady state:-b. Steady state:-Derivation: Catalytic efficiency and Turnover number. Chapter-6 Factor affecting the rate of catalysis 58 Substrate concentration Effect of temperature and pressure Effect of pH on Enzyme Chapter-7 Enzyme inhibition 68 A. Competitive inhibition B. Uncompetitive inhibition C. Noncompetitive inhibition Determination of Vmax and Km Chapter-8 Multisubstrate enzymes 83 Enzyme Catalysis

Mechanisms of Chemical Catalysis one-substrate enzymes: Linear plots for enzyme kinetic studies FYI - The Eadie-Hofstee Plot ENZYME KINETICS AND INHIBITION What's exciting about enzyme inhibition? Multi-substrate Enzymes Chapter-9 ISOLATION of ENZYME 102 INTRODUCTION strategies for enzyme purification. Method of separation. Test of purity is done by following method Source of enzyme Media for enzyme production Cell can be break down byfollowing methods A. Cell breakage by osmotic shock B. Use of enzymic lytic methods c. Ultrasonic cell disruption d. Cell lysis by High pressure homogenisers Method of separation of enzyme from lysed cell; CentrifugationB. ULTRACENTRIFUGATION A. Differential centrifugation Filtration of enzyme after separation Separation of enzyme in Aqueous biphasic systems Preparation of enzymes from clarified solution; Ultrafiltration Chapter-10 Concentration and purification of enzyme 123 Concentration by precipitation A. Nucleic acid removal B. Heat treatment 1. Enzyme purification by Chromatography 2 Affinity chromatography .3 Hydrophobic interaction chromatography (HIC) or affinity elution CHOICE OF MATRIX Electrophoresis 2.11 Maintaining Enzyme Activity Chapter-11 Techniques used in Enzyme characterization 132 HOW TO KNOW THE PROPERTY OF ENZYME Introduction A. Chromatography Principles of chromatography Adsorption Chromatography COLUMN CHROMATOGRAPHY History Principle Mechanism of separation by size in gel permeation chromatography ADVANTAGE OF GEL –CHROMATOGRAPHY Application of GPC or gel permeation chromatography PAPER CHROMATOGRAPHY Ion –exchange chromatography There are two type of ion exchanger Choosing an Ion Exchanger Gas / Liquid Chromatography Reversed Phase Chromatography HIGH PRESSURE LIQUID CHROMATOGRAPHY [HPLC] Partition Chromatography

127

Affinity chromatography Principle METHOD OF LIGAND IMMOBILIZATION 1. CNBR activated agarose 2. 6-AMINO-HEXENOIC ACID & 1,6 DIAMINO HEXANE. 3. Carbomyl diimidazole activated agarose. Applications Thin layer chromatography Principle ADVANTAGE OF TLC OVER OTHER CHROMATOGRAPHY TECHNIQUE HPTLC and HPPLC Selection of chromatographic system Electrophoretic technique a. Agarose Gel Electrophoresis b. Polyacrylamide Gel Electrophoresis c. SDS-PAGE f. Iso-electric focusing: IEF gel: g. Chromatofocussing 2D-PAGE DETECTION, ESTIMATION AND RECOVERY OF PROTEIN GELS. i. AGAROSE GEL ELECTROPHORESIS OF DNA Preparation of the gel Polyacrylamide gel Chapter -12 Enzyme Immobilization 160 Introduction Aim of Enzyme Immobilization [a] Physical properties [b] Chemical properties [c] Stability [d] Resistance [e] Safety [f] Economic Limitations of Immobilized Enzyme Advantages of Immobilizations Methods of immobilizations Carrier matrices Adsorption of enzymes Some disadvantages of adsorption include Covalent coupling Functional groups that affects the covalent coupling A. cyanogen bromide (b) Ethyl chloroformate (c) Carbodiimide (d) Glutaraldehyde (e) 3-aminopropyltriethoxysilane Entrapment and Encapsulation Entrapment of enzymes Membrane confinement Encapsulation of Enzymes Crosslinking Chapter-13 Introduction to Industrial biotechnology 171 Application of Immobilised-Enzyme Processes Introduction 172 1-High-fructose corn syrups (HFCS) 2-GLUCOSE ISOMERASE a Treatment with activated carbon.

3-Use of immobilised raffinase 4-Use of immobilised Invertase 5-Production of amino acids 6- Use of immobilised lactase .7 Production of antibiotics Preparation of acrylamide Chapter-14 Application of Enzyme in clinics 233 Introduction Determination of enzyme activities 1- Clinical Enzymology of liver disease. 2- In Heart disease α-amylase Creatine Kinase and fructose bisphosphate aldolase. Alkaline phosphates Acid phosphatase. In Treatment of cancer Enzyme deficiencies Enzyme inhibitors and drug design 183 Use of enzyme in Dignosis Blood glucose Disadvantages of Using Enzyme as Therapeutic Agents Thermozymes The large-scale use of enzymes in solution The use of enzymes in detergents Chapter-15Application of Enzyme in Industries different sector of industry where enzymes are used Food Textile and other indsutries Chapter-16 Enzyme Reactors all type of reactors detailed dscription kinetics involved in reactors Chapter-17 Protein Engineering 215 Artificial enzymes Chapter-18 Biosensors and Immunosensors 225 Chapter-19 Enzyme and protein STABILITY 246 I Definition of Stability II The Unfolded State III Major Factors Affecting Protein Stability The Hydrophobic Effect Hydrogen Bonds Conformational Entropy of Unfolding IV Other Factors Affecting Protein Stability Salt Bridges Aromatic-Aromatic Interactions Metal Binding Disulphide Bonds V Chemical Degradation VI Conclusions Chapter -20 Study of drug and molecule binding stratgies 260 TYPES OF DNA BINDING DRUGS NON COVALENT INTERACTIONS Minor Groove binders INTERCALATORS COVALENT INTERACTIONS DNA-Drug Interaction : Chapter-21 Technique to study protein 284

Nuclear Magnetic Resonance(NMR)Spectroscopy Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin. Structure of bacteriorhodopsin: Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin Chapter-22 Enzymology of DNA folding and unfolding 290 WHAT IS DNA Major and minor grooves Replication Enzyme involved in DNA folding and unfolding Invitro-DNA folding and unfolding Folding unfolding kinetics Chapter-23 Safety and regulatory aspects of enzyme use 305

Module I Enzymes: Introduction and scope, Nomenclature, Mechanism of Catalysis. Chapter- 1 Enzymes an introduction Definition Calorimetry: Measurement of heat released or taken up in a chemical reaction or physical process to derive thermodynamic data (e.g., dissociation constant, freeenergy change, enthalpy change, entropy change) for molecular interactions. Two kinds of calorimetry important to biomolecular studies are isothermal titration calorimetry (ITC), which can be used to detect the number of binding sites on an enzyme; and differential scanning calorimetry (DSC), which monitors. Enzyme: Protein that acts as a catalyst, speeding the rate of a biochemical reaction but not altering its direction or nature. Electrophoresis: Method of separating large molecules (such as DNA fragments or proteins) in a sample. An electric current is passed through a medium containing the sample; each molecule travels through the medium at a different rate, depending on its electrical charge, shape and size. Agarose and acrylamide gels are commonly used media for electrophoresis of proteins and nucleic acids. In onedimensional (1D) gel electrophoresis, proteins and nucleic acids are separated in one direction on a gel, primarily by size. Two-dimensional (2D) gel electrophoresis is used in proteome analyses to separate complex protein mixtures using two separation planes (e.g., vertically down a gel by net charge and horizontally by molecular mass). Each unique protein mixture produces a characteristic pattern or fingerprint of protein separation on a 2D gel.

Flow Cytometry: Analysis of biological material by detection of light-absorbing or fluorescing properties of cells or subcellular components (e.g., chromosomes) passing in a narrow stream through a laser beam. An absorbance or fluorescence profile of the sample is produced. Automated sorting devices, used to fractionate samples, separate successive droplets of the analyzed stream into different fractions depending on the fluorescence emitted by each droplet. Kinetics: Field of study that deals with determining the rates of biological, chemical, and physical processes (e.g., how quickly reactants are converted into products) under various conditions. Metalloprotein: Protein that incorporates one or more metals into its molecular structure by binding individual metal ions [e.g., iron (Fe2+ or Fe3+), zinc (Zn2+), or magnesium (Mg2+)] or nonprotein organic compounds containing metals. Metalloprotein are important components of electron transport chains. Steady State: Growth state in which the concentration of bacterial cells is in equilibrium with the concentration of nutrients or substrates (i.e., the concentrations remain constant over time). X-ray crystallography: Technique used to obtain structural information for a substance (e.g., protein, molecular complex) that has been crystallized. A beam of X rays is focused on the crystals, and the scattering pattern of the X rays is used to create 3D representations of the crystal with atomic resolution.

Introduction Enzymes are the wonderful creations of the biological system. Various biological reactions go on smoothly without single mistakes. Enzymes have specificity not only toward the substrate but also recovered in the end of the reactions. They are also regulated very precisely. Normal monomeric enzyme like trypsin and pepsin are regulated by removing or adding a small piece of peptide that make them inactive or active while the complex enzymes are regulated by end product , or by allosteric mechanism. Homones in several cases also play important role in regulations like in formation of lactose by progestron and prolactin before and after the birth of baby. The system (complex enzymes are termed as system) specifically and precisely binds to the substrate and convert them into the required product that sometimes becomes the substrate for next reaction for next enzyme to act on. Total activity of enzyme depends on several factors like acid base, structural orientations and strain, electrostatic interactions (by metal ions) and formation of covalent bond. In several cases certain organic molecule or metals termed as co-enzyme or cofactor that either or electron or proton acceptor or by electrostatic mechanism are needed to convert a substrate to product. Actually enzymes activity depends on the precise function of a small part called as the active site that is a small pocket like structure that contains various helper amino acid residues that by electron or proton transport make bond of the substrate molecule weaker and rearrange them into a stable product at normal temperature and pressure. Any deficiency in the formation of these pockets like structure may render the whole enzyme to become inactive that leads to certain deficiency diseases. Modern techniques like MOLDI-TOF, x-ray crystallography along with sequencer helped a lot to scientist in deciphering the mechanism of enzyme activity. Knowledge of enzyme activity and its detailed mechanism made it possible to design artificial enzyme in the lab to cop up with many enzyme related metabolic problems or complex viral enzyme like AIDS. Historical aspects in enzyme discovery 1860 Bertholate disrupted yeast cell and isolated extract that catalyzed

e. and the second digit denotes the subclass. e. Therefore they divided the enzyme into six major classes. 1958 and 1960 1961 1965 1986 Koshland proposed induced fit model to account for enzyme catalytic power specificity.1. Urea hydrolysis is done by the Urease. 1894 sucrose to glucose and fructose.C.1. 1920 Sumner crystallized Urease first time that hydrolyze the urea into CO2 and NH3. Here S lactate is the substrate and NAD+ is electron acceptor/ hydrogen acceptor and the reaction is of type oxidation reaction. Ribonuclease was artificially synthesized from amino acid precursor. For clear understanding we can take example of S-latate NAD+ oxidoreductase (E. Classes of enzyme: there are six classes of enzyme according to reaction type they performed 1.U.conversion of 1878 Word enzyme first used by kuhne. RNase was found to work as catalyst by Cech. For example 1-for NAD+ or NADP+ 2. substrate they act. oxidases.B.27). In EC number 1 digit denotes type of reaction i. Fisher proposed lock and key hypothesis to explain the mechanism of Enzyme operating between substrate and enzyme. Enzymes such as dehyrogenases. while 4th digit is the actual substrate. position of group or functional group and number of amino acid. Each enzyme has assigned 4 digit classification number and a systematic name. 1. Oxidoreductases. EC 2.g. There are major 6 classes and each with the subclass. For oxygen. three dimensional structure of Lysozyme was deduced. Its third digit refers to the hydrogen or electron acceptor. such as alcohol dehydrogenase. . 1897 Buchner isolated yeast extract used for formation of ethanol. third and four digits is the group transfer or accepted as shown in figure. For e. Its second digit has been denoted in the table. In 1955 International Union of Biochemistry classified the enzyme on the basis of kind of reaction they performed. system also specifies a textual name for each enzyme. most enzymes are still called by their common name. and peroxidases. Oxidation means transfer of oxygen atom and reduction means transfer of H atom. The enzyme's name is comprised of the names of the substrate(s). are widely known in the scientific community by their common names.1 means first digit indicates the class transferases.approved nomenclature has been slow. In everyday usage.g. Ribonuclease amino acid was sequenced. Suffix added in the enzyme is –ase to the name of substrate or to word. Ribonuclease –P Classification of Enzymes. 3rd digit denotes NAD as electron acceptor NAD+.7. the change to I.1. oxidoreductase 2nd digit is the functional group of lactate which is alcohol therefore it is denoted by subclass 1. the product(s) and the enzyme's functional class. For Fe+3 3.These are enzymes that catalyze oxidations or reductions. The I.B.U. Because many enzymes. They were called as ribozymes.

Its third digit denotes the group transferred.Lactate dehydrogenase CH3CH(OH)COO.4 Acting on the CH-NH2 group of donors EC 1.9 Acting on a heme group of donors EC 1.3 Acting on the CH-CH group of donors EC 1.7 Acting on other nitrogenous compounds as donors EC 1.These enzymes catalyze the removal of groups and create double bond in non-aqueous media. Phosphotransferases are given trivial name as kinase for example hexokinases (E.2 Acting on the aldehyde or oxo group of donors EC 1.These are also called synthetases which are enzymes that catalyze condensation reactions where smaller molecules are connected with the resulting removal of a water molecule. transaminases. 3 for monoester bond. Isomerases. amylase. Its second digit has been provided in the table. 2 for thiol ester bond.Enzymes that catalyze the isomerization of molecules means L isomer will be D isomer after catalysis and cis isomer will be trans isomer after the catalysis for Examples L alanine will be converted into the D alanine by alanine racemases.6 Acting on NADH or NADPH EC 1.+ NADH+ + H+ 2. 3 for ether bond. Subclass Name EC 1 Oxidoreductases EC 1. third digit denotes the type of bond hydrolysed like 1 for hydrolysis of ester bond.C. 4 for peptide bond etc. Its second digit denotes the bond broken and third digit denotes type of group removed like 1 for carboxyl group and 2 for aldehyde group and 3 for ketoacid group. Lyases. 2.7. 4. Examples such as Phosphatases. Transferases.1 or D-hexose-6-phosphotransferase. 6. For example alkaline phophatases is a hydrolase that hydrolyse variety of substrate at alkaline pH. maltase.5 Acting on the CH-NH group of donors EC 1. Thus 1 denotes transfer of CH3 group while 2 denotes the transfer of CH2OH and 3 denotes the carboxyl or carbamoyl transferases. An example would be the amino acid RNA Ligases.+ NAD+ ---------. An example would be the decarboxylases. They are classified according to the type of bond hydrolysed 1 for ester 2 for glycosidic bond.1. and lactase. 4 for phosphoric diester hydrolases.10 Acting on diphenols and related substances as donors . and transmethylases.8 Acting on a sulfur group of donors EC 1. D-hexose-6-phosphotransferase (hexokinase) D hexose + ATP-----------------------------------phosphate + ADP D hexose-6- 3. L histidine carboxy lyase histidine ------------------------------------ histamine + CO2 5. This is accompanied with the formation of a high energy Phosphate link that stores energy.CH3C(O)COO.1 Acting on the CH-OH group of donors EC 1. Ligases.These enzymes catalyze the transfer of a group from one molecule to another. 7 for phosphate group. 2nd digit refers to the type of reaction like 1 for recemization or epimerization (if inversion occurs at assymitric carbon having four different attached group. Hydrolases-These enzymes catalyze hydrolysis reactions. 1 for single carbon unit) that catalyses the transfer of phosphate from ATP to D hexose resulting the D hexose-6-phosphate. Examples are the digestive enzymes such as sucrase.

2 cis-trans-Isomerases EC 5.2 Transferring aldehyde or ketonic groups EC 2.9 Acting on phosphorus-nitrogen bonds EC 3.99 Other isomerases EC 6 .2 Glycosylases EC 3.11 Acting on carbon-phosphorus bonds EC 3.12 Acting on hydrogen as donor EC 1.2 Carbon-oxygen lyases EC 4.99 Other lyases EC 5 Isomerases EC 5.15 Acting on superoxide radicals as acceptor EC 1.6 Phosphorus-oxygen lyases EC 4.4 Acting on peptide bonds (peptidases) EC 3.8 Acting on halide bonds EC 3.3 Acting on ether bonds EC 3. with incorporation or reduction of molecular oxygen EC 1.3 Acyltransferases EC 2.14 Acting on paired donors. other than methyl groups EC 2.19 Acting on reduced flavodoxin as donor EC 1.3 Intramolecular isomerases EC 5.7 Transferring phosphorus-containing groups EC 2.16 Oxidising metal ions EC 1. other than peptide bonds EC 3.5 Carbon-halide lyases EC 4.13 Acting on single donors with incorporation of molecular oxygen (oxygenases) EC 1.21 Acting on X-H and Y-H to form an X-Y bond EC 1.4 Carbon-sulfur lyases EC 4.8 Transferring sulfur-containing groups EC 2.1 Transferring one-carbon groups EC 2.4 Intramolecular transferases (mutases) EC 5.5 Transferring alkyl or aryl groups.13 Acting on carbon-sulfur bonds EC 4 Lyases EC 4.6 Transferring nitrogenous groups EC 2.4 Glycosyltransferases EC 2.7 Acting on carbon-carbon bonds EC 3.17 Acting on CH or CH2 groups EC 1.1 Racemases and epimerases EC 5.12 Acting on sulfur-sulfur bonds EC 3.20 Acting on phosphorus or arsenic in donors EC 1.EC 1.1 Carbon-carbon lyases EC 4.6 Acting on acid anhydrides EC 3.97 Other oxidoreductases EC 2 Transferases EC 2.9 Transferring selenium-containing groups EC 3 Hydrolases EC 3.11 Acting on a peroxide as acceptor EC 1.3 Carbon-nitrogen lyases EC 4.18 Acting on iron-sulfur proteins as donors EC 1.5 Intramolecular lyases EC 5.5 Acting on carbon-nitrogen bonds.10 Acting on sulfur-nitrogen bonds EC 3.1 Acting on ester bonds EC 3.

Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligase Electron transfer Group transfer Hydrolysis Addition of group to double bonds Transfer of group to yield isomeric forms.3 EC 6.C-O.Ligases EC 6.C-S.4 EC 6. Cytochrome oxidase Carbonic anhydrase Alcohol dehydrogenase Hexokinase Glucose -6-phosphate .2 EC 6.C-N.6 SN 1 2 3 4 5 6 Forming Forming Forming Forming Forming Forming carbon—oxygen bonds carbon—sulfur bonds carbon—nitrogen bonds carbon—carbon bonds phosphoric ester bonds nitrogen—metal bonds Class Reaction catalyzed.5 EC 6. Some metalloenzymes and their cofactor Enzyme Cofactor Cytochrome oxidase Catalase Peroxidase. Formation of C-C.1 EC 6.

and RNAase.Pyruvate kinase. Simple enzymes They are composed solely of protein. single or multiple subunits. • hydrogenases. Alpha Amylase. Chapter-2 Types of Enzymes A. Other enzymes have retained common names attributed to them. Lactase. If they consist of single polypeptide unit they are termed as monomeric enzyme ( sometime like in chymotrypsinogen they are of three polypeptide and are inctive zymogen form and . Catalase.Enzymes that catalyze oxidations In addition each enzyme has a specific name which usually consists of part of the name of the substrate molecule. • Hydrolases-Enzymes that catalyze hydrolysis reactions. and others. Chymotrypsin. Trypsin. Maltase. Arginase Ribonucleotide reductase Pyruvate kinase. Urease Dinitrogenase Glutathione peroxidase Fe+2 or Fe+3 Cu+2 Zn+2 Mg+2 Mn+2 K+ Ni+2 Mo Se In addition to the classification name most enzymes have group names. Lysozyme.Enzymes that catalyze the addition of Hydrogen atoms • Oxidases. Pepsin. Enzymes such as. galactosidase. Examples are sucrase.

it is often useful to consider coenzymes to be a special class of substrates. the coenzymes donate the carried chemical grouping to an acceptor . which are composed of protein plus a relatively small organic molecule. Those have a lower affinity for metal ion. H2M2. Thus they contain four subunit and substrate can bind to each of four subunit and they are termed as isozyme. but still require the metal ion for activity. when the binding between the apoenzyme and non-protein components is non-covalent. while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds. In all cases. They form the integral part of the enzyme and remain adjacent to the catalytic center to provide coordination for interacting with substrate functional group. Entire complex called “holoenzyme”. In this terminology the protein component is known as the apoenzyme. B. or second substrates. that are termed as serine proteases because of an active serine residue. Non-protein part called “coenzyme” or “prosthetic Group”. and M4. which lack sufficient cofactors derived from vitamins to maintain homeostasis. H3M.000 kd (kilodalton) like trypsin . Enzymes composed completely of protein are known as simple enzymes in contrast to complex enzymes. Thus a slight decrease in entropy make free energy negative that is essential for stabilization of intermediate transition state). Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions that is with very high affinity. are known as metal-activated enzymes. Serine residue become active due to relay mechanism of electron transfer (adjacent amino acid can transfer electron in series of pathway and make the serine active sufficient to active at electron deficient or carbocation center of substrate making breaking of bond possible. Lactate is formed from pyruvate in aneorbic condition and thus pyruvate accumulation are avoided. Since coenzymes are chemically changed as a consequence of enzyme action. Lactate arev then transported to other organs. The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. Monomeric enzymes are of few 200-300 amino acid and molecular weight ranges from 15. Role of Coenzymes The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. Complex enzymes are also known as holoenzymes. Like alcohol dehydrogenase (contains two polypeptide and Zn+2 and act by electrostatic meechansim) and lactate dehydrogenase that requires cofactor NAD+ or NADP+ for its activity and occurs in multiple unit like H4. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. These rules give each enzyme a unique number. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes. The detailed mechanism will be discussed in next topic under the head of enzyme specificities. Complex enzymes Complex enzymes They involve protein plus small organic molecule(s) or metal.000 kd to 35. HM3.after cleavage from pie to delta and finally to alpha trypsinogen they become active and consist of two polypepide and still they are termed as monomeic enzyme since they are read in continous sequences). which are common to many different holoenzymes. The chemical groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD. Actually Ogstien considered three sites on enzyme one or two binding site along with one catalytic subunit. the small organic molecule is called a coenzyme. Enzymes are also classified on the basis of their composition. Protein part called “apoenzyme”. Therefore multiple substrates can interact with enzyme and react with them. elastases. Other enzyme consisting of two or more polypeptide are termed as oligomeric enzyme. or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate.

Almost every significant life process is dependent on enzyme activity. Nickel. Indeed. B. the assay and pharmacological regulation of enzymes have become key elements in clinical diagnosis and therapeutics. Acids and Bases in solution serve as catalysts in a wide variety of Organic reactions. RNA) protein enzyme. Intracellular enzymes (enzyme located inside the cell and can be isolated after breaking the wall of the cell) catalyze the reactions of metabolic pathways. There are two types of catalysts: 1. except for a class of RNA modifying catalysts known as ribozymes. One enzyme molecule might be responsible for converting thousands of reactant molecules called substrate molecules into product. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst. since (unlike the usual substrates. Most industrial catalysts are responsible for more than one catalysis among reactants and are considered relatively non-specific in what they catalyze. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the Phosphodiester bond of other RNAs. The macromolecular components of almost all enzymes are composed of protein. and enzymes of the circulatory system are responsible for regulating the clotting of blood. Heterogeneous Catalysts 2. most enzyme molecules catalyze only one specific reaction but it does so in a phenomenally efficient manner. This is sometimes referred to as surface catalysts. . Enzymes are found in all tissues and fluids of the body. The Biological Catalysts are efficient in lowering down the activation energy of the reaction and thus they occur at very low temperature and pressure. Enzyme mediates rearrangement of the bond in substrate molecule by breaking of one covalent bond and formation of other covalent bond. and Iron serve as industrial catalysts that catalyze a wide variety of reactions such as Hydrogenation. Because of their role in maintaining life processes. All the chemical reactions inside the cell take place with the help of enzyme. Homogeneous Catalysts Heterogeneous catalysts are those that provide a surface for the reaction to proceed upon. Enzymes increase the rate of reactions without involving themselves being altered in the process of substrate conversion to product They mediate the formation of several biomolecules like Polynucleotide (DNA. Certain transition state metals like Palladium. The catalyst and the reactant molecules are not in the same phase. starch in plants. A. Platinum. Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. All current research suggests that enzymes are extremely specific in what a given enzyme catalyzes. The bond of the substrate molecule comes under strain due to presence of several functional group of different type over amino acid residue present inside the active site of the enzyme. Biocatalyst or Enzymes Enzymes are the protenaceous catalyst of the biosphere.molecule and are thus regenerated to their original form. which are used up during the course of a reaction) coenzymes are generally regenerated. Catalysts Catalysts are substances that increase product formation by lowering the energy barrier (activation energy) for the product to form and increase the favorable orientation of colliding reactant molecules for product formation to be successful. Homogeneous catalysts are catalysts that exist in the same phase as the reactant molecules usually in a solution. Plasma membrane enzymes (membrane bound enzyme) regulate catalysis within cells in response to extracellular signals (signal may be any chemical or any substrate molecule). Enzymes are very different. ATP etc.

Molecule is bounded by the active sites and acted upon by the enzyme is called as substrate. vacuoles. found inside the cell. These biological catalysts are physiologically important because they speed up the rates of reactions many folds (up to 10 6 to 10 15 times than normal one) that would otherwise be too slow to support life. the taq polymerase isolated from the thermus aquaticus. How enzymes speed up the reaction without taking part? In cells and organisms most reactions are catalyzed by enzymes. and their supernatant is utilized for further purification and isolation by NH4 SO4. Transition state. Actually an enzyme decreases the free energy between the ground state and the transition state. They have pocket like structure called as active sites ( is only small portion of total protein and contains substrate binding site and catalytic site. and aqueous environment. Catalyst or enzymes decreases this requirement by lowering down the activation energy. This difference of energy is called as activation energy. Enzymes are active at certain optimum temperature. An enzyme provides specific environments found inside the cells. mild temp. like cell cytoplasm. which are regenerated during the course of a reaction. E P Reaction coordinate R= reactant P = product ∆G = ∆H – T ∆S this is the thermodynamical term used to explain the stability of reactions. A negative free energy stabilizes the system and makes the reaction possible. They are specific towards the substrate and remains in inactive form inside the cell. In recent years Enzymes are in more demand due to their application in various biotech and pharmacy industry. pH. Enzymes never disturb the equilibrium. In unstabilized or uncatalysed system ∆S is positive and formation of transition complex renders them lowering of entropy leading to make overall free energy negative. The rate of reaction can be increases by increasing the temperature.sometimes by as much as one millionfold. Their isolation involves the breaking of cell membrane. In old days renine (new name chymosine) were utilized for the formation of cheese from milk. ∆H denotes the enthalpy and ∆S denotes the entropy. Catalysts speed up the . One such enzyme is utilized in the PCR polymerase chain reaction. Most of the study done regarding the ES complex since this complex decides overall rate of reactions means formation of products. A more ordered system also has lower entropy for example free amino acid has more entropy than the amino acid linked with the peptide bond. ES Free energy G Enzyme lowers the activation energy. but more typically by about one thousand fold. Catalytic site contains various amino acid residue which reacts with substrate and form a complex structure). They are found in all the living system except the viruses.Enzyme may occur anywhere inside the cell. Enzymes increase reaction rates--. Higher activation energy reflects the slower reaction. and cell organelle. Enzymes mostly mediate formation of specific isomer either L (+) or D (-). Most of the biological molecules are stable in the neutral pH. See graph in the figure. Enzymes can be active at higher temperature and pH if isolated from the extremophiles bacteria living in the harsh condition.

The amount of energy required to achieve the transition state is lowered. at any instant a greater proportion of the molecules in the population can achieve the transition state. This complex is known as the activated state or transition state complex for the reaction. Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. ethanol is the preferred substrate. For example. Enzymes and other catalysts accelerate reactions by lowering the energy of the transition state. The enzymes known as racemases . the ratio of the rate constants remains the same in the presence or absence of enzyme. In the case of ADH. while succinic dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its substrate is invariably succinic acid. ranging from methanol to butanol. the same is not always true of substrates they attack. Unit of activity Micromole substrate consumed or product formed per minute is referred as IU international unit. Enzymes that attack D sugars will not attack the corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. alcohol dehydrogenase (ADH) always catalyzes oxidation-reduction reactions but attacks a number of different alcohols. Enzymes increase reaction rates by decreasing the amount of energy required to form a complex of reactants that is competent to produce reaction products. enzymes having broad substrate specificity are most active against one particular substrate. SI unit is ‘katal’ that is defined as Micromole substrate consumed or product formed per second. Generally. Chapter 3 Enzyme Specificity Although enzymes are highly specific for the kind of reaction they catalyze. The free energy required to form an activated complex is much lower in the catalyzed reaction.forward and reverse reactions proportionately so that. Since the equilibrium constant is equal to a ratio of rate constants. consequently. The result is that the reaction rate is increased. although the magnitude of the rate constants of the forward and reverse reactions is are increased. it is apparent that enzymes and other catalysts have no effect on the equilibrium constant of the reactions they catalyze.

The detection of specific LDH isozymes in the blood is highly diagnostic of tissue damage such as occurs during cardiac infarct. Therefore further cleavage occurs to make them active exposing the catalytic center.provide a striking exception to these generalities. the role of racemases is to convert D isomers to L isomers and vice versa. etc. each of which uses the same substrate(s) and produces the same product(s). alkaline phosphatase. This is the reason why sometime in presence of non-polar solvent like DMSO dimethylsulphoxide some enzyme are more reactive. ie. For example of several seine proteases like chymostrypsinogen.g. or carboxypeptidase which snips the C-terminal amino-acid off many polypeptide chains. As enzymes have a more or less broad range of substrate specificity. The best studied set of isozymes is the lactate dehydrogenase (LDH) system. or act over by specific substrate like glucokinase that transfer phosphate from ATP to glucose. They may be of product specific or substrate specific or stereospecific act on specific stereo molecules like alanine recemase acts on only L–alanine to convert them into Dalanine.. and C4 alcohols.g. Active site most often contains both polar as well as non-polar amino acid thus facilitates the interaction with both hydrophilic and hydrophobic amino acid. Specificity May Be Very Strict Or Relatively Broad Broad specificity is shown by many degradative enzymes e. Specificity is actually decided by amino acid residue present in the active site of enzyme that is capable of binding to the substrate molecule. but they exhibit differing degrees of efficiency. LDH is a tetrameric enzyme composed of all possible arrangements of two different protein subunits. alcohol dehydrogenase. The all H isozymes is characteristic of that from heart tissue. Therefore both the subunit of enzyme like binding and catalytic site is the active center of the enzymes. These subunits combine in various combinations leading to 5 distinct isozymes. in fact. alcohol (ethanol) dehydrogenase of E. Aspartase catalyses the interconversion of aspartate and fumarate: L-Aspartate ↔ Fumarate + NH3 . These are the products of genes that vary only slightly. Other examples include chymotrypsin. The amino acids that are not involved in binding to the substrate do not have any contribution towards the specificity. They exhibit absolute specificity. which removes phosphates from a wide range of molecules. often. In Elastases substrate are blocked from binding due to presence of two bulky side chain valine and threonine while chymostrypsin and trypsin have no bulky group but instead contains glycine amino acid that facilitates the substrate to approach easily. and character and must not block the substrate from binding. Intermediate specificity is most common e.Some enzymes are absolutely specific. Thus enzyme may be group specific if they act over on several closely related substrate for example alcohol dehydrogenase on which several alcohol are substrate. Thus racemases attack both D and L forms of their substrate. These isozymes all catalyze the same chemical reaction. Replacement of glycine by alanine make the enzyme more active. and the all M isozymes is typically found in skeletal muscle and liver. For proper catalytic activity their side chains must be of suitable size. trypsin and elastases having almost similar structure but differ in specificity due to variations in the side chain amino acids. The individual members of a set of enzymes sharing such characteristics are known as isozymes. the subunits are known as H (for heart) and M (for skeletal muscle). For example chymotrypisn are inactive because their zymogen form don’t have accessible catalytic center. it follows that a given substrate may be acted on by a number of different enzymes. a variety of enzymes from different classes have been shown to function in nonaqueous media. C3. various isozymes of a group are expressed in different tissues of the body. pyruvate dehydrogenase will also use the C4 homolog of pyruvate etc. shape. They are only small part of the total enzyme and they must be accessible to the substrate. coli will act on C2.

An active site is an indentation or cavity whereby a reactant molecule (substrate) is attached to. 3Transition state stabilization ENZYME MECHANISM MODELS There are two major considerations . showing asymmetric binding site in the enzymes. and the product molecule can disengage itself from the active site thus freeing the site for another incoming substrate molecule. The actual reaction occurs in the active site. This has the effect of inhibiting enzyme activity. Such enzymes follows michaelis behaviour while others having complex and separate site behave in more complex way as allosteric site. A. Furthermore. The analogy is like a hand .Fig . small temperature changes and small changes in pH will not result in the enzyme being inhibited from catalyzing its intended reaction. 1Lock and key hypothesis 2Induced fit hypothesis. a small region of the enzyme where the substrate is bound. One of the first theories or models that help to explain this phenomenally efficient catalytic efficiency of enzymes is called the "lock and key" model. Evidence does not support these assumptions. Enzymes must bind the correct substrate and position it correctly relative to catalytically active groups in the active site. Nor will it add NH3 to maleate. The model of enzyme structure was proposed by fisher as three point one catalytic site and others are binding site. Lock and Key Model for Enzyme Activity The high specificity and efficiency of enzymes can be explained by the manner that they associate with the reactant molecules called the substrate. This does not seem to support the ridged active site assumption in the lock and key model. Once the product molecule has been formed the electrical attractions that made the substrate molecule adhere to the active site no longer are present. This model assumes that molecules that lock into the active site must form a perfect fit. Modification of the lock and key model is necessary to account for the occurrence of pH and temperature ranges of optimum enzyme activity and to explain why other molecules can effectively block the active site. Three theory were proposed to explain the specificity of enzyme but all of them are correct and none of them are alone operative in the enzyme specificity. The polar and non-polar groups of the active site attract compatible groups on the substrate molecule so that the substrate molecule can effectively lock into the cavity and position itself for the necessary collisions and bond breaks and formations that must take place for successful conversion to a product molecule. According to this model. This is called the enzyme-substrate complex. This process occurs in a highly efficient manner hundreds or even thousands of times in a short time span. Induced Fit (Hand and Glove) Model of Enzyme Activity Modification of the lock and key model assumes that the active site has a certain amount of elasticity whereby the active site can expand or contract in a limited way in order to accommodate the substrate molecule. Most enzymes are very large relative to their substrates. each enzyme molecule may have as few as one active site on the surface of the enzyme molecule itself. Sometime enzymes have both catalytic as well as binding site as same site. It will only use aspartate (not similar amino acids such as glutamate) and only the L-isomer. For example. certain molecules can lock into the active site even though the bogus molecules have a different shape compared to the true substrate molecule.substrate specificity and catalytic power. B. Also the assumption is that the active site conformation is ridged. The occurrence of pH and temperature ranges of optimum enzyme activity does not support the assumptions made by the lock and key model of ridged active site cavities. the cis isomer of fumarate.

They are engaged in the catalytic turn-over of the substrate. rather large.the induced fit. Mechanisms of Enzyme Catalysis Enzymes are proteins. induced fit. The two substrates are brought together in the active site by a conformational change. Small changes in temperature would distort the active site conformation but not so much that the active site could not still accommodate the substrate molecular size. Its uniqueness is caused by the sequence and nature of its amino acid side chains. which again are the basis for the spatial arrangement (conformation) of the molecule and which help to maintain this structure by stabilizing it. This tolerance would explain why bogus molecules of slightly different size compared to the true substrate molecule can still be accommodated by the elastic active site. Phosphoglycerate kinase catalyzes the synthesis of ATP. That explains the specificity of catalysis and the selectivity for a certain substrate (and also that for additional regulatory factors). Lipases are enzymes that hydrolyze esters of long chain fatty acids.fitting into a glove. It is this structure that allows an enzyme to perform its catalytic activity. In that way. Enzyme molecules are. compared to most of their substrate molecules. Often. The activation of the lipases at the interface is a result of a conformational change. why reactions take place at an enzyme's surface that occur in solutions only after a considerable amount of activation energy has been . the onedimensional information that was stored in the genome as a DNA sequence is first transcribed into mRNA and after translation into an amino acid sequence finally transformed into a three-dimensional structure. pockets. Such enzymes will have a lower value of kcat/KM (a numerical value to compare the catalytic efficiency of enzyme). Numerous enzymes depend on additional factors that are necessary to perform the catalytic reaction. the binding is either reversible (achieved through weak interactions) or irreversible (covalent bonds). because some of the binding energy must be used to support the conformational change in the enzyme. Induced fit assumes that the active site of an enzyme is not complementary to that of the transition state in the absence of the substrate. grooves. hollows etc. Induced fit example Hexokinase catalyzes the ATP-dependent phosphorylation of glucose. The binding of glucose to the enzyme induces a major conformational change . Induced fit increases KM without increasing kcat. the enzyme's surface is structured in a way that the coenzyme is bound to a specifically shaped pocket. In other words: the enzyme binds first to a coenzyme like NAD or FAD. PH changes which would also change the active site conformation but not so much that the active site could not flexibly accommodate the substrate molecule. caused by the enzyme binding to the interface. Atoms or ionized groups of the coenzyme take part in the catalytic reaction. Furthermore. These enzymes show virtually no activity against water-soluble substrates and only hydrolyze substrates present in the interface between water and lipid interfacial activation. This conformational change exposes the active site of the enzyme. These properties explain. certain amino acid side chains are exposed at the active centre. The glove adjusts in shape and size to fit various sized hands within a certain range. Depending on the type of molecule. The Induced Fit Model seems to explain why there is some flexibility in the abilility of the active site to accommodate other molecules and at limited temperature and pH ranges. They form a number of weak interactions. Their surface is not evenly structured but displays dent-ins. The shape of the holoenzyme (= apoenzyme [protein] + coenzyme) causes the substrate selectivity. An induced fit at an interface. The part that binds to a substrate molecule is termed the active centre or substrate-binding site and is characterized by a shape complementary to that of the substrate molecule. Every protein is determined by its amino acid sequence (its primary structure) and its tertiary structure (the three-dimensional folding of the polypeptide chain).

The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity. 5. . but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to be altered. 3. The possible mechanisms of catalysis are five in number: 1. there is no loss in translational or rotational energy in going to the transition state. the induced structural rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds. and DS‡ would therefore be negative.state complexes and reaction products. b. • This is paid for by binding energy. one or more mechanisms of catalysis generate transition. Chapter 4 Enzyme-Substrate Interactions The favored model of enzyme substrate interaction is known as the induced fit model. The collision theory states that the event of two molecules meeting in solution in a way that a bond can form between them is rather improbable.supplied. temperature). • If DG‡ is the change in free energy between the ground state and the transition state. The binding sets reactants into a state of enhanced reactivity recognizable by the close vicinity and the right orientation towards each other. Therefore. Catalysis by Bond Strain In this form of catalysis. the transition state would be significantly more ordered than the ground state. In addition to inducing strain. 2. existing substrate bonds. The new conformation often forces substrate atoms and bulky catalytic groups. • The formation of a transition state is accompanied by losses in translational entropy as well as rotational entropy. into conformations that strain. After binding takes place. then DG‡=DH‡–tDS‡. In solution. and their proximity and orientation toward the substrate thus favors their participation in catalysis. which more easily attain the transition state. This model proposes that the initial interaction between enzyme and substrate is relatively weak. Enzymatic reactions take place within the confines of the enzyme active-site wherein the substrate and catalytic groups on the enzyme act as one molecule. general base catalysis Catalysis by electrostatic effects Covalent catalyis (nucleophilic or electrophilic) Catalysis by strain or distortion a. 4. Catalysis by approximation General acid. Catalysis by Proximity and Orientation • Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another. groups such as aspartate are frequently chemically reactive as well. such as aspartate and glutamate. though the probability can be considerably increased by the supply of energy (pressure.

thereby causing hydrolysis of the peptide bond. One of the best-known examples of this mechanism is that involving proteolysis by serine proteases. Amino acids with extra carboxyl or amino groups can obviously act as acids or bases. • General acid catalysis involves donation of a proton by the catalyst. in order to have appropriate concentrations of each buffer species. • General acid-base catalysis is a commonly employed mechanism in enzyme reactions. Most hydrolytic enzymes use acid/base catalysis. histidine reacts very fast (protonation/deprotonation of imidiazole ring has a reaction half-time of less than 10-10 seconds at neutral pH). Histidine is rarely found in proteins except at catalytic sites.g. Concerted acid-base catalysis Providing both acid and base simultaneously is impossible in free solution. or contain an electronegative atom . e. His. phosphate group reactions. Tyr and Lys can be involved in general acid-base catalysis.. the leaving group must be protonated. the buffer aids in stabilizing the transition state via donation or removal of a proton. the use of glutamate as a general acid catalyst (proton donor). d. –OH or H+ accelerates the reaction. The reaction rate is dependent on pH only. Cys. addition to carbonyl • groups. • Specific acid–base catalysis means specifically. In the second step (collapse of the tetrahedral intermediate). Also. or contain unfilled valence electron shells.c. the substrate is oriented to active sites on the enzymes in such a way that a covalent intermediate forms between the enzyme or coenzyme and the substrate. which donate or remove protons to (or from) the transition state intermediate. The general acid–base is best when its pKa is near that of the pH of the solution. Glu. General acid-base catalysis is due to proton donors or proton acceptors. In addition. These proteases contain an active site serine whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond. for example. Therefore. which include both digestive enzymes (trypsin. the rate of the reaction is dependent on the buffer concentration.0) can act as either an acidic or basic catalyst depending on whether or not it is protonated. • General acid-base catalysis is involved in a majority of enzymatic reactions. yet enzymes can do this. chymotrypsin. General acid–base catalysis needs to be distinguished from specific acid–base catalysis. histidine (pK around 7. as well as the appropriate protonation state. and elastase) and several enzymes of the blood clotting cascade.ions which temporarly donates proton or accept proton. • General base catalysis involves abstraction of a proton by the catalyst. • Specific acid-base catalysis is due to H+ or OH. • In General acid–base catalysis. • The side chains of Asp. Covalent Catalysis: • In catalysis that takes place by covalent mechanisms. Biologically important nucleophiles are negatively charged or contain unshared electrons • Biologically important electrophiles generally are either positively charged. An acidic group in one part of the active site donates a proton and a basic group in another part of the active site removes a second proton from the reaction intermediate. and not on buffer concentration. etc. hydrolysis of ester/ peptide bonds. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases) • Other mechanisms also contribute significantly to the completion of catalytic events initiated by a strain mechanism.

Serine type . Serine proteases b. Aspartate proteases d. Sometime nucleophilic catalyst form intermediate more rapidly than normal catalysis. subtilisin are serine enzymes. Phosphoenzymes usually attach the phosphate to serine or histidine. Acyl enzymes usually attach the acyl group at an active serine or cysteine residue.Covalent Intermediates Here the strategy is to lower the transition state energy "hump" by taking an alternative reaction pathway therefore sometime it is also called as alternative pathway catalysis. cleaves the . Metallo proteases This classification uses the functional group within the enzyme. Chymotrypsin and trypsin. and Elastase. They catalyze the hydrolyses of peptide bonds in proteins. and a serine. which we’ve talked about. aspartate) or a metal as co-enzyme at the active site of the enzyme. acyl group or glycosyl group. This reaction usually involves a nucleophilic group on the enzyme and an electrophilic group on the substrate Uncatalysed: BX + Y BY + X Catalysed: BX + Enz Enz-B + X Enz-B + Y Enz + BY Where. stabilizes S* over S). proteases can be characterized by their substrate affinity and the catalytic rate of the reaction. phosphotransferase system. water soluble proteins that function as enzymes. The proteases are globular. and they all have three important conserved residues–a histidine. hydrolyzes peptide bond • The serine proteases are a class of enzymes that degrade proteins in which a serine in the active site plays an important role in catalysis. • All three enzymes are similar in structure. B is usually some chemical group such as a phosphate group. a covalent bond is transiently formed between the substrate and the enzyme (or coenzyme). 4 families of proteases have been defined: a.g. The name of the protease family refers to an amino acid (e. In covalent catalysis. Being enzymes. glyceraldehyde phosphate dehydrogenase and most enzymes using acyl CoA derivatives. • The family includes among many others. Histidine type . Thrombin is crucial in the blood-clotting cascade • Subtilisin is bacterial protease • Plasmin breaks down the fibrin polymers of blood clots • Tissue plasminogen activator (or TPA). an aspartate. sequence specificity Oxyanion whole stabilizes S* over S in enzyme The catalytic triad forms tetrahedral intermediate (transition state. This analysis showed that four major functional groups are found in the catalytic site of proteases and based on this functional groups. Structural element of active site Function The main chain substrate binding unspecific binding of polypeptide segment Specificity pocket semi-specific binding of side chains. alkaline phosphatase. Using proteases to study the effects of single amino acid substitutions (mutations) on catalytic rate and substrate affinity demonstrated that these two properties are linked and that this linkage can be explained by analyzing the conformation of the catalytic or active site of the enzyme. Cysteine enzymes include papain (a protease).phosphoglucomutase. elastase. Most proteases (eg trypsin. and does not relate to the substrate specificity of the proteases themselves. Cystein proteases c.glucose 6 phosphatase.

• The enzyme-inhibitor adduct is very stable. Peptide released while H57 donates -H (from water molecule) to S195 (E+P) • Chymotrypsin cleaves after mainly aromatic amino acids. oxyanion hole stabilization. and proximity/orientation catalysis • The tetrahedral intermediate in chymotrypsin. hydrophobic pocket that binds the sidechain of the amino acid N-terminal to the site of bond cleavage (S1) – binding of appropriate sidechains to this pocket positions the adjacent peptide bond into the active site for cleavage Many serine .• proenzyme plasminogen. electrostatic. acid-base catalysis. one of which bears a negative charge. Upon hydrolysis of the protein (6 N HCl. is considered to be the transition state intermediate in the chymotrypsin reaction. It diffuses into the active. 110°C) and amino acid analysis on the hydrolysate. covalent catalysis. Chymotrypsin is a protease that cleaves peptide bonds on the carboxyl side of aromatic or large hydrophobic amino acids – the mechanism of chymotrypsin’s cleavage reaction includes covalent. proximity and orientation effects Substrate specificities in proteases are due to active site binding pockets. peptide with new N-term released from enzyme taking -H from H57 (originally S195. releasing fluoride anion. It inhibits the reaction because it blocks entry of normal substrates. Notice how their active sites are suited for these tasks. general acid-base. a novel amino acid was isolated. and cleaves after small neutral amino acids. A water molecule placed next to H57 and acyl-enzyme intermediate at S195 (EP*) 5. Upon formation of the tetrahedral intermediate. covalent bond formation between -C=O and S195 hydroxyl (nucleophilic attack). • How is it that the enzyme stabilizes this transition state intermediate? The backbone amides of gly193 and ser195 form an oxyanion hole. • Chymotrypsin substrate specificity is due almost entirely to one deep. tetrahedral intermediate as transition state. wherein a nucleophilic amino acid attacks the phosphate. • Crystal structures of chymotrypsin reveal the reason for the high reactivity of Ser 195 – Ser 195 is in a H-bonding interaction with His 57. H57 binds the -released H+ from S195 (ES*) 3. They loosely hydrogen bond to the carbonyl oxygen under attack. This results in a covalent bond between the nucleophile and the inhibitor. Substrate attaches to enzyme at the main chain binding site and specificity pocket with the scissile (peptide) bond exposed to the triad (ES formation) 2. see step 2) (EP*) 4. It was the diisopropylphosphoryl derivative of serine. the water molecule initiates a nucleophilic attack and undergoes a reaction with one -H attaching to H57 and -OH to acyl-enzyme intermediate forming again a tetrahedral transition state of the new C-terminal peptide fragment (EP*) 6. while trypsin cleaves after basic amino acids. Elastase is fairly nonspecific. acyl-enzyme intermediate and peptide bond hydrolysis. • Diisopropylflurophosphate is an inhibitor of chymotrypsin. which consists of the Ser195 adduct before departure of the leaving group. and the negatively charged oxygen is better accommodated in the oxyanion hole. It is high energy because there is a carbon surrounded by 3 electronegative atoms. which is H-bonding with Asp 102 (catalytic triad) – the catalytic triad serves to activate Ser 195 through general acid-base catalysis – catalytic triads are common in serine proteases Conformation changes from trigonal to tetrahedral in the peptide upon nucleophilic addition allow the oxyanion formed to move into a hole with two backbone amides – inside the oxyanion hole the transition state anion can form two hydrogen bonds with the amide hydrogens as donors – the enzyme cannot form H-bonds with the amide hydrogens in the oxyanion hole when the peptide is in trigonal conformation • The mechanism of chymotrypsin utilizes stabilization of the transition state. electrostatic. the resulting carbon-oxygen single bond is longer. yielding plasmin The six step reaction mechanism of chymotrypsin: 1.

It would not be possible to determine the X-ray structure in the presence of the true substrate. but ES cannot be efficiently converted to EP. S). There are 6 subsites within the crevice. enzyme covalent intermediates are very difficult to isolate. OH and SH respectively) are good electron donors the intermediates they form are unstable and react easily with the final acceptors. • Although crystal structures of other proteins had been determined previously. lysozyme was the first enzyme to have its structure determined. particularly those found in the peptidoglycan cell wall of bacteria. Look at the many hydrogen bonding contacts between the substrate and enzyme active site that enables the ES complex to form. H2O enters HO. S1 has bulky Val residues Histidine.attacks CO2 Methylation prevents H-bonding with DNA substrate • Lysozyme is a small globular protein composed of 129 amino acids. cystein prtoease Renin .An Aspartyl Protease A Metalloprotease with Substrate Carbonic Anhydrase Mechanism His64 assists in H+ removal HCO3. • It is found in many body fluids. serine and cysteine operate by nucleophilic catalysis. each of which is where hydrogen bonding contacts with . Since the whole point is that covalent intermediates should rapidly break down to release the reaction product. Their nucleophilic groups (imidazole ring. In particular.released. This analog binds tightly in the enzyme active site to form the ES complex. • The active site consists of a crevice or depression that runs across the surface of the enzyme. and is one of the body’s defenses against bacteria. The X-ray crystal structure of lysozyme has been determined in the presence of a nonhydrolyzable substrate analog. because it would be cleaved during crystal growth and structure determination. • The best studied lysozymes are from hen egg whites and bacteriophage T4. such as tears. • It is also an enzyme which hydrolyzes polysaccharide chains. it hydrolyzes the glycosidic bond between C-1 of N-acetyl muramic acid and C-4 of Nacetyl glucosamine.proteases have high similarity to chymotrypsin – trypsin and elastase are approximately 40% identical in primary sequence to chymotrypsin and their overall structures are very close • Substrate specificity of these other proteases compared to chymotrypsin can be attributed to their S1 binding pockets – Trypsin: cleaves after positively charged residues. S1 has Asp – Elastase: cleaves after small side chains (A.

• The binding of NAM4 in the chair conformation is unfavorable.-.and S-glycosyl (EC 3.2. enzymes hydrolyzing O. 18O ends up at the C-1 hydroxyl group at site D. Glu can act as a general acid to protonate the leaving group in the transition state. This distortion raises the energy of the ground state. • But the binding of residue 4 in the half-chair conformation is favorable (preferential • transition state binding).-) with number 17 (EC 3. Lysozyme belongs to the Glycosylases family (EC 3. it is known that the C-1 carbon is located between two carboxylate residues of the protein (Glu-35 and Asp-52).the sugars are made. Here .-). • Within the class of hydrolases. The lysozyme mechanism illustrates: • Transition state stabilization • Acid-base catalysis • Several residues in the protein participate in substrate binding.2.e. the conformation of the sugar is distorted in order to make the necessary hydrogen bonding contacts. while Glu-35 is protonated.17) in this group Lysozyme Lysozyme catalyzes the hydrolysis of the (1-->4) linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in bacterial cell wall polysaccharides and (1 4)-linked poly NAG (chitin). Lysozyme reaction is the hydrolysis of the beta (1-4) glycosidic bond between N-acetylglucosamine sugar (NAG) and N-acetylmuramic acid sugar (NAM) and therefore it is possible classify it as Glycosidases. Asp-52 exists in its ionized form. i. • At what position does water attack the sugar? When the lysozyme reaction is run in the presence of H218O. • Hydrolysis involves acid-base catalysis (Glu35 serves as a proton donor to the • oxygen of the leaving alcohol.1. From the X-ray structure. Asp can function to stabilize the positively charged intermediate. Glu then acts as a general base to deprotonate water in the transition state. bringing the substrate closer to the transition state for hydrolysis. In site D. The resulting carbonium ion (+) is stabilized by • the ionized side chain of Asp52 until it can react with water) • Many glycosidases utilize a covalent intermediate in their mechanism.2. This suggests that water adds at that carbon in the mechanism.1.

Binding substrates in the proper orientation. SUMMARY Metal • • • • Mn+2. inducing charge separation. This causes the local dielectric constant to be lower. Cu+2. Mn2+. In this case. Metal ions can also function to make potential nucleophiles (such as water) more nucleophilic.7 to 6-7 when it is coordinated to Zinc or Cobalt. • Ca+2. Sometimes these are used as redox centers. nitrate reductase etc.is a good • example of transition state stabilization using a bacterial glycosidase. or more susceptible to nucleophilic attack. Metalloenzymes contain tightly bound metal ions: Fe+2. Metal ions that are bound to the protein (prosthetic groups or cofactors) can also aid in catalysis. or Ca2+ Many enzymes have metal ions at the active site. • The inhibitor is a naturally occurring transition state analog of a protein substrate. Mg2+. e) Metal Ion Catalysis Nearly 1/3 of all known enzymes require the presence of metals for catalytic activity • Metalloenzymes . Which enhances charge-charge interactions in the active site. ion catalysis is widely used. The hydroxide ion is 4 orders of magnitude more nucleophilic than is water. Zn2+. • Another excellent example that clearly shows how transition state stabilization works is • found in the structure of this transferase. K+. and making the carbon more electrophilic. but acts to stabilize negative charges on the reaction intermediate. Zn+2. water isusually excluded from the active site. • Pancreas contains a small protein (6 kDa) that is a potent inhibitor of trypsin. • The three dimensional structure of the enzyme can bring several reactive side chains into close proximity in the active site. In carboxypeptidase. Fe3+. Mg+2. so allowing the hydride ion (H–) to be added to the carbon atom. often the metal ion does not get reduced or oxidized. the pka of water drops from 15. Electrostatically stabilizing or shielding negative charges. Zinc is acting as a Lewis acid. • Chloroketones are inhibitors of serine proteases. The ions are usually Na+. Co2+ • Metal Activated enzymes – only loosely bind the metal ions. . Metal-activated enzymes contain loosely bound metal ions: Na+.contain tightly bound metal cofactors such as Fe2+. It coordinates to the non-bonding electrons of the carbonyl. However. which bind in the active site and react with • His 57. Electrostatic interactions are much stronger in organic solvents than in water due to the dielectric constant of the medium. Fe+3. Electrostatic catalysis refers to the fact that when a substrate binds to an enzyme. Zn2+ polarizes the C=O of the peptide bond which is about to be broken. In alcohol dehydrogenase Zn2+ polarizes the C=O group of acetaldehyde. The interior of enzymes have dielectric constants that are similar to hexane or chloroform. Cu2+. For example. K+. Proximity and orientation effects are important in enzymatic reactions. as in cytochromes. Mediating oxidation-reduction reactions.

• Binding of the substrate in the active site can orient the substrate for most efficient interaction with these side chains. Catalytic Power Enzymes lower the energy of the transition state by stabilizing the original reaction intermediate or by providing an alternative reaction pathway. • We might think that the substrate is under stress meaning that it is subjected to forces but not distorted by them. • When the active site is complementary to the transition state the full substrate binding energy is only realized as the reaction reaches the transition state. • Having the active site bind the transition most strongly also means that products are bound weakly. as for example in induced fit. Rate increases by enzymes range from 108 to 1020 relative to the uncatalysed. becoming first the transition state and then products. which states that it is not the substrate that is distorted but rather that the transition state makes better contacts with the enzyme than the substrate does. • It is possible that the substrate and enzyme interact unfavorably and this unfavorable interaction is relived in the transition state. so the full binding energy is not achieved until the transition state is reached. Preferential Transition State Binding is probably the most important rate enhancing mechanism available to enzymes. which lowers the activation energy of the reaction by the magnitude of the binding energy. Utilization of enzyme-substrate binding energy in catalysis The maximum binding energy between an enzyme and a substrate will occur when there is maximal complementarity between the structure of the binding site and the structure of the substrate. • It is more likely that the enzyme is strained. • Transition state stabilization is a more modern concept. then binding increases as the reaction proceeds. . therefore Go‡ is lowered. which lowers the activation energy. • Strain or stress? It is unlikely that there is enough energy available in substrate binding to actually distort the substrate toward the transition state. which favors dissociation of the products from the enzyme once the reaction is complete. Molecular Mechanisms for the Utilization of Binding Energy • Strain is a classic concept in which it was supposed that binding of the substrate to the enzyme somehow caused the substrate to become distorted toward the transition state. • If the structure of the active site is complementary to the transition state. • The structure of the enzyme can only be complementary to one form of the substrate. • The structure of the substrate changes during the reaction. • Weak interactions between the enzyme and substrate are optimized in the transition state. • This means that the enzyme binds the transition state of the reaction more tightly than either the substrate or product.

the relative rate gets greater as the reacting groups are brought closer together. For a typical bimolecular reaction in free solution about 1/100 collisions between molecules of sufficient energy actually leads to a reaction. the total protein concentration in cytoplasm is 1 to 10mM at most).g. It increases their concentration and and reduces entropy loss for subsequent formation of a transition state. 1995.) f. Carrea et al. 109 for alcohol dehydrogenase. Catalysis in organic solvents Another approach that has generated some interest in enzyme technology is catalysis in non-aqueous or nearly anhydrous media (Bell et al.binding of the substrate(s) to the enzyme aligns the reactive groups so that the relevant molecular orbitals overlap. Some of these are illustrated in overheads for porcine pancreatic lipase (Zaks & Klibanov 1984. 1992).spontaneous reaction (e. In addition. Since chemical reaction rates are proportional to the concentrations of the reactants a rate enhancement of up to 106 may be generated by local concentration effects. 1985). As the five examples show. They found startling changes to enzyme properties as the water content of the reaction mixture decreases. Some examples include: 1.up to 106 fold. f) Distortion of the substrate . Factors involved in enzyme rate increases: a) Proximity . rather than the total water content of the system. . 1016 for alkaline phosphatase). (Ar = methoxyphenyl group. They concluded that it is the water bound to the enzyme that is critical for activity. This effect is counteracted to some extent by the fact that the concentration of enzyme active sites is low (the active site is a small portion of a very large molecule. his colleagues and other researchers have opened up novel opportunities in enzyme technology and this field has changed from a mere curiosity to a serious alternative to chemical reactions. Orbital steering hypothesis . Gupta. Therefore it appears that enzyme bound transition state is the single most in determining whether the reaction should proceed. This has been called as proximation effect or approximation effect. The functions of a number of enzymes under non-aqueous conditions have been studied.i. we will link R1 and R2 together. The question arises as to how much water is really needed by the enzyme to function. holding them in the correct orientation also helps. Consider the attack on an aryl ester by a carboxylic acid. Alexander Klibanov & his colleagues at MIT initiated much of the work in this area. Calculations indicate that the local concentration of substrate in the active site may be as much as 50M whereas its concentration in the cytoplasm may be less than 1mM. This led them to test how enzymes function in various amount of organic solvents. The reacting groups must be properly oriented. b)Orientation .up to 100 fold c) Covalent enzyme-substrate intermediates around 1010 fold d) General acid-base catalysis . Research by Klibanov. b) Orientation Proximity alone is insufficient.e) Metal ion catalysis . altered substrate specificity and a propensity towards synthetic reactions in non-aqueous media. Thus the maximum effect of orientation would be 100-fold.up to 108 fold (but largely hypothetical) a) Proximity The enzyme binds the substrate so that the susceptible bond is very close to the catalytic group in the active site or in close proximity in the active site.around 1010 fold. Lipases.around 1010 fold . 1995. Traditionally.e. Now let's put both reacting groups on the same molecule . Such systems are applicable to enzymes of varying reaction types. This increases the probability of forming the transition state. enzyme reactions are carried out in aqueous media. which show increased thermostability.

They have been used throughout the ages in leather tanning. Can use enzymes directly within a chemical process. High catalytic rate possible. 3. Conceptually. The whole biotechnology field depends on the activities of enzymes. 5. Optimizing 1 or 2 enzyme catalyzed reactions is easier than optimizing the whole pathway in a whole cell. Enhanced thermo stability of enzymes. Competing side reactions from other enzymes. 8. Exploitation of enzymes is not a recent development. Reduction in water-dependent side reactions. Importance and relevance of whole cell enzymes in biotechnology Enzymes are biological catalysts. There may be some drawbacks to using whole cells in catalysis. some with high stereospecificity. it is easy to decide whether one should use whole cells or isolated enzymes in a biotechnological process to produce or transform compounds. ie. 8. 1986). new catalytic activity seen with lipases or horse radish peroxidase). 6. These include 1. along with the requirement for co-factor regeneration in several steps. interferons. in the absence of water. With the exception of ribozymes. eg. 10.2. in cheese-making. Non-polluting catalysis. because enzymes are insoluble in organic solvents. 2. water is required to inactivate enzymes at high temperature. enzymes can be superior because their use will eliminate some of the potential problems with using whole cells. and can be recovered by simple filtration. Less by-products formed. Their main task is to decrease the activation energy (Eact) of a chemical reaction. enzymes are more stable. Glucose isomerase for conversion of glucose to fructose to produce high fructose corn syrup (HFCS). Elimination of microbial contamination. Therefore. either acting alone or in concert. 5. Less problem with sterility. Immobilization is unnecessary. dehydrogenation. Increased solubility of nonpolar substrates. 7. . 4. Other examples include chymotrypsin. Formation of by-products. 4. Some advantages to using enzymes in non-aqueous solvents include (Dordick. These transformations involve a number of enzymes acting sequentially. 9. 3. alcohol dehydrogenase. 3.. reduction. Enzymes catalyze a wide range of reactions. 11.. in the preparation of malted barley for beer brewing & the leavening of bread. with one-step or two-step stereospecific transformations. Sterility problems often arise when one deals with whole cells. etc. 4. 12. and may also possess lipid or sugar moieties. 7. 2. Ease of product recovery from low boiling organic solvents. The advantages of using enzymes include: 6. enzymes are mainly proteins in nature. 1989): 1. Shifting thermodynamic equilibria to favor synthesis over hydrolysis. Novel enzyme reactions possible (eg. Some processes require multi-step transformations. such as oxidation. Altered activity and affinity (Km) of the enzyme. the synthesis of antibiotics. Functions in moderate conditions. or ethanol production from glucose. monoclonal antibodies. dehalogenation. Some conditions may lead to cell lysis. On the other hand. These processes use enzymes in the form of whole cells. They are clearly candidates for using whole cells. 10. and this can result in loss of biocatalysts or severely decrease reaction rate depending on the nature and type of enzyme reaction. a variety of enzymes from different classes have been shown to function in nonaqueous media. 9. Horse radish peroxidase (HRP) which is able to depolymerize lignin in 95% dioxane (Dordick et al.

Many useful enzymes are intracellular. Enzyme by forming the active site similar to the transition state. enzyme have not been used widely as compared to chemical catalysis. etc. where as the actual transition state exists only fleetingly. the presence of metal ions. dehydration. This complementarity in shape and charge to the transition state is readily demonstrated by the effectiveness of transition state analogue is a phosphonate containing molecule that mimics the transition state of the hydrolysis of the corresponding acyl derivative. old ones are replaced by new ones. 17. Most enzymes are water-soluble. They are able to perform this task because they have been elicited against antigen known as transition state analogues. This may include poor stability with respect to temperature. Enzyme observed to bind transition complex but not the substrate. 15. it is chemically stable. are capable of catalyzing specific chemical reactions.hydration. an enzyme that catalysed the hydrolysis of this ester would also bind tightly to phosphonate analogue. This is because the use of enzymes also suffers from some serious drawbacks: 14. cofactor. etc. In cells. In this example. Describe the six classes of enzyme with examples? 2. Define holoenzyme ? write the role of apoenzyme. and the product. When an ester is hydrolysed the central carbonyl group changes from planer sp2 structure to tetrahedral sp3. 1 Exercises LONG TYPE 1. may be difficult to separate from reactants or products. ie. Therefore. The general strategies behind the production of catalytic antibody is to (i) design and synthesize a molecule whose shape is closely resemble to that of transition state of the reaction one wishes to catalyze. Define Enzyme ? Classify the Enzyme on the basis of enzyme commission? 4. (3) elicit an immune response to this conjugate. and therefore. This is not surprising considering that most enzymes have evolved to function in a biological milieu where the conditions may differ from those used in industry. The effect is to accelerate the reaction because the activation barrier is more easily overcome. This is difficult to do with isolated enzymes in vitro. Define enzyme specific activity? Write the different method of enzyme . Most isolated enzymes are not stable enough for use under industrial conditions. the scope of enzyme technology is very broad. 13. Abzymes Catalytic antibodies are also known as abzymes. (4) screen the resultant monoclonal antibodies for catalytic activity of the type desired. 16. Antibodies that are elicited against transition state analogue and therefore have the ability to bind them will be chemically and sterically complementary to the transition state and will therefore be potentially capable of catalyzing the reaction. these are molecules that closely resemble in shape and charge the reaction transition state (the highest energy spices in reaction pathway known as activated complex). the phosphonate containing molecule analogue is able to reproduce the shape of the transition state as well as the partially negative charged oxygen. Despite all these striking characteristics. (2) tether this molecule to larger molecule. pH. all enzymes are subject to turnover. and metalloenzymes on enzyme activity. In addition. Table 7. enzyme stabilizes or lowers down the energy of this species. Define Biocatalyst ? Describe how biocatalyst lower down the activation energy? 3. and hence are difficult to isolate. 5.

monomeric and oligomeric enzymes. Enzyme kinetics The study of the rate at which an enzyme works is called enzyme kinetics. two different polypeptide chains will be generated. g. it was shown in numerous examples that A may be favourable under certain environmental conditions while a. Metalloenzymes. Chapter-5 ENZYME KINETICS & INHIBITION TERMS Enzymes are protein catalysts of biological origin that. Holoenzyme. They may also differ in their rate of substrate turnover. Module II Specificity of enzyme action. like all catalysts. Enzyme specificity c. may be inactive while that of A is fully active. Furthermore. They achieve their effect by temporarily binding to the substrate. Noncompetitive inhibition. They may be separated by gel electrophoresis under favourable conditions. Cofactor f. d. when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency. write short notes on following a. e. If a gene exists in a heterozygous state. Enzyme activity b. for example. . But all states in between are also possible.substrate interaction in brief? Short type 1. Enzyme inhibition. speed up the rate of a chemical reaction without being used up in the process. Coenzyme. The gene product a. when the substrate and inhibitor compete for binding to the same active site. Enzyme commission. Competitive inhibition. Alloenzymes.

For example.Velocity refers to the change in concentration of substrate or product per unit time. By duplication of the genetic material a gene may be present within a haploid genome for two or more times. Therefore initially the order is first and later on it becomes zero order. Noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power. Initial velocity is the change in reactant or product concentration during the linear phase of a reaction. the reaction in which A is converted to B is written as follows: A B----------------------------1 The rate of this reaction is expressed algebraically as either a decrease in the concentration of reactant A: -[A] = k[B]-----------------------2 or an increase in the concentration of product B: [B] = k[A]--------------------3 In the second equation (of the 3 above) the negative sign signifies a decrease in concentration of A as the reaction progresses. The rate of reactions depends on the order and molceulcarity. Therefore maximum velocity is affected by the occupying all space of the active site by the substrate.is favourable under other conditions (more in the section about evolution). Each chemical reaction has characteristic . Therefore. Isoenzymes. brackets define concentration in molarity and the k is known as a rate constant. It is not important whether the gene loci are on one chromosome or distributed over several chromosomes. Reaction rate is always dependent on the concentration of the chemicals involved in the process and on rate constants that are characteristic of the reaction. Rate in the itegarl form is shown by the rate = dX/ dt. Chemical Reactions and Rates According to the conventions of biochemistry. the rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into product(s) in a specified time period.but are not changed by the enzyme. This is the important step in understanding the mechanism of catalysis. it is necessary to study the factors affecting the rate of catalysis. If this is occupied by the inhibitor as in competitive inhibition the v max remain same since space is same and inhibitor has similar structure as the substrate. Polypeptides (enzymes) that are encoded by different alleles are called alloenzymes. INTRODUCTION Kinetics is the study of the rate of change of reactants to products . This time reaction rate become slower and the product formation starts. Rate constants are simply proportionality constants that provide a quantitative connection between chemical concentrations and reaction rates. This assumption is true for single substrate and single enzyme. Rate refers to the change in total quantity per unit time. Initially substrate consumes and product is released. The gene products (enzymes) of the different gene loci (pseudoalleles) are called isoenzymes. Competitive inhibitors are molecules that bind to the same site as the substrate preventing the substrate from binding as they do so . When enzyme concentration is small and substrate concentration increases then first the reaction occurs rapidly but maximum rate is achieved only when all the space in the active site is filled and no more substrate can be filled. Study of rate of the catalyzed reaction is called as kinetics.

Empirically. 2 For this reaction the forward reaction rate would be written as: ATP + H2O---------8 . At equilibrium. Chemical Reaction Order Order of reaction is refers to the number of molecules involved in forming a reaction complex that is competent to proceed to product(s). we state that the rate of the forward reaction [v forward] is equal to the product of the forward rate constant k+1 and the molar concentration of A. A reaction with two substrates forming two products would a second-order reaction.and higher. At equilibrium the rate (v) of the forward reaction (A B) is by definition is equal to that of the reverse or back reaction (B A). To put the relationships of the two equations into words. The exponent on the substrate concentration in the rate equation for this type of reaction is 1. the reactants in second. The rate of the reverse reaction is equal to the product of the reverse rate constant k-1 and the molar concentration of B. Thus. not to an actual negative value for the constant.---------------7 This equation demonstrates that the equilibrium constant for a chemical reaction is not only equal to the equilibrium ratio of product and reactant concentrations. However. The rate constant for the forward reaction is defined as k+1 and the reverse as k-1. k+1 and k-1 represent rate constants for the forward and reverse reactions. rate constants and the equilibrium constant for this simple case. the rate of the forward reaction is equal to the rate of the reverse reaction leading to the equilibrium constant of the reaction and is expressed by: [B]/[A] = k+1/k-1 = K eq. a relationship which is algebraically symbolized as: V forward = v reverse---------------4 Where. 1 FIGURE 10. An example of a second order reaction is the formation of ATP through the condensation of ADP with orthophosphate: ADP + H2PO4 FIGURE 10. order is easily determined by summing the exponents of each concentration term in the rate equation for a reaction. respectively.values for its rate constants. The negative subscript refers only to a reverse reaction. these in turn directly relate to the equilibrium constant for that reaction. reaction can be rewritten as an equilibrium expression in order to show the relationship between reaction rates. A reaction characterized by the conversion of one molecule of A to one molecule of B with no influence from any other reactant or solvent is a first-order reaction. but is also equal to the ratio of the characteristic rate constants of the reaction. for the forward reaction: V forward = k+1[A]---------------5 and for the reverse reaction: V reverse = k-1[B]----------------6 In the above equations.order reactions need not be different chemical species.

11 k-1 The ES complex then breaks down in a slower second step to yield the free enzyme and the reaction product P: k2 ES -------. The maximum initial rate is observed when the entire enzyme E0 is present as ES complex and the concentration of E is very small. Between the binding of substrate to enzyme. most of the enzyme will be in uncombined form E. a series of complex events must take place. and the reappearance of free enzyme and product. This site. called the catalytic site of the enzyme. overall rate of enzyme-catalyzed reaction must be proportional to the concentration of species that reacts in the second step. At low [S]. The latter is finally competent to dissociate into product and free enzyme. The series of events can be shown thus: E + S ES P-------------------10 and E+S K1 ES ES* EP E + ………. At a minimum S. when substrate conc. Under these state the enzyme is “saturated” with its substrate.V forward = k1 [ADP][H2PO4]---------------9 L.. The catalytic event that converts substrate to product involves the formation of a transition state. Is increased then initially rate become faster and at maximum velocity where enzyme saturates the rate become steady as shown in figure. here the rate will be proportional to the [s] because the equilibrium of equation will shift for the formation of more ES as S is increased. The complex that forms when substrate(s) and enzyme combine is called the enzyme substrate (ES) complex. has been evolutionarily structured to provide specific. The Michaelis derived an equation to explain the fact as Initial velocity Michaelis-Menton constant In typical enzyme-catalyzed reactions. and the transition state complex must advance to an enzyme product complex (EP). Consequently. This is the condition when [S] is sufficiently high that essentially all the free . the enzyme exists in the combined form E or uncombined form ES. an ES complex must be formed. and it occurs most easily at a specific binding site on the enzyme. At any time the enzyme-catalyzed reaction. Reaction products arise when the ES complex breaks down releasing free enzyme. high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. As we know at fixed enzyme concentration. In biochemical reactions. Menten in 1913 postulated that enzyme first combine reversibly with the substrate to form an enzyme substrate complex in a relatively fast reversible step and then product is released along with free enzyme.E+P ……………. Michaelis and M. which is ES. reactants are commonly known as substrates. It means that.(12) Since the second step is slower and therefore it limits the rate of overall reaction. each enzyme molecule catalyzes the conversion to product of many reactant molecules. reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. this complex must pass to the transition state (ES*). so that further increase in [S] have no effect on rate.

enzyme catalyzed reaction and is denoted by the Km. …. 5=6 k1( [Et]—[ES] [S] [ES]-----------------------(17) or k1[Et][S]--.enzyme is converted into the ES form. It is too difficult to observe because of very short time. [S] / Km + [S]-------------------22 What will happen if the initial velocity is exactly one-half of maximum velocity? . the rate equation of one substrate. because [S] is greater than Et.K1[ES] [S] [ES]----------------------------(18) = = k-1[ES] + k2 (k-1 + k2) To Simplify the equation add the term k1[E] [S] to both side. b. which is determined by the [ES]: V0=k2 [ES]-----------------------------------------------------(14) TOTAL enzyme concentration can be represented by [Et]. (15) K1 denotes the rate of formation of ES= k1( [Et]—[ES] [S] ………… k-1 denotes the rate of ES breakdown= k-1[ES] + k2 [ES]-------------------------(16) So in the steady state. eq. So free enzyme can be represented by [Et]—[ES]. Also. we finally get K1 [Et][S] k2)[ES]-------------------------(19) Solving for the ES we get. Putting value of ES in equation 21 from equation 13 gives new final equation that is Vo= V max . After the ES complex breaks down to yield product P the enzyme is free to catalyze another reaction. Derivation: E+S k-1 k1 k2 ES E+P ………………(13) VO can be determined by the breakdown of ES to give product. [ES] = --------------------------------(20) or [ES] = --------------------------------(21) [Et][S] -----------------[S] +( k-1+ k2)/ k1 K1[Et][S] = (k1 [S]+k-1+ -----------------k1[S] + k-1+ k2 The term ( k-1+ k2)/ k1 is called as Michaelis—Menten equation.. Steady state:-Here ES complex is constant over a period of time. the amount of substrate bound by the enzyme at any given time is negligible compared to total [S]. Pre-steady state:-The duration in which ES formation takes place is called as pre-steady state. this defines the rate of initial velocity and maximum velocity. a.

So if V0 = Vmax/2. thus k2<<k-1 So Km reduces to Km=k-1/k1. Km denotes the affinity of enzyme. See figure 10. The kcat is a direct measure of the catalytic production of product under saturating substrate conditions. The lower the Km. 4. 2. 3 showing rate of reaction and Km at ½ Vmax Figure 10. Catalytic efficiency is denoted by the ratio of Kcat/ Km. Km can be very complex in the multistep enzyme catalysis. On writing the Michaelis Menten constant in reciprocal form 1 / v = (kM / vmax) x 1 / [S]--------------------25 This representation is also known as the Lineweaver-Burk equation. 3. Km can change from one enzyme to other. and it is defined as dissociation constant. the turnover number. Note that Km has unit of molarity. FIGURE 10. the higher is the enzyme's affinity for its substrate. other enzyme that requires large amount of substances for their activation and conversion into product is said to have as High Km value but low affinity. is the maximum number of substrate molecules converted to product per . This property is efficiently utilized by the LDH enzyme that has different Km value in the heart and muscle. high value of Km denotes low enzyme activity. then Vmax/2= V max [S]/ km+[S]. we get Km+[S] = 2[S]. the dimension of kM is Mol.l kcat. 1.5 1 / kM and 1 / vmax are the points of intersection with the co-ordinates. In muscle LDH have High Km. 5. K cat is the turn over number of enzyme . Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. while in the heart it has low Km essential for the survival.3. k2 is the rate limiting . the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate). This shows that the MichaelisMenten constant equals the substrate concentration at half-maximal reaction velocity. Consequently. An enzyme that acts on very low concentration of substrate has very low Km. See from the table below the enzyme having very high value of Kcat have high capacity to convert substrate into the product The turnover number is a measure of catalytic activity. Note :for the Michaelis-Menten reaction. but it does not tell anything about the nature of enzyme.4 & 10. Its advantage is in the favourable graphic depiction of the quantities. Ks for the ES complex. or --------------------24 This represent that Km is equivalent to the substrate concentration at which Vo is one half Vmax.-----------------23 ½= [S]/ km+[S] solving for km. This plot has advantage that it allows more accurate determination of Vmax.4 Catalytic efficiency and Turnover number. The smaller the value of kM . A double recessive depiction yields a straight line with a gradient of kM / vmax. Low Km enzyme can efficiently covert very low amount of substrate into the product such enzyme is said to have high affinity of enzyme while. 4 Graphical method to find the value of Km by plotting the graph between the 1/Vo verses 1/[S] 10. See figure 10.

best is the enzyme for maximum conversion of substrate into the product. 7 The kinetics of simple reactions like that above was first characterized by biochemists Michaelis and Menten. respectively. Note that high the TON of an enzyme. 6 The constant Kcat has unit S-1. 5 showing turn over number of different enzyme. ES. therefore K cat /Km is a second order rate constant. We can therefore write a simple kinetic scheme as follows: We will work within an approximation called the steady-state approximation. we wil work within the approximation that we can neglect the backreaction. The rate constants for formation and breakdown back to reactants are k+1 and k-1. and to further simplify our treatment. can be written as . in multistep enzymatic reaction. According to M-M model. The first thing we need to realize is that the total concentration of enzyme in all of its forms should be equal to the original free enzyme concentration: 2. one step may be rate limiting that rate limiting step is equivalent to Kcat. It also denotes about the reaction between the enzyme and substrate since at low K cat. so the initial velocity. E. It is also useful to write down two other definitions and assumptions before we continue: 1.e. S. FIGURE 10. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today. Equation 22 can be written as by placing the value of Vmax = Kcat x Et -------------------26 figure 10. in which the rate of formation of the enzyme-substrate complex is equal to its rate of decay. Vo depends on both Et and S. Note that two enzymes catalyzing different reaction may have the same Kcat value (turnover number) yet the rate may be different. The rate constant for formation of the product is k+2. Many enzymes have K cat /Km value in the range 108 to 109 .. vo. and maximum possible velocity. k-2. reciprocal of time. k cat = Vmax /Et. or Vmax. From equation 26. ENZYME KINETICS We start with a very simple expression for formation of the enzyme-substrate complex.enzyme molecule per unit of time. Values of k cat range from less than 1/sec to many millions per sec. and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states. obtained when all the enzyme is substrate-bound. i. Therefore Kcat /Km is the best way to compare the catalytic efficiency of the enzyme. The second point is that the rate of the reaction should be equal to formation of products. and is therefore zero. from free enzyme. and free substrate. Km value will also be unsatisfactory. FIGURE 10.

vo. or Km. Now we have an even simpler expression: We are almost done. Concentration of substrate molecules (the more of them available. the quicker the enzyme molecules collide and bind with them). i. . 1234Substrate concentration. 1. Therefore we can write and from the steady-state approximation. Therefore the rate of decay of the enzyme-substrate complex will be the sum of two rate constants.Now. if the steady-state approximation holds. then you can rearrange the equation to look like this: Now group all of the rate constants together We can define this collection of rate constants as something called the Michaelis Constant. that equation is insoluble.. To get the equation into that form. this has to equal zero. we can look at the kinetic scheme above and deduce that the rate of change in the concentration of the enzyme-substrate complex should be equal to the rate of formation of the complex minus the rate of decay of the complex. . [S]. The rate of decay involves two possible pathways. One is to form products. which in turn is the same as the starting concentration of enzyme. we need two things from above. before making the steady-state approximation. The rate of formation is dependent upon the rate constant k+2 and the enzyme and substrate complexes. Now. however. The first is our statement that the total enzyme concentration is equal to the concentration of enzyme in all of its forms. you can solve it fairly simply (despite what I did in class). and the other is to reform the reactants. Temperature pH Inhibitors The rate at which an enzyme works is influenced by several factors.e.E total = Eo = [E] + [ES] or conveniently [E] = [Eo] – [ES] more. k+2 and k-1. and the initial velocity. The concentration of substrate is designated [S] and is expressed in unit of molarity. However. Before making this approximation. First. we want an expression in terms of constants and measurable quantities such as the substrate concentration. Note that this is not an equilibrium constant. Then we can substitute that into our previously simplified equation to obtain Solve for ES Since initial velocity V0 = K2 [ES] and max velocity Vmax = K2 [E total] Chapter-6 Factor affecting the rate of catalysis.

But as enzymes are proteins. low enough then the graph between the velocity of reaction and substrate utilization.1 Substrate concentration As we know that substrate react with the enzyme and it is converted into the product. This is the reason why ethyl alcohol is given to person that has consumed the toxic alcohol (methyl alcohol) so that methyl alcohol can be competitively replaced by ethyl alcohol. Toxicology is the study of how toxicological substances can interfere with life sustaining enzymes via inhibition. Many heavy metals like Lead. Once locked into position. 9 10. Outside that temperature range the enzyme is rendered inactive and is said to be totally inhibited. coma or even death of the organism. All in all the use of inhibitors can be used for the benefit of mankind or its destruction. This effectively blocks the active site. this causes a change in the secondary and tertiary levels of protein structure. Many toxic substances owe their toxic properties to their ability to act as inhibitors (slow down the rate of reactions by different mechanism) to important enzymes responsible for catalyzing important biochemical processes.2 Effect of temperature and pressure Every enzyme has a temperature range of optimum activity. If the concentration of substrate becomes more than the inhibitor they may replace the inhibitor later on. will give almost a linear graph. Biological warfare owes its success to enzyme inhibition but so does the life giving chemotherapeutic treatment of cancerous tumor growths with agents that inhibit important cancel cell enzymes. As the temperature rises.4. FIGURE 10. Other inhibitors latch themselves not to the active site itself but to some portion of the enzyme molecule close to the active site which results in the changing of the shape of the active site. 10. The temperature. and the active site is altered in its conformation beyond its ability to accommodate the substrate molecules it was intended to catalyze.and hence collisions between enzyme and substrate . the blocker molecule prevents the true substrate molecule from getting into position. The presence of inhibitors. See the graph 10. For example.2. But if the substrate concentration is high enough then we will obtain the graph that will be parabolic for some time and then straight in term of velocity. The pesticide and herbicide industries make use of competitive and Non-Competitive Inhibitors. dipole-dipole attractions) as well as the Hydrophobic forces between non-polar groups within the protein structure. This occurs because as the temperature changes this supplies enough energy to break some of the intramolecular attractions between polar groups (Hydrogen bonding. 3. Most enzymes (and . cyanide poisoning is due to the cyanide ion competitively inhibiting the active site of the cytochromases enzymes responsible for catalyzing the Oxidation and Reduction processes of the Electron Transport System which is responsible for cellular respiration. If the substrate concentration is. and Chromium will function as non-competitive inhibitors. When these forces are disturbed and changed.4 . The study of the rate at which an enzyme works is called enzyme kinetics. This is referred to as non-competitive inhibition or mixed inhibition. Mercury. Competitive Inhibition occurs when a molecule that is close enough to the shape of the true substrate will fit into the active site. there is an upper limit beyond which the enzyme becomes denatured and ineffective. Once the enzyme is inhibited the process cannot take place. The molecule competes for the active site with the true substrate molecule which is concentration dependent. molecular motion . 8 FIGURE 10.9.speed up. and a toxicological symptom occurs that often leads to paralysis.

not only alters the equilibrium constant. Of all most important factor is the active site. increases from 1. ΔG* =EA is the standard free energy of activation (kJ M-1) which depends on entropic and enthalpy factors. The reverse holds for endothermic reactions such as that of glucose isomerase where the ratio of fructose to glucose. because active site contains amino acid residues inside the pocket. —— short incubation period. rise with increase in temperature in accordance with the Arrhenius equation. including those catalyzed by enzymes. It may be due to covalent changes such as the deamination of asparagine residues or non-covalent changes such as the rearrangement of the protein chain. In general.6). there is a rapid rate of loss of activity (Figure 8. ________________________________________ ________________________________________ FIGURE 10. It follows that. K = A e –ΔG*/RT --------------27 OR log K = log A –EA /2.36) which means that. Inactivation by heat denaturation has a profound effect on the enzymes productivity (Figure 8. Note that the temperature at which there appears to be maximum activity varies with the incubation time.3 RT where k is the kinetic rate constant for the reaction. At some temperature activity becomes zero and at some temperature enzyme efficiency of conversion of substrate into product becomes high. the reverse reaction (having higher activation energy) increases more rapidly with temperature than the forward reaction. Enzymes. R is the gas law constant and T is the absolute temperature. but also reduces the optimum temperature for maximum conversion as the reaction progresses. 11 A schematic diagram showing the effect of the temperature on the activity of an enzyme catalyzed reaction. These denaturing reactions have standard free energies of activation of about 200 . and the ability of these residue to interact with the substrate functional group and capacity to breaking and making bonds is certainly going to be effected by the . Typical standard free energies of activation (15 . This.e. This can happen if body temperature gets too low (hypothermia) or too high (hyperthermia).. Rates of all reactions.00 at 55°C to 1. Temperature is an important factor in the regulation of enzyme activity. The actual loss of activity is the product of this rate and the duration of incubation (Figure 8.2 and 2.7). conformational alteration entailing a loss of biological activity) at temperatures above those to which they are ordinarily exposed in their natural environment.5). 12 .e. in an exothermic reaction. it would be preferable to use enzymes at high temperatures in order to make use of this increased rate of reaction plus the protection it affords against microbial contamination. also known as the frequency factor. however.there are hundreds within the human organism) within the human cells will shut down at a body temperature below a certain value which varies according to each individual. causing changes in both Km and Vmax.long incubation period. A is the Arrhenius constant. ________________________________________ FIGURE 10.5). above a critical temperature.300 kJ mole-1 (Q10 in the range 6 . Q10 is within the range 1. All the rate constants contributing to the catalytic mechanism will vary independently. are proteins and undergo essentially irreversible denaturation (i. in this case. ----.2 .2.17 at 80°C.70 kJ M-1) give rise to increases in rate by factors between 1. This factor for the increase in the rate of reaction for every 10°C rise in temperature is commonly denoted by the term Q10 (i. 10 FIGURE 10. at equilibrium.5 for every 10°C rise in temperature.

g. it may be seen that it is prudent to err on the low temperature side. overhead costs). The optimum productivity is seen to vary with the process time. kd1 and kd2).d[E] / dt =Kd1[E] -----------------31 Integrating equation 31 using the boundary condition in equation 28 gives: [E] = [E]0 e (-Kdt) = ---------------32 From the reaction scheme . [E] = [E1] = [E2] = [E0] ----------30 It follows from the reaction scheme [27. inactivation data should. which may be determined by other additional factors (e. appear to follow this seriestype deactivation scheme.e.A schematic diagram showing the effect of the temperature on the productivity of an enzyme catalyzed reaction. in general. —— 65°C. at the beginning of the reaction: [E] = [E]0 -----------28 and: [E1] = [E2] = 0 ----29 At time t. . Assuming. the half-life of the enzyme is shown to be inversely proportional to the rate of denaturation. This model allows for the rare cases involving free enzyme (e. substituting for [E2] from equation 30. It is not surprising that such data can be made to fit a model involving four determined parameters (A1. Af = 0. tyrosinase) and the somewhat commoner cases involving immobilised enzyme where there is a small initial activation or period of grace involving negligible discernible loss of activity during short incubation periods but prior to later deactivation. t1/2 = 0.e. The thermal denaturation of an enzyme may be modeled by the following serial deactivation scheme: --------27. the simple first order deactivation rate expression results Af = e (-Kdt) -----------39 The half-life (t1/2) of an enzyme is the time it takes for the activity to reduce to a half of the original activity (i. be assumed to be rather error-prone. —— 60°C.e (-Kd2t) ) -----------35 If the term 'fractional activity' (Af ) is introduced where. Many enzyme preparations.693 / Kd1 --------------41 In this simple case. A2. both free and immobilised.1]. Af = [E] + A1 [E1] + A2 [E2] /[E0]---------36 then. Despite this possible . E1 may have higher or lower activity than E) whereas A2 is normally very small or zero. If the enzyme inactivation obeys equation 39. be an equilibrium mixture of a number of species. However because reliable and reproducible data is difficult to obtain.1 Where kd1 and kd2 are the first-order deactivation rate coefficients.d[E1] / dt =Kd2[E1]-Kd1 [E] ---------------33 Substituting for [E] from equation 32 . It is often difficult to get precise control of the temperature of an enzyme catalyzed process and. distinct in structure or activity.d[E1] / dt =Kd2[E1]-Kd1 [E]0 e (-Kdt) -------------34 Integrating equation 33 using the boundary condition in equation 29 gives: [E1]=Kd1 [E]0 / Kd2-Kd1 = (e (-Kd1t) .5). E is the native enzyme which may. —— 55°C.g. the half-life may be simply derived. under these circumstances. and E1 and E2 are enzyme molecules of average specific activity relative to E of A1 and A2. gives: Af = [E] + A1 [E1] + A2 ([E0]-[E]-[E1]) /[E0]-------------37 therefore: ------38 When both A1 and A2 are zero. or may not. A1 may be greater or less than unity (i. Ln (1/2) = =Kd1 t 1/2 -----------------------40 Therefore.

g. may improve the thermal stability in particular cases. Figure 10. The practical effect of this is that usually kd1 is apparently much larger than kd2 and A1 is less than unity. such as de amidation of one or two asparagine or glutamine residues. in turn. they remain active for considerable periods even at temperatures above 100°C.reservation. This will affect the total net charge of the enzymes and the distribution of charge on their exterior surfaces. solutions. if rather small. The equilibrium position of the reaction will also be shifted due to any difference in molar volumes between the reactants and products.1). where the enzyme preparation consists of a heterogeneous mixture of a large number of closely related structural forms. Some enzyme-reactant mixtures may undergo reductions in volume amounting to up to 50 ml mole-1 during reaction due to conformational restrictions and changes in their hydration. rather than dilute. Changes in the pH or acidity of the environment can take place that would alter or totally inhibit the enzyme from catalyzing a reaction. Cooling below 0°C in the presence of additives (e. These effects are especially important in the neighborhood of the active sites. limited proteolysis or disulphide interchange. which prevent freezing. glycerol). Most enzymes are stable for months if refrigerated (0 . Clearly any reaction involving dissolved gases (e. Freezing enzyme solutions is best avoided as it often causes denaturation due to the stress and pH variation caused by ice-crystal formation. according to their acid dissociation constants. and/or a halving in the Km for a 1000 fold increase in pressure. This property has great technological significance and is currently being exploited by the use of organic solvents. in addition to the reactivity of the catalytically active groups. influence is due to the volume changes which occur during enzymic binding and catalysis.4. equations 38 and 39 remain quite useful and the theory possesses the definite advantage of simplicity.4°C). 13 Figure showing optimum pH for two separate enzymes. the larger the variability the more apparent will be the series-type inactivation kinetics.g. such as the presence of thiols anti-oxidants. can generally increase this storage stability even further. [S] >> Km). oxygenases and decarboxylases) will be particularly affected by the increased gas solubility at high pressures.g. especially at concentrations where little free enzyme exists (e. In order to minimise loss of activity on storage. Taken together. They act slowly on the low pH or high pH. This. However an additional. structural stability and solubility of the enzyme. 10. Either proton is removed or added. even moderate temperatures should be avoided. This is because of change in the ionization pattern of the amino acid residue inside the active site of the enzyme. with the pH of their environment (Table 1. In a dry or predominantly dehydrated state. The charges on these groups will vary. These may have been formed during the past history of the enzyme during preparation and storage due to a number of minor reactions. enzymeinhibitor and enzyme-product complexes which helps to explain the substantial stabilizing effects of suitable ligand. So an optimum pH is needed for its full activity. The first order deactivation constants are often significantly lower in the case of enzyme-substrate. In some cases the series-type deactivation may be due to structural micro heterogeneity. This change in the pH will affect . Pressure changes will also affect enzyme catalyzed reactions. the changes in charges with pH affect the activity. Other factors. Alternatively it may be due to quaternary structure equilibrium or the presence of distinct genetic variants. Therefore all enzymes are affected by the change of pH. They are generally more stable in concentrated. The relative effects on kcat and Km depend upon the relative volume changes during binding and the formation of the reaction transition states.3 Effect of pH on Enzyme Enzymes are amphoteric molecules containing a large number of acid and basic groups. It has been found that the heat denaturation of enzymes is primarily due to the proteins' interactions with the aqueous environment. may lead to a doubling of the kcat. mainly situated on their surface. In any case.

1. In a similar manner to the effect on enzymes.8. the charge and charge distribution on the substrate(s). If the environment was too basic the acid groups would be deprotonated. their complete removal from the enzyme. the extent of which depends on the heats of ionization of the particular groups concerned.5 Maltase 6.0 Lipase (castor oil) 4. Other enzymes like alpha amylase found in the saliva of the mouth operate most effectively at near neutrality.0 Lipase (stomach) 4. If the pH climbs to an unacceptably high value called alkalosis then enzymes ceases to function effectively.8 . Correcting pH or temperature imbalances will usually allow the enzyme to resume its original shape or conformation. Still other enzymes like the lipases will function most effectively at basic pH values.5 .0 Amylase (malt) 4. Normally. Some enzymes like many of the hydrolytic enzymes in the stomach such as Pepsin and Chymotrypsin effective operate at a very low acidic pH. characteristic of each enzyme. Buffers are a substance or mixtures of substances that resist any change in the pH. at high hydroxyl concentrations. and the chemical change would be inhibited from taking place as efficiently or not at all. The temperature also has a marked effect on ionizations. increase the successful competition of hydrogen ions for any metal cationic binding sites on the enzyme. There are many buffer systems found in the body to adjust the pH so that enzymes might continue to catalyze their reactions.2 Catalase 7. The relationship between the change in the pKa and the change in temperature is given by a derivative of the Gibbs-Helmholtz equation. at which the net charge on the molecule is zero. If the pH drops in the blood called acidosis then enzymes in the blood will be inhibited outside their optimal pH range. . leads to increasing hydroxyl ion concentration which competes against the enzymes' ligand for divalent and trivalent cations causing their conversion to hydroxides and.5. at which the enzyme generally has minimum solubility in aqueous solutions. on the other hand. Table II pH for Optimum Activity Enzyme pH Optimum Lipase (pancreas) 8. This is also called denaturation. these conditions do not take place because of the highly efficient buffers found in the blood that restrict the pH of the blood to a very narrow range. reducing the bound metal cation concentration.0 There will be a pH.6 Trypsin 7.7.7 .0 . Many toxic substances will break covalent bonds and cause the unraveling of the protein enzyme. This would alter the electrical attractions between polar groups. Decreasing hydrogen ion concentration.0 Invertase 4. Other toxic substances will precipitate enzymes effectively removing them from the solution thus preventing them from catalyzing the reaction.6.7 Urease 7.7 Pepsin 1. Increasing hydrogen ion concentration will.the polar and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme and the active site as well to the point where the substrate molecule could no longer fit.6 . product(s) and coenzymes (where applicable) will also be affected by pH changes. In an acid solution any basic groups such as the Nitrogen groups in the protein would be protonated.8 Amylase (pancreas) 6. This is called the isoelectric point (pI). The enzyme is said to be irreversably denatured. Every enzyme has an optimum pH range outside of which the enzyme is inhibited.5. Some substances when added to the system will irreversibly break bonds disrupting the primary structure so that the enzyme is inhibited permanently. additionally.1 .

It is possible to alter the pH-activity profiles of enzymes. dioxan. the variation must be determined under the industrial process conditions. essentially irreversible. These changes. The ionisation of the carboxylic acids involves the separation of the released groups of opposite charge. I=0. there may be partial destruction of cystine residues due to base catalysed b-elimination reactions whereas. In alkaline solution (pH > 8). or by immobilisation. These include the variation of solubility of substrate(s) and product(s). This method is sometimes useful but not generally applicable to enzyme catalysed reactions as it may cause a drastic change on an enzyme's productivity due to denaturation (.5 x (0. may occur. within a range of 2-3 units each side of the pI. suppression of the ionization of a product to facilitate its partition and recovery into an organic solvent. k-1 and kcat (k+2 in the Michaelis-Menten mechanism). however. only likely to exert a small influence on the enzyme's . protonated basic groups which are stabilised by neighbouring negatively charged groups will be stabilised (i. In simple terms. how much substrate it is capable of converting to product). At higher solution ionic strength. Changes in the ionic strength (I) of the solution may also have some effect.g. changes in the position of equilibrium for a reaction. in acid solutions (pH < 4). is normally a reversible process.e. a number of other factors may mean that the optimum pH in the Vmax-pH diagram may not be the pH of choice in a technological process involving enzymes. assumption that only one charged form of the enzyme is optimally catalytic and therefore the maximum concentration of the enzyme-substrate intermediate cannot be greater than the concentration of this species. The variation of activity with pH. increases the pKa of carboxylic acid groups. is 0.303) is the natural logarithm of 10. sometimes found next to aspartic acid residues.1 x 22 + 0.e. ethanol).3 M. therefore. The ionic strength is defined as half of the total sum of the concentration (ci) of every ionic species (i) in the solution times the square of its charge (zi). The variation of productivity with pH may be similar to that of the Vmax-pH relationship but changes in the substrate stream composition and contact time may also make some contribution. denaturation. This relationship is further complicated by the variation in the effect of the pH with both the duration of the process and the temperature or temperature-time profile. plus any consequent structural alterations. Δ H is the heat of ionisation and the numeric constant (2.314 J M-1 K1). R is the gas law constant (8. cause a timeand temperature-dependent. have little effect on the overall charge on the enzyme molecule at neutral pH and are.where T is the absolute temperature (K).1 M solution of CaCl2 . reducing the dielectric constant of an aqueous solution by the addition of a co-solvent of low polarity (e. may be reflected in changes in the binding of the substrate. and the reduction in susceptibility to oxidation or microbial contamination. This process is encouraged within solutions of higher polarity and reduced by less polar solutions. The importance of the knowledge concerning the variation of activity with pH cannot be over-emphasized. Both Vmax and Km will be affected due to the resultant modifications to the kinetic rate constants k+1. However. This variation is sufficient to shift the pI of enzymes by up to one unit towards lower pH on increasing the temperature by 50°C. i.5Σ(CiZi2) . the catalytic efficiency and the amount of active enzyme.e. Generally. These charge variations. However. The pKa of basic groups are not similarly affected as there is no separation of charges when basic groups ionise. and the variation in the concentration of active enzyme. the ionic strength of a 0. assume EH. Extremes of pH will. charge separation is encouraged with a concomitant lowering of the carboxylic acid pKas. The important parameter derived from these influences is the productivity of the enzyme (i. as pKa's are based on logarithms with base 10. The effect of pH on the Vmax of an enzyme catalysed reaction may be explained using the. Thus.For example. extensive as they may be. The major such factor is the effect of pH on enzyme stability. have lowered pKa) by solutions of lower polarity. generally true. hydrolysis of the labile peptide bonds.2 x 12) = 0.is the only active form of the enzyme.

000 o From: Daniel Koshland.000006 2700 > 450 million Creatine Kinase Creatine Creatine Phosphate <.003 40 > 13. Loss of activity may be either reversible. respectively. which generally act in a fairly specific manner. The ionic strength of the solution is an important parameter affecting enzyme activity.g. Molecular geometry in enzyme action. 1956.g. It is found that a single change in charge has little effect on the pH-activity profile. FIGURE 10. it is clearly important to control the ionic strength of solutions in parallel with the control of pH. Thus both the binding of charged substrates to enzymes and the movement of charged groups within the catalytic 'active' site will be influenced by the ionic composition of the medium. If the charges are opposite then there is a decrease in the reaction rate with increasing ionic strength whereas if the charges are identical. by reaction with succinic anhydride) or if all the carboxylates are converted to amines (e. Chemical derivatisation methods are available for converting surface charges from positive to negative and vice-versa. unless it is at the active site.g.isoelectric point. The cause of these shifts is primarily the stabilisation or destabilisation of the charges at the active site during the reaction. and the effects are most noticeable at low ionic strength.g. Others. Journal of Cellular & Comparative Physiology 47: 217-234. are known as inhibitors. 57histidine+ and 145arginine+ causing a significant increase in kcat on increasing the ionic strength of the solution). Chapter-7 Enzyme inhibition A number of substances may cause a reduction in the rate of an enzyme catalysed reaction. urea) are non-specific protein denaturants. by coupling to ethylene diamine by means of a carbodiimide the profile can be shifted about a pH unit towards higher or lower pH. where activity may be restored by the removal . However if all lysines are converted to carboxylates (e. 14 showing molecule activation at high and low temperature Enzyme Substrate Product Rate without Enzyme µmoles/L per min Rate with Enzyme µmoles/L per min Acceleration due to Enzyme Hexokinase Glucose Glucose 6-Phosphate <. the rate controlling step in the catalytic mechanism of chymotrypsin involves the approach of two positively charged groups.000000005 1600 > 320 billion Alcohol Dehydrogenase Ethanol Acetaldehyde <. Some of these (e. Even if a more complex relationship between the rate constants and the ionic strength holds. This is especially noticeable where catalysis depends on the movement of charged molecules relative to each other. Jr. an increase in the reaction rate will occur (e.0000001 1300 > 13 billion Phosphorylase <.

--------------------42 where I represents the reversible inhibitor and the inhibitory (dissociation) constants Ki and Ki' are given by --------------------43 and.of the inhibitor. lower Vmax). Noncompetitive inhibition 3. Heavy metal ions (e.e. In order to simplify the analysis substantially.19a). A number of simplified cases exist that are reversible. Uncompetitive inhibition. gives: --------------50 therefore --------------51 If the total enzyme concentration is much less than the total inhibitor concentration (i.e. These are generally discussed in terms of a simple extension to the Michaelis-Menten reaction scheme. [E]0<< [I]0). it is necessary that the rate of product formation (k+2) is slow relative to the establishment of the equilibria between the species. it . ( 44) and (46). Competitive inhibition. A. Therefore: -----------46 also ----------------------------47 where: -----------48 therefore ---------------49 Substituting from equations (43). Competitive inhibition This occurs when both the substrate and inhibitor compete for binding to the active site of the enzyme. In the presence of a competitive inhibitor.. 1 showing effect of concentration of inhibitor on enzyme activity on different time interval. --------------------------44 For the present purposes. The inhibition is most noticeable at low substrate concentrations but can be overcome at sufficiently high substrate concentrations as the Vmax remains unaffected (Figure 10. then: --------------------52 This is the equation used generally for mixed inhibition involving both EI and ESI complexes (Figure 10. 1. or irreversible. involving incomplete inactivation. 2. as discussed earlier for substrate binding. where the loss of activity is time dependent and cannot be recovered during the timescale of interest. followed by simplification. and result in the independent variation of both Ki and Ki' with pH. More important for most enzyme-catalysed processes is the effect of reversible inhibitors. mercury and lead) should generally be prevented from coming into contact with enzymes as they usually cause such irreversible inhibition by binding strongly to the amino acid backbone. Figure 11.19b). it is assumed that neither EI nor ESI may react to form product. Normal Vmax can be observed when substrate is sufficiently present.g. but makes no net contribution to the rate equation as it must be equivalent to the equilibrium established through: ------45 Binding of inhibitors may change with the pH of the solution. in other cases. irreversible inhibition behaves as a time-dependent loss of enzyme concentration (i. If the inhibited enzyme is totally inactive.There may be time-dependent changes in both Km and Vmax. Equilibrium between EI and ESI is allowed.

S2) but uncompetitive with respect to another (e. in a competitive situation using the same enzyme and with both substrates at the same concentration: --------------59 Where and > in this simplified case. where the reaction scheme may be represented by. note that on increasing inhibitor concentration 1/Vmax remain unchanged while Km value changes. the Km (apparent Km) will increase in presence of inhinitor. all with different kinetic parameters. The rate equation for product inhibition is derived from equations (52) and (53). some hydrolyses): ---------------60 therefore --------------------------61 B. The rate equation is given by: ---------------------52 where Kmapp is the apparent Km for the reaction. If the rates of product formation are much slower than attainment of the equilibria (i. therefore. Uncompetitive inhibition This occurs when the inhibitor binds to a site which only becomes available after the substrate (S1) has bound to the active site of the enzyme. 2 figure showing competitive inhibition 1/Vo (1/ M/min) verses 1/[S] (1/ mM). which may cause a substantial loss of productivity when high degrees of conversion are required. their binding is mutually exclusive and they behave as competitive inhibitors of each others reactions. and especially relevant to macromolecular hydrolyses where a number of different substrates may coexist. e. If both reactions produce the same product (e. In double reciprocal plot this can be obsereved. S1).g. Ki is much greater than the total inhibitor concentration and the EI complex is not formed. This inhibition is most commonly encountered in multi-substrate reactions where the inhibitor is competitive with respect to one substrate (e. ---------------55 Both substrates compete for the same catalytic site and. and is given by -----------------53 Normally the competitive inhibitor bears some structural similarity to the substrate. the rate of formation of P1 is given by: ---------56 and the rate of formation of P2 is given by ---------------57 If the substrate concentrations are both small relative to their Km values: --------------------58 Therefore. inhibition of lactase by galactose). The relative rates of reaction are in the ratio of their specificity constants. one-half Vmax requires a higher [S] than before and thus Km is larger. The reaction involving two co-substrates may be modelled by the scheme. Figure 11.takes a higher substrate concentration to achieve the same velocities that were reached in its absence.g. -----54 Figure 11. But at [S] at which Vo=1/Vmax. k+2 and k+4 are very much less than k-1 and k-3 respectively).g. Ki' is much greater than the total inhibitor concentration and the ESI complex is not formed but EI is formed. and often is a reaction product (product inhibition. So while Vmax can still be reached if sufficient substrate is available.e. quite a common state of affairs in industrial conversions. --------61 . 3 showing effect of concentration of inhibitor on enzyme activity. A similar effect is observed with competing substrates.g.

5 Vmax). enzyme rate (velocity) is reduced for all values of [S].20). It is primarily caused by more than one substrate molecule binding to an active site meant for just one. again. (b) —— competitive inhibition ([I] = Ki). ________________________________________ Figure 11. at high substrate concentrations. —— KS/Km = 100. is often a reaction product. The inhibitor binding causes inactivation of enzyme due to which apparent Vmax (Vmax = Kcat [Et] ) lower down whether or not substrate is bound. ________________________________________ A special case of uncompetitive inhibition is substrate inhibition which occurs at high substrate concentrations in about 20% of all known enzymes (e. KS/Km >> 100. 5 figure showing case of uncompetitive inhibition In this case the specificity constant remains unaffected by the inhibition. higher Kmapp (= 2 Km). ________________________________________ C.e. —— KS/Km = 1.The inhibition is most noticeable at high substrate concentrations (i. 4 Figure 11.6 A schematic diagram showing the effect of reversible inhibitors on the rate of enzyme-catalysed reactions. invertase is inhibited by sucrose). lower Vmaxapp (= 0. It may be modeled by the following scheme ---------------65 where ---------------------66 The assumption is made that ESS may not react to form product. higher Kmapp (= 2 Km). If the resultant complex is inactive this type of inhibition causes a reduction in the rate of reaction. Noncompetitive inhibition This occurs when the inhibitor binds at a site away from the substrate binding site. often by different parts of the substrate molecules binding to different sites within the substrate binding site. Vmaxapp unchanged (= Vmax).g. lower Vmaxapp (= 0. and lower KS values cause substantial inhibition (Figure 10.5 Vmax) and Kmapp (= 0. Normally the uncompetitive inhibitor also bears some structural similarity to one of the substrates and. 7 The effect of substrate inhibition on the rate of an enzymecatalysed reaction. (c) —— uncompetitive inhibition ([I] = Ki'). S1 in the scheme above) and cannot be overcome as both the Vmax and Km are equally reduced (Figure 1. —— KS/Km = 10. (d) —— noncompetitive inhibition ([I] = Ki = Ki').8c).5 Ki'). (a) —— mixed inhibition ([I] = Ki = 0. —— no inhibition. ________________________________________ figure 11.67 Vmax). unchanged Kmapp (= Km). —— no inhibition. It follows from equation (52) that: -------------------------67 Even quite high values for KS lead to a leveling off of the rate of reaction at high substrate concentrations. By the nature of the binding causing this inhibition. A comparison is made between the inhibition caused by increasing KS relative to Km. lower Vmaxapp (= 0. including Vmax and one-half Vmax but Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged Therefore there is no . it is unlikely that KS/Km < 1. causing a reduction in the catalytic rate.5 Km). The rate equation is: ----------------------62 where Vmaxapp and Kmapp are the apparent Vmax and Km given by --------------------63 and --------------------64 Figure 11.

on integration. be determined. the optimum conditions for the reaction. There are two approaches to this problem using either the reaction progress curve (integral method) or the initial rates of reaction (differential method). so that [S] = [S]0. Both the EI and ESI complexes are formed equally well (i. too low).e. Alternatively the direct linear plot may be used (Figure 10. the programming skill involved is usually fairly low. may be considered as a special case of noncompetitive inhibition. Figure 11. Equation (70) can be utilised directly using a computer program.e. therefore. usually by use of a computer-aided analysis involving a weighted least-squares fit. The rate equation is given by: Figure 11. where the parameters for determining the hyperbolic relationship between the initial rate of reaction and initial substrate concentration (i. where -----------------------72 then equation (72) may be simplified to give: ----------73 Use of equation (70) involves the determination of the initial rate of reaction over a wide range of substrate concentrations. described earlier..e. ------------------70 which. involving a weighted least-squares fit. at least approximately. It is common practice to show the data . becomes ------------71 If the fractional conversion (X) is introduced. The fractional inhibition is identical at all substrate concentrations and cannot be overcome by increasing substrate concentration due to the reduction in Vmax . and the assumption is made that the errors inherent in the practically determined data are normally distributed about their mean (error-free) value. Many such computer programs are currently available and. if they are to be used most efficiently. It is quite rarely found as a special case of mixed inhibition. the predetermined and accurately known substrate concentration at the start of the reaction. Km and kcat/Km should. Km and Vmax) are chosen in order to minimise the errors between the data and the model. the inhibitor being the hydrogen ion on the acid side of the optimum or the hydroxide ion on the alkaline side. if not. using the boundary condition that the product is absent at time zero and by substituting [S] by ([S]0 . too high) as negative (i.effect on Km. initial attempts are usually made to fit the data to the Michaelis-Menten kinetic model. If the mechanism is not known.25). This is a powerful non-parametric statistical method which depends upon the assumption that any errors in the experimentally derived data are as likely to be positive (i. The initial rates are used.[P]). Use of either method depends on prior knowledge of the mechanism for the reaction and. If the mechanism is known and complex then the data must be reconciled to the appropriate model (hypothesis). 6 (b) Determination of Vmax and Km It is important to have as thorough knowledge as is possible of the performance characteristics of enzymes. Ki equals Ki').e. Its use also ensures that there is no effect of reaction reversibility or product inhibition which may affect the integral method based on equation (73). The kinetic parameters Vmax. 8 figure showing Noncompetative inhibition slope is Km /Vmax ---------------------------68 where Vmaxapp is given by: --------------------------------69 The diminution in the rate of reaction with pH. 9 Figure 11.

1. 4. 2. 1.91.94..89.91. It can be seen that outlying estimates have little or no influence on the results. 8 (a) Lineweaver-Burk (double-reciprocal) plot of 1/v against 1/[S]0 giving intercepts at 1/Vmax and -1/Km Figure 11.98. derived from equation (70) (Figure 10.99. the double reciprocal plot is preferred to test for the qualitative correctness of a proposed mechanism. 2.14.03. The Km must lie between the 4th (1. 1. 21 estimates for both Km and Vmax. ----------76 The progress curve of the reaction (Figure 10.89. the width of the subrange dependent on the accuracy required for the error and the number of data points in the analysis.obtained by the above statistical methods on one of three linearised plots.65.85 M. 3. 1. 3.12. is utilised in the analysis. The intersections are separately ranked in order of increasing value of both Km and Vmax and the median values taken as the best estimates for these parameters.28) can be used to determine the specificity constant (kcat/Km) by making use of the relationship between time of reaction and fractional conversion (see equation (73). 4.01.13. 4.87.25). possibly only one progress curve is necessary. This has the advantage over the use of the initial rates (above) in that fewer determinations need to be made. 9 (b) Eadie-Hofstee plot of v against v/[S]0 giving intercepts at Vmax and Vmax/Km (75) Figure 11.45. and the Eadie-Hofstee plot is preferred for discovering deviations from linearity. 4.y) points (-[S]0.1. If only the early part of the progress curve.70.96. 3. with a median value of 3.26.40. 1. 3. ________________________________________ Three ways in which the hyperbolic relationship between the initial rate of reaction and the initial substrate concentration can be rearranged to give linear plots (a) Lineweaver-Burk (double-reciprocal) plot of 1/v against 1/[S]0 giving intercepts at 1/Vmax and -1/Km --------------74 Figure 11. These estimates are determined from the intersections of lines passing through the (x. 1978).98.min-1) and 18th (4. 3. 4.0) and (0. with a median value of 1.min-1) estimate at a confidence level of 97%. The Vmax must lie between the 4th (3. 4. 3.85. 2.96. The error in these estimates can be simply determined from sub-ranges of these estimates.min-1) is ranked separately as 3. 1.68. Every pair of data points may be utilised to give a separate estimate of these parameters (i.29.81.18. 4.85. therefore.96. The ranked list of the estimates for Km (mM) is 0.35. 2.05. 2. 1.98 mM.38. 1.06. 10 c Hanes-Woolf (half-reciprocal) plot of [S]0/v against [S]0 giving intercepts at Km/Vmax and Km. 1.16.70 mM) and 18th (2.min-1.59. 2. In this example there are 7 data points and.v). 1. 3. each intersection forming a separate estimate of Km and Vmax.98. this procedure may even be used in cases where there is competitive inhibition by .21. 3. 1. 2.03. Of these.25 mM) estimate at a confidence level of 97% (Cornish-Bowden et al. 2.25.18 M. A plot of the initial rate of reaction against the initial substrate concentration also showing the way estimates can be directly made of the Km and Vmax. and sometimes the initial rate of reaction is rather difficult to determine due to its rapid decline. or its derivative. 4.98 M.94.80. The list of the estimates for Vmax ( M. 7 The direct linear plot. This is a major advantage over the least-squared statistical procedures where rogue data points cause heavily biased effects. n(n-1)/2 estimates from n data points with differing [S]0).87.e. 3. 3. ________________________________________ Figure 11. 4.

Sigmoidal kinetics and Allosteric enzymes Chapter-8 Multisubstrate enzymes Many Enzymes react with two or more substrates simultaneously (includes cofactors : ATP. Linear plots. A schematic plot showing the amount of product formed (productivity) against the time of reaction. Inhibitors and activators. ping-pong mechanism. King-Altman’s method.the product. or where the reaction is reversible. NADP. Multisubstrate systems. FAD etc) Michaelis Menten kinetics is observed when only one substrate is varied with the other(s) held constant (usually the cofactors) Common observed Mechanisms: Sequential – All substrates are bound by enzyme before the any products are formed and released Ordered : Substrates bind and products released in specific order Random : no order Ping Pong – When one or more product(s) are released before all substrates are bound by enzyme (acyl/phosphoryl enzyme intermediate) . 15 . Alberty equation. Table -2 Module III Enzyme Kinetics: Single substrate steady state kinetics. in a closed system. Michaelis Menten equation. The specificity constant may be determined by a weighted least-squared fit of the data to the relationship given by equation (73). ________________________________________ Figure 11. FADH. 11 FIGURE 10. NADPH.

electron microscopy.sequential means all substrates on before any products off. one substrate is obligated to bind to the enzyme before a second substrate. • In ordered sequential reactions. • Within the realm of sequential reactions lies ordered sequential and random sequential at the extreme ends. while ping pong has products off before all substrates on . • In practice. and then a second reactant interacts with the modification. in which both substrates must be bound before any product is released. enzyme molecules) are available • The kinetic mechanism of an enzyme is simply the sequence in which substrates bind to..can be ordered or random . in which one reactant modifies the enzyme.g. In practice. there is usually some degree of order in binding. Then. • Lineweaver-Burk (double-reciprocal) analysis should yield a family of lines that intersect at the left of the y-axis of the graph. DG= refers to the difference in free energy between the transition state and the substrate Enzymes work by decreasing DG= to facilitate more rapid formation of P Think of it as enzymes helping the chemical reaction get over the “hump” in order to form product (P) • ES complex demonstrated in a variety of ways including X-ray crystallography. vary the concentration of the second substrate and repeat. sequential and ping-pong (also known as double displacement) mechanisms. and spectrophotometry • Enzymes derive much of their catalytic power by bringing in a substrate molecule at a “favorable” orientation (this is how enzymes reduce the free energy required to convert S into P) • Leonor Michaelis (1913): noticed that at constant [enzyme] the rate of a reaction increases with increasing [S] until a “maximal velocity” (Vmax) is achieved • This “saturation effect” is an important distinction versus uncatalyzed reactions • Interpretation of data: ES complexes formed until substrate saturation occurs at which point no more substrate binding sites (i. In random sequential mechanisms there is no preference. • This is distinguished from what is termed ping-pong kinetics. Kinetic mechanisms can be broadly divided into two main types. It should not be confused with the chemical mechanism which is concerned with the chemical interactions between the enzyme and its substrate(s) which results in the creation of product(s). measure initial rates as a function of one substrate while holding the other constant.e. • Sequential kinetics can be distinguished from ping-pong kinetics by initial rate studies. • Sequential mechanisms • Sequential reactions are ones in which all reactants bind to the enzyme . and products are released from the enzyme.: ________________________________________ • Adenylate kinase displays sequential kinetics.e.

buffers. Thus if we can find a way to stabilize the transition state (lower Ea) then the reaction rate will be enhanced. because of their own chiral nature enzymes can often hold substrates in such a way that on one chiral product is made. • Example enzymes to demonstrate these mechanisms in enzyme catalysis. o Example . o Example . o • Plots of vi = d[P]/dt vs.3rd order • First Order: r = k[S] for S P.before the first product is released. • Ping-pong mechanisms • Ping-pong reactions are ones in which at least one product is released before all the substrates have bound. Enzyme Catalysis We will look at catalysis in two types of systems: • Model systems in organic chemistry to elucidate probable mechanisms of chemical catalysis. Prochirality. • To clarify these distinctions we'll look at each of these mechanisms in turn using a typical bi bi enzyme: • A + B P + Q • We'll start by looking at an ordered sequential reaction.) • Covalent o nucleophilic o electrophilic • Proximity/orientation • Stabilization of Transition State Conformation (Strain/distortion. Types of Catalysis: • Acid/Base o Specific (H+ & OH. in which the reactants and products are bound and released in an obligatory sequence. Types of specificity: o Geometric specificity: shape Chiral specificity: most chirally specific enzymes are absolutely stereospecific.in water) o General (Bronsted acid definition: proton donor/acceptor pairs. increase the concentrations of intermediates 3. distinguishing between seemingly identical groups. stabilize transition states 2.SN2 from organic chemistry: A + B C + D as 2nd order reaction. [S] for 0 . which is perhaps the simplest in kinetic terms.SN1 from organic chemistry: A + B C + D as 1st order reaction. They can be further subdivided into ordered reactions. Generally we will be looking at three ways to increase rates 1. So how does catalysis work? Recall that the slow step of a reaction is reaching the transition state. o Chemical specificity: functional groups. Charge neutralization) • Metal/Metal ion. in which there is no obligatory binding sequence. and random reactions. types of chemical reaction. or can have r = k[A]2 . use a different reaction pathway. which in turn depends only on [R3CCl] • Second order: r = k[A][B] for A + B P. as in the hydroxylation of primary alkylchlorides: . & r =-d[S]/dt = d[P]/dt. as in the hydroxylation of t-alkylchloride: First order because rate depends only on the formation of the carbocation. Mechanisms of Chemical Catalysis Look at some examples of catalysis in model systems (organic chemistry) and how they might operate in enzymes.

important in enzyme catalysis. S P get rectangular hyperbola type plot for vi vs [S] (text Figure 6-11).k3[ES]. This was first done by Michaelis and Menten for an equilibrium model. • • • Let's look at a mathematical model and attempt to generate curve. For simple. then vi = Vmax/2 This is definition of KM. 0 order also only occurs above a minimum [A]. so take reciprocals of both sides and have . Linear plots for enzyme kinetic studies Double Reciprocal or Lineweaver-Burke Plot: Need in form: y = ax + b . which we will derive. with a strong influence on the slope and the KM intercept ¬ this is not as much of a concern now with computer statistical packages. The rate constant (First order) for the breakdown of the [ES] complex. but uncommon. Then at the limit of high concentrations have a horizontal line.(reverse of figure above) are involved in slow step. then vi = Vmax and get Zero order (r = k) • [S] << KM. Note that the data points are distributed much more evenly over the plot giving better statistics for the slope.The Eadie-Hofstee Plot One common plot is shown below. so have • • Now vi = d[P]/dt = k3[ES] (Note that kcat is often used instead of k3). So. while low precision points [low concentration] are more spread out.Now 2nd order because both RH2CCl and OH. Better is the steady state model of Haldane and Briggs (more general). the formula for a rectangular hyperbola: (text Figure 6-12) Turnover Number. and are better in terms of statistics (L-B one of worst. (text Table 6-08) At very low concentrations of [S] can find the second-order rate constant for the conversion of E + S E + P: vo = (kcat / KM)([E][S]. (text Box 6-01 figure 6-1) Other linear plots are also available. • Zero order: r = k: Only occurs with catalysts. the substrate concentration at half-saturation. In addition the . • For S P assume • • And for initial reaction conditions [P] = 0 & therefore k4 = 0. We will see others in the laboratory discussion. • Higher order reactions occur. • Note consequences for a plot: start off with approximately linear slope with y = kx. • Assume steady state (steady state assumption: d[ES]/dt= 0): • d[ES]/dt= 0. Which is known as the Michaelis-Menten Equation. Thus: 0 = d[ES]/dt= k1[E][S] . kcat (k3). best quality points [high concentration] have least influence on slope. have Michaelis-Menten Equation as a model for enzyme activity. is also known as the turnover number. that is the maximum number of substrate molecules processed/active site (moles substrate/mole active site): kcat=Vmax / [E]total. but you still have to understand the statistics). Note predicted consequences of model: • [S] >> KM. and have a large moment. and get First order (r = k [S]) • [S] = KM. one-substrate enzymes then. similar to Mb binding curve. This is exactly what we expect if we look at the general form of the equation: . FYI .k2[ES] . then . Note that this is best determined under saturating conditions. one-substrate enzymes: For simple enzyme.

as in the equation above. giving better precision. o Understanding drugs and toxins to counter etc. two off). S & I do not bind to the same sites! (text Figure 6-15c) Model: . Classically assume binding to same site. leading to more complex behavior. Note: A must bind first. 3. they may be the same. 4. Plots: (text Box 6-2 figure 1) We can model this inhibition with chemical equations. Model: . 2. keeping in mind that S & I are mutually exclusive. one off. Noncompetitive: the inhibitor can bind to either E or ES. P or Q may be released last. where . one off). or could be different. overlapping sites for S & I. Three major types of inhibition: • Competitive inhibition • Noncompetitive inhibition • Uncompetitive inhibition Competitive Inhibition: S & I are mutually exclusive. but other possibilities also.value of KM is obtained from the slope. with a L-B plot with an intersection on the plot above the 1/vo axis. E can bind to one OR the other. • Potential uses as drugs and toxins. For double reciprocal plots get parallel lines! (text Box 6-2 figure 2) This is not generally found for single substrate enzymes. steric hindrance between S & I in different sites. Note: A or B may bind first. Note that will have two inhibitor binding constants. two off). Model: . and but with different values of Ki.5): Mixed: Like non-competitive above. and . Conformational change of enzyme with binding of either such that other can not bind. Partial sharing of sites. simple situation (overhead MvH 11. and: . one on. This is in fact the more common situation. Two-substrate Enzyme Product Inhibition Patterns . and ." • Ordered Sequential Bi Bi mechanism (two on. but is found in multi-substrate systems. (text Box 6-2 figure 3) Uncompetitive: In uncompetitive inhibition the inhibitor binds ONLY to the ES complex (text Figure 6-15b). E can bind to one OR the other: (text Figure 6-15a) Model: . 1. ENZYME KINETICS AND INHIBITION What's exciting about enzyme inhibition? • Potential to tell us about enzyme. • Ping Pong Bi Bi (one on. Plots for classic. We will use Cleland Nomenclature and "Kinetic mechanism diagrams. • Multi-substrate Enzymes Look at three common and easily understood types. Q is released last. Note: have some sort of modified enzyme intermediate (often covalent intermediate) • Random Sequential Bi Bi (two on.

in this case the free enzyme. The active site is full as this substrate will be converted to product before the second substrate can bind. but it's actually easily explained and quite a common mechanism. This might seem slightly unlikely at a first glance. etc.(Based on: E. since they both bind to the E-X complex. The process starts by binding of the enzyme to the first substrate in the usual way: E + A (EA) Notice that I've used parentheses around the EA complex to indicate that it is a central complex. Biochemistry: Mechanisms of Metabolism. In this case we also see a competitive inhibition between the second substrate and the first product. The removed section has become covalently bound to the enzyme to create a new form of the enzyme. ed. converting it to product P. enzyme F.) The ping-pong (double displacement) mechanism ________________________________________ The distinguishing feature of these enzymes is that at least one product is released from the enzyme before all of the substrates have bound. Cleland. Purich.) and the amino transferases work in this way. The next reaction is the key to the whole process: (EA) (FP) In this reaction a part of the substrate has been removed from substrate A. Here we see across the board noncompetitive since in each case the substrates (and products) can each bind to more than one substrate form. McGraw-Hill Book Company. so competitive inhibition will not be possible! (Think of the product as competing with one order of binding but not the other. for instance the serine proteases (trypsin. Similarly for the Ping pong mechanism the first substrate and last product should be competitive as the both bind the free enzyme." Thus for the Ordered Sequential mechanism only the first substrate and last product bind to the same form. chymotrypsin. The Random Sequential mechanism is a bit more subtle. and W. (Daniel L. New York (1978). The active site has no room for the second substrate so it is full. Cunningham. New york (1983)) Kinetic Mechanism Variable Substrate Product Type of Inhibition Ordered Sequential Bi Bi Ordered Sequential Bi Bi A B Q Noncompetitive A P Noncompetitive B P Noncompetitive Random Sequential Random Sequential B Q A P B P Ping pong Bi Bi Ping pong Bi Bi B Q A P B P Bi Bi Bi Bi A Q Noncompetitive Noncompetitive Noncompetitive Q Competitive Noncompetitive A Q Competitive Noncompetitive Noncompetitive Competitive Note that in each case we can predict/explain the pattern of inhibition on the basis of the substrate and inhibitor binding to the same "enzyme form. B. "Substrate Inhibition: in Contemporary Enzyme Kinetics and Mechanism. Some very familiar enzymes. The first product of the reaction is now released and the second substrate binds: (FP) F + P F + B (FB) Now the stored section of the first substrate is transferred to the second .) Academic Press.

which is then released: (FB) (EQ) (EQ) E + Q The animation should help to clarify this. while we vary the concentration of the other substrate (the variable substrate). Since you're increasing the concentration of a substrate you would expect the velocity to rise and in fact the reaction would be faster at any given concentration of the variable substrate than it was in the previous experiment. An increase . the reaction would be the short supply.substrate to create the second product. enables us to distinguish between sequential and ping-pong enzymes. To start with we'll examine the effects of changes in substrate concentration on multisubstrate reactions. A typical Lineweaver-Burk plot obtained as a result of this type of experiment can be seen below. So you'd end up with a second set of data which you could use in your chosen plot. The actual pattern of lines obtained will vary according to the way in which the enzyme interacts with the two substrates and. Remember in our discussion of enzyme inhibition we found that the slope was the rate constant at low substrate concentrations. We'll start by considering the expected results of this experiment when carried out with a sequential enzyme. Substrate concentration assays with sequential enzymes ________________________________________ Consider a bi bi ordered sequential reaction: A + B P + Q The Cleland plot for such a reaction would be: We'll discuss the results of a set of enzyme assays in as the variable substrate and substrate B as the fixed At very low A concentrations the rate limiting step of binding of A to the enzyme as the substrate is in very which substrate A is used substrate. as we'll see in the next couple of pages. We simply need to keep one substrate (the fixed substrate) at a constant concentration in all our assays. The results would be exactly the same as you'd expect in a single substrate system and you could use any of the methods that we've studied to calculate the kinetic parameters. In discussing graphs of this type we'll be considering changes in the Vmax and slope of the line. let's say a typical bi bi enzyme: A + B P + Q we can carry out exactly the same experiment. Effects of Substrate Concentration in Multisubstrate Systems ________________________________________ With a multisubstrate enzyme. The kinetic parameters would change to reflect the change in velocity. A change in Vmax indicates the effect that a change in the concentration of the fixed substrate on the reaction speed at very high concentrations of the variable substrate. What would happen though if you repeated the experiment with an increased concentration of the fixed substrate. Now that we're familiar with the principal types of kinetic mechanism we need to think about experimental techniques to distinguish between them. Remember that Vmax is the velocity of the reaction under those conditions. Substrate A was used as the variable substrate and substrate B as the fixed substrate. A change in slope indicates the effect of a change in concentration of the fixed substrate on the speed at very low concentrations of the variable substrate. The Cleland plot for a ping-pong enzyme is quite distinctive: as the first upward arrow (product P) is to the left of the first down arrow (substrate B). If you repeated this at a variety of concentrations of the fixed substrate you would get a series of lines.

As we're looking at effects at low A concentrations this would be seen as a change in the slope of the Lineweaver-Burk plot. Inhibition kinetics helps in functional study of the enzyme.in the concentration of B would reduce the concentration of the EA complex in the reaction mixture by binding to it to form EAB complex. So the increase in B has increased the speed of the rate limiting step. 1 strategies for enzyme purification. It helps in increasing the sensitivity of the diagnosis test in the clinics. At very high A concentrations the rate limiting step would be the EAB EPQ. or EA + B EAB at low B levels. You might like to demonstrate that yourself as an exercise. Other need of the enzyme purification is the study of kinetics that is rate of enzyme and its specificity towards the substrate. and therefore of the overall reaction. And also isolation of enzyme gives the maximum understanding of the behavior of the enzyme in complex system. FIGURE 8. source of enzyme Method of enzyme isolation (cell lysis by osmotic. In summary then. Reducing the concentration of the product of the E + A EA reaction will pull it to the right by the Law of Mass Action. This would be seen as a change in Vmax as we are considering effects at high A concentration. Purification of enzyme is aimed to get maximum turn over number that is to increase the activity of enzyme in other world to increase its efficiency of conversion of per mole substrate into the product. An increase in B would increase the speed of either of these as a reactant in the second reaction and by the knock on effect of generating more EAB for the central reacton to occur. During the disturbance in the metabolic pathway some enzyme may not form and it results in the formation of unwanted product like in the phenylketonuria disease homigenistic acid secretion in the urine results. a change in the concentration of the fixed substrate would bring about a change in both the slope and intercept of the Lineweaver-Burk plot and the graph would be similar to that suggested on the previous page. which is the inherently slowest reaction. sonication. . Often during purification enzyme yield is decreases many folds. for an ordered sequential reaction. A similar result would be obtained if the assay was reversed and B used as the variable substrate or if the enzyme used a random sequential mechanism. PURIFIED enzyme is needed for laboratory work and less purified enzyme is needed for commercial purpose. enzyme. its regulation mechanism. [i] Based on size and mass ( centrifugation. Therefore for the diagnostic purpose much pure enzyme is needed. Purifying enzyme is much labor intensive process and main aim is to separate other protein that are not the part of the enzyme. Chapter-9 ISOLATION AND PURIFICATION OF ENZYME INTRODUCTION ENZYME is isolated and purified to obtain maximum desired product because they are not utilized during the product formation. GPC gel permeation chromatography. or homogenization method) Method of separation.

Affinity elution. A very wide range of sources are used for commercial enzyme production from Actinoplanes to Zymomonas. Attempts are being made to overcome some of these difficulties by the use of animal and plant cell culture. Some important industrial enzymes and their sources. [ii] Based on polarity (Ion –exchange. Test of catalytic activity c).1. Non-microbial sources provide a larger proportion of these. Electrophoresis (Examining enzyme composed of non identical subunit) 3.11. at the present time. reliable supplies of raw material of constant composition are more easily arranged. (4). Capillary Electrophoresis 5. Isoelectric –focusing ( very sensitive method) 6. isoelectric focusing. Microbes are preferred to plants and animals as sources of enzymes because: 1. SDS-PAGE ( good method for detecting impurities and for detecting damage of non-identical subunit. they are generally cheaper to produce. over a half are from fungi and yeast and over a third are from bacteria with the remainder divided between animal (8%) and plant (4%) sources (Table 8.1. their enzyme contents are more predictable and controllable. 3. endogenous enzyme inhibitors and proteases. hydrophobic interaction chromatography). Enzyme a EC number b Source Intra/extra -cellular c Scale of production d Industrial use Animal enzymes Catalase 1. 5.1 Pancreas Lipasee 3. Test of purification a).21.1. Ultrapurification (for impurities < 5%) 2. A very much larger number of enzymes find use in chemical analysis and clinical diagnosis. electrophoresis. Dye –ligand chromatography.( power full method ) Source of enzyme Biologically active enzymes may be extracted from any living organism.4. Immuno-adsorption chromatography.Dialysis and ultracentrifugation).3 Pancreas E Food Rennetf Food E - Leather . plant and animal tissues contain more potentially harmful materials than microbes. [iv] Based on change in dielectric strength by adding organic solvent. including phenolic compounds (from plants). [iii] Based on changes in solubility ( by change of pH.1).) 4. Affinity chromatography. Covalent chromatography. 2.1. change in ionic strength (Salting in or salting out). and 4. Active site titration Test of purity is done by following method 1. ________________________________________ Table 2. test of purity b). Mass –spectrometry. from spinach to snake venom. Of the hundred or so enzymes being used industrially. [v] Based on specific binding site.6 Liver I Chymotrypsin 3.

14 Bacillus E +++ Detergent Pullulanasej 3.1.1 Aspergillus E ++ Baking Aminoacylase 3.2.4.1 Escherichia coli I Health Glucose isomeraseh 5.3.2.3 Aspergillus E +++ Starch Catalase 1.1.1. Where appropriate.14 Aspergillus I Pharmaceutical Glucoamylasek 3.1.22.2.11.4.1.2.22 Mortierella I Food Yeast enzymes Invertasep 3.22.21.6 Mucor miehei E ++ Cheese Pectinasen 3.22 Saccharomyces I Food a The names in common usage are given.1 Malted barley E +++ Brewing -Amylase 3.10 Aspergillus E Drinks Proteasem 3. the recommended names of this principal .2.3.15 Aspergillus E ++ Drinks Pectin lyase 4.6 Aspergillus E + Baking Raffinaseo 3.11 Penicillium E Food Glucose oxidase 1.1.1.2 Malted barley E +++ Brewing Bromelain 3.4 Pineapple latex E Brewing Glucanaseg 3.4.1.2.41 Klebsiella E Starch Fungal enzymes Amylase 3.4 Abomasum E + Cheese Trypsin 3.4.1.4.1.3 Candida E Food Raffinaseo 3.21.22.4 Pancreas E Leather Plant enzymes Actinidin 3.23.1.1.23.4.1.2.1.4 Trichoderma E Waste Dextranase 3.3 Fig latex E Food Lipoxygenase 1.23 Kluyveromyces I/E Dairy Lipasee 3.5.1.1.2 Pawpaw latex E ++ Meat Bacterial enzymes Amylase 3.2.26 Saccharomyces I/E Confectionery Lactasel 3.2 Bacillus E + Starch Asparaginase 3.1.12 Soybeans I Food Papain 3.5 Bacillus I ++ Fructose syrup Penicillin amidase 3.1.2.3.14 Kiwi fruit E Food Amylase 3.11 Bacillus I Pharmaceutical Proteasei 3.6 Aspergillus I Food Cellulase 3.1.2.3 Rhizopus E Food Rennetm 3.2. these names may vary from the recommended names of their principal component.5.5. As most industrial enzymes consist of mixtures of enzymes.1.2.23 Aspergillus E Dairy Lipasee 3.13.6 Malted barley E ++ Brewing Ficin 3.4.2.1 Bacillus E +++ Starch -Amylase 3.23.2.1.4.1.4 Aspergillus I Food Lactasel 3.4.1.2.2.22.1.2.11.1.1.2.

f chymosin. g Endo-1. b The EC number of the principal component. sometimes refined but often simply as ground cereal grains. it may be economical to use a more expensive defined medium of easily reproducible composition. e triacylglycerol lipase. carbohydrate feedstock change.component is given below.-glucosidase.-glucanase. only other enzymes and material likely to interfere with the process . ++ > 10 ton year-1. In contrast. corn steep liquor. the great majority of microbial enzymes come from a very limited number of genera. .4. Less-defined complex media are composed of ingredients selected on the basis of cost and availability as well as composition. distillers soluble and wheat bran is important components of fermentation media providing carbohydrate. The effects of changing feedstock must be considered in relation to downstream processing.6. Soybean meal and ammonium salts are frequently used sources of additional nitrogen. E . Most of these materials will vary in quality and composition from batch to batch causing changes in enzyme productivity. Extra carbohydrate is usually supplied as starch. frequently below 10% of the activity of the original material (Table 2.intracellular enzyme. for instance. Details of components used in industrial scale fermentation broths for enzyme production are not readily obtained. of which Aspergillus species. n polygalacturonase. This is not unexpected as manufacturers have no wish to reveal information that may be of technical or commercial value to their competitors. Thus molasses. Media for enzyme production Detailed description of the development and use of fermentors for the large-scale cultivation of microorganisms for enzyme production is outside the scope of this volume but mention of media use is appropriate because this has a bearing on the cost of the enzyme and because media components often find their way into commercial enzyme preparations. h xylose isomerase. Also some components of media may be changed from batch to batch as availability and cost of. In practice. i subtilisin. l -galactosidase. Often the enzyme may be purified several hundred-fold but the yield of the enzyme may be very poor. If such variability is likely to significantly reduce the efficiency of the standard methodology. m microbial aspartic proteinase. Shining examples of such developments are the production of high fructose syrup using glucose isomerase and the use of pullulanase in starch hydrolysis. industrial enzymes will be purified as little as possible. Clearly defined media are usually out of the question for large scale use on cost grounds but may be perfectly acceptable when enzymes are to be produced for high value uses.2). + > 1 ton year-1.-glucosidase. c I . j dextrin endo-1. p -fructofuranosidase.extracellular enzyme. Bacillus species and Kluyveromyces (also called Saccharomyces) species predominate.< 1 ton year-1. d +++ > 100 ton year-1. Such changes reveal themselves in often quite profound differences in appearance from batch to batch of a single enzyme from a single producer. There are very few examples of the industrial use of enzymes having been developed for one task. nitrogen and some vitamins. Waste materials and byproducts from the food and agricultural industries are often major ingredients.3(4). such as analysis or medical therapy where very pure preparations are essential. k glucan 1. Most of the strains used have either been employed by the food industry for many years or have been derived from such strains by mutation and selection. minerals. o -galactosidase.

or inhibit. but the problem is less when preparing thermophilic enzymes.g. oxidation. manpower and loss of enzyme activity. As a result. Flow diagram for the preparation of enzymes. Cell can be break down byfollowing methods 1. irreversible inhibitors and loss of cofactors or coenzymes. Solid/liquid separation is generally required for the initial separation of cell mass. denaturants. Single 'nicks' by proteases in these circumstances may have little immediate effect on protein conformation and. It is also the major reason for enzyme inactivation by microbial contamination.1. A. may severely reduce the conformational stability of the enzyme to heat or pH variation so greatly reducing its operational stability. The content of the required enzyme should be as high as possible (e. This may be achieved by developing the fermentation conditions or. Before this can be achieved it is important to keep enzyme preparations cold to maintain their native conformation and slow any protease action that may occur. It should have maximum possible activity. this together with associated pH effects is probably the most significant. plus additives to stabilize the enzyme's activity. enzymes have highly structured domains which are resistant to attack by proteases because many of the peptide bonds are mechanically inaccessible and because many proteases are highly specific. 3. Osmotic shock 2. sub-optimal pH. Enzymic lysis. 10% w/w of the protein) in order to ease the downstream processing task. by genetic engineering. Proteolysis is most likely to occur in the early stages of extraction and purification when the proteases responsible for protein turnover in living cells are still present. Ultracentrifugation. Clearly the best way of preventing proteolysis is to rapidly remove. however. Enzyme inactivation can be caused by heat. The chances of a susceptible peptide bond in a structured domain being available for protease attack are low. Some intracellular enzymes are used commercially without isolation and purification but the majority of commercial enzymes is either produced extracellularly by the microbe or plant or must be released from the cells into solution and further processed (Figure 2. specific. protease may destroy it.1). therefore. Cell breakage by osmotic shock Various intracellular enzymes are used in significant quantities and must be . activity. Of this heat inactivation. the whole polypeptide chain may be available for proteolysis and the same. filtration or aqueous biphasic systems are used to remove unwanted cells or cell debris whereas centrifugation is the preferred method for the collection of required solid material. Unnecessary purification will be avoided as each additional stage is costly in terms of equipment. It is important that the maximum activity is retained during the preparation of enzymes. The main objective of the purification is To obtained maximum possible yield. The effect. Homogenization 4. the removal of cell debris after cell breakage and the collection of precipitates. It should have maximum possible purity. proteolysis. It may well be economically viable to spend some time cloning extra copies of the required gene together with a powerful promoter back into the producing organism in order to get 'over-producers' (see Chapter protein engineering).which the enzyme is to catalyze. centrifugation or aqueous biphasic partition. often more dramatically. some commercial enzyme preparations consist essentially of concentrated fermentation broth. will be removed. Figure 2. If the domain is unfolded under these changed conditions. In general. protease activity. This can be achieved by filtration. It is likely to occur during enzyme extraction and purification if insufficient cooling is available. In their native conformations.

The particular hazards to enzyme activity relevant to cell breakage are summarized in Table 2.7) As the protein released from the cells (Pr) is given by (2. of reinforced concrete. such as EDTA. Chemical Some enzymes may suffer conformational changes in the presence of detergent and/or solvents. The amount of energy that must be put into the breakage of cells depends much on the type of organism and to some extent on the physiology of the organism. . ________________________________________ Media for enzyme extraction will be selected on the basis of cost-effectiveness so will include as few components as possible. substrate analogues or polyols may also help stabilise the enzyme. fungal mycelia and some gram-positive bacteria which have cell wall and membrane structures capable of resisting internal osmotic pressures of around 20 atmospheres (2 MPa) and therefore have the strength. green algae. ________________________________________ Table 2. iron.3.6) (2. Foaming The gas-liquid phase interfaces present in foams may disrupt enzyme conformation. Heavy-metal toxicity Heavy metal ions (e. substrate analogues or polyols may also help stabilise the enzyme. (2. particularly in the presence of heavy metal ions and/or an air interface. are heating and shear. pH Buffered solutions may be necessary.9) It is most important in choosing cell disruption strategies to avoid damaging the enzymes. The most significant of these. and by the use of ascorbic acid to reduce polyphenol oxidase action. t is the time and k is a release constant dependent on the system. copper and nickel) may be introduced by leaching from the homogenisation apparatus. Polyphenolics derived from plants are potent inhibitors of enzymes.g.g.5) where P represents the protein content remaining associated with the cells. mercaptoethanol and dithiothreitol) may be necessary. in general. Some types of cell are broken readily by gentle treatment such as osmotic shock (e. Hazards likely to damage enzymes during cell disruption. primarily factors causing denaturation (Table 2. Enzymes may be protected from irreversible inactivation by the use of chelating reagents. animal cells and some gram-negative bacteria such as Azotobacter species). Shear Shear forces are needed to disrupt cells and may damage enzymes.1). ascorbic acid. Proteases Disruption of cells will release degradative enzymes which may cause serious loss of enzyme activity. inexpensive protein) or inhibitors in the extraction medium. The presence of substrates. This problem may be overcome by the use of adsorbents. These include yeasts.g.3). Heat All mechanical methods require a large input of energy. Such action may be minimised by increased speed of processing with as much cooling as possible.released from cells and purified (Table 2.3. This may be improved by the presence of an excess of alternative substrates (e. The presence of substrates. Other components will combat other hazards to the enzyme. generating heat. Cooling is essential for most enzymes. Integrating from P = Pm (maximum possible protein releasable) at time zero to P = Pt at time t gives (2.8) the following equation for cell breakage is obtained (2. Oxidation Reducing agents (e. Consequently a variety of cell disruption techniques have been developed involving solid or liquid shear or cell lysis. whilst others are highly resistant to breakage. Media will usually be buffered at a pH value which has been determined to give the maximum stability of the enzyme to be extracted. The rate of protein released by mechanical cell disruption is usually found to be proportional to the amount of releasable protein. such as polyvinylpyrrolidone.g. weight for weight.

surfactants and solvents can be effective in releasing enzymes. freezing followed by thawing. Where applicable. The constant (k) is independent of cell concentrations up to high levels and approximately proportional to the input acoustic power above the threshold power necessary for cavitation. Detergents. The collapse of the bubbles converts sonic energy into mechanical energy in the form of shock waves equivalent to several thousand atmospheres (300 MPa) pressure. Each method has its drawbacks but may be particularly useful under certain specific circumstances. This energy imparts motions to parts of cells which disintegrate when their kinetic energy content exceeds the wall strength. such as guanidine HCl. The rate of drying is very important in these cases. Use of enzymic lytic methods The breakage of cells using non-mechanical methods is attractive in that it offers the prospects of releasing enzymes under conditions that are gentle. Ultrasonication produces cavitation phenomena when acoustic power inputs are sufficiently high to allow the multiple production of microbubbles at nucleation sites in the fluid. enzymic lysis and chemical lysis. provided that the enzymes are sufficiently robust. Much of the energy absorbed by cell suspensions is converted to heat so effective cooling is essential. are effective in releasing membrane-bound enzymes. Yeast-lytic enzymes from Cytophaga species have been studied in some detail and other lytic enzymes are under development. On collapse. then are collapsed during the compression phase. gentle and convenient method of releasing enzymes but has not apparently been used on a large scale. The whole process of gas bubble nucleation. a violent shock wave passes through the medium. slow methods being preferred to rapid ones like lyophilisation.000 g). The amount of protein released by sonication has been shown to follow Equation 2.1 MHz). This would be a cheap. c. Ultrasonic cell disruption The treatment of microbial cells in suspension with inaudible ultrasound (greater than about 12 kHz) results in their inactivation and disruption. It is characterised by high frequency (12 kHz . It has often used to lyse Gram positive bacteria in an hour at about 50. and are quiet to the user. Some types of cell may be caused to autolyse. If significant markets for lytic enzymes are identified. Where costs are reduced by the use of the relatively inexpensive. Disintegration is independent of the . However. Autolysis is a slow process compared with mechanical methods. desiccation. desiccation may be very useful in the preparation of enzymes on a large scale. The methods that are available include osmotic shock. growth and collapse due to the action of intense sound waves is called cavitation.000 U Kg-1 (dry weight). the scale of their production will increase and their cost is likely to decrease. The chief objection to its use on a large scale is its cost. Lysozyme. from hen egg-white. is the only lytic enzyme available on a commercial scale. Ultrasonication utilises the rapid sinusoidal movement of a probe within the liquid. small displacements (less than about 50 m). Certain types of cell can be caused to lyse by osmotic shock. Yeast invertase preparations employed in the industrial manufacture of invert sugars are produced in this manner. do not subject the enzyme to heat or shear. An additional factor which increases cell breakage is the microstreaming (very high velocity gradients causing shear stress) which occur near radially vibrating bubbles of gas caused by the ultrasound. cold shock. may be very cheap. Enzymic lysis using added enzymes has been used widely on the laboratory scale but is less popular for industrial purposes. moderate velocities (a few m s-1). Lysozyme-rich. a major separation problem may be introduced.9. and microbial contamination is a potential hazard. The bubbles grow during the rarefying phase of the sound wave. steep transverse velocity gradients (up to 4. alkali. dried egg white.B. such as Triton X-100. used alone or in combination with certain chaotropic agents. but it can be used on a very large scale if necessary.000 s-1) and very high acceleration (up to about 20. in particular yeasts and Bacillus species. such materials are costly and may be difficult to remove from the final product.Lysis by acid.

N2O) have been shown to reduce this inactivation. very fine cell debris particles may be produced which can hinder further processing. showing the flow of material.000 L hr-1. however. The release of proteins can be described by Equation 2. disruption being more rapid at higher temperatures. homogenisers . the higher the operating pressure. they decrease the throughput productivity rate and because the further passage of already broken cells results in fine debris which is excessively difficult to remove further downstream.2. Cells are subjected to impact. In practice. The main disruptive factor is the pressure applied and consequent pressure drop across the valve.sonication frequency except insofar as the cavitation threshold frequency depends on the frequency. the more efficient is the disruption process. Cell lysis by High pressure homogenisers Various types of high pressure homogeniser are available for use in the food and chemicals industries but the design which has been very extensively used for cell disruption is the Manton-Gaulin APV type homogeniser. k=k'P2. Consequently. The model depicted is the 'CD Valve' from APV Gaulin. At an operating pressure of 50 MPa. Multiple passes are undesirable because. The cell suspension is pumped at high pressure through the valve impinging on it and the impact ring. through an adjustable discharge valve which has a restricted orifice (Figure 2.2 in Escherichia coli. Unbound intracellular enzymes may be released by a single pass whereas membrane bound enzymes require several passes for reasonable yields to be obtained. it is unsuitable for the disruption of mycelial organisms but has been used extensively for the disruption of unicellular organisms. The shape of the exit nozzle from the valve seat varies between models and appears to be a critical determinant of the homogenisation efficiency. Reasons for this may be the conformational lability of some (perhaps most) enzymes to sonication and the damage that they may realize though oxidation by the free radicals. C. Sonication remains. This causes the impact and shear stress which are proportional to the operating pressure. This consists of a positive displacement pump which draws cell suspension (about 12% w/v) through a check valve into the pump cylinder and forces it.10) In the commonly-used operating range with pressures below about 75 MPa. the release constant (k) has been found to be proportional to the pressure raised to an exponent dependent on the organism and its growth history (e.g. useful and simple small-scale method for cell disruption. ________________________________________ As narrow orifices which are vulnerable to blockage are key parts of this type of homogeniser. a popular.9 but normally a similar relationship is used where the time variable is replaced by the number of passes (N) through the homogeniser. of course. the location of an enzyme within the cells can influence the conditions of use of an homogeniser. In addition to the fragility of the cells. The protein release rate constant (k) is temperature dependent.g. Equipment for the large-scale continuous use of ultrasonic has been available for many years and is widely used by the chemical industry but has not yet found extensive use in enzyme production. (2. Use of radical scavengers (e. Different growth media may be selected to give rise to cells of different cell wall strength. A cross-section through the Manton-Gaulin homogeniser valve. shear and a severe pressure drop across the valve but the precise mechanism of cell disruption is not clear. As with most cell breakage methods. singlet oxygen and hydrogen peroxide that may be concomitantly produced. this advantage cannot be used since the temperature rise due to adiabatic compression is very significant so samples must be pre-cooled and cooled again between multiple passes. Clearly. at high pressures of up to 150 MPa (10 tons per square inch) and flow rates of up to 10. d.5). where P represents the operating pressure and k' is a rate constant). the temperature rise each pass is about 12 deg. Figure 2.9 in Saccharomyces cerevesiae and k=k'P2.

11 reduces to give the simplified relationship of Equation 2. may be disrupted by bead milling but. For the same volume of beads. Heat production is the major problem in the use of bead mills for enzyme release. Equation 2. a large number of small beads will be more effective than a relatively small number of larger beads because of the increased likelihood of collisions between beads and cells. if cooling is effective there is little damage to the enzymes released. Any type of biomass. Use of bead mills for cell lysis When cell suspensions are agitated in the presence of small steel or glass beads (usually 0.9 with respect to the time (t) that a particle spends in the mill. both of which consist of a cylindrical vessel containing a motor-driven central shaft equipped with impellers of different types. the protein release rate constant (k) is a function of temperature.9 at low (near zero) values of i. The valve unit is prone to erosion and must be precision made and well maintained. Glass Ballotini or stainless steel balls are used. 2. particularly on a large (e. Under these circumstances the relationship can be shown to be (2. the size range being selected for most effective release of the enzyme required.6 Method of separation of enzyme from lysed cell. The kinetics of protein release from bead mills follows the relationship given by Equation 2. Bead mills are available in various sizes and configurations from the Mickle shaker which has a maximum volume of about 40 ml to continuous process equipment capable of handling up to 200 Kg wet yeast or 20 Kg wet bacteria each hour.1. High pressure homogenisers are acceptably good for the disruption of unicellular organisms provided the enzymes needed are not heat labile. to release membrane-bound enzymes from bacteria.0 mm diameter) they are broken by the high liquid shear gradients and collision with the beads. being equipped with devices which retain the beads within the milling chamber. Smaller vessels may be cooled adequately through cooling jackets around the bead chamber but larger mills require cooling through the agitator shaft and impellers.2 -. Both can be operated continuously. Impeller speeds can be increased with advantage until bead breakage becomes significant but heat generation will also increase. impeller rotational speed and cell loading. Thus 1 mm diameter beads are satisfactory for the rapid release of periplasmic enzymes from yeast but 0. The rate and effectiveness of enzyme release can be modified by changing the rates of agitation and the size of the beads. increased bead loading increases the rate of protein release but also increases the production of heat and the power consumption. In addition to bead size. the efficiency of the equipment declines with throughput as the degree of backmixing increases. In general. i = 0 under ideal plug flow conditions and i = 1 for ideal complete backmixing). Unfortunately. in general.will be used at the highest pressures compatible with the reliability and safety of the equipment and the temperature stability of the enzyme(s) released.11) where i represents the degree of backmixing (i.g.e. for a longer period. There will be an optimum impeller tip speed at which the increases in disruption are balanced by increases in backmixing. filamentous or unicellular. The bead mills that have been studied in most detail are the Dyno-Mill and the NetschMolinex agitator. The shear forces produced are not capable of damaging enzymes free in solution. Centrifugation . as well as the dimensions of the equipment.25 mm diameter beads must be used. This is more noticeable at low flow rates (high average residence times) and when the proportion of protein released is high. 20 liters) scale. bead loading. e. when continuously operated there will be a significant amount of backmixing which reduces the efficiency of the protein released with respect to the average residence time (τ) . It may be counteracted by designing the bead mill to encourage plug flow characteristics. however well designed these mills are. At a constant impeller speed. the larger sized cells will be broken more readily than small bacteria. However.

After the centrifugation. and density of the particle. polysomes. They are related with the study of macromolecular structure rather than the collection of particle fractions. lipoprotein and viruses 2. A. Optical system records the change in refractive index. Most of the biological particles having same size are having narrower density range. Analytical centrifugation: it is devoted to study of pure or virtually pure or macromolecular or particles. shape. The Schleiren optical system plots the refractive index gradient against distance along the analytical cell. So . sealed and evacuated to minimized excess rise in temperature. This is type of preparative ultracentrifuge. and is equipped with the ultraviolet light absorption system. the chamber is surrounded by the refrigerated. Particle having similar density can be separated by the differential centrifugation or the rate zonal method. chromatin. its fabrication is of same type. In centrifugation the larger particle are sedimented first. Light of suitable wavelength is passed through moving analytical cell containing the solution under analysis e. ribosome. It can also measure the refractive index. Note that there must be equal loading of sample. and density. since particles of varying sizes and densities were distributed homogeneously at the start of centrifugation. Centrifugation is continued long enough to pellet all the largest class of particles. the denser particle is sedimented first and less dense will sediment later. thus increasing the rate at which the particle settles. It has speed 70.g. protein or nucleic acid and intensity of light is measured on the photographic plate. The particle having the same mass but different density. so pellet will not be homogeneous but will contain a mixture of all the sediment components.000 rev per min (500. The particle moves against respective sedimentation rates. For safety purpose ultracentrifuge are always enclosed in heavy armour plating. the basis of centrifugation separation techniques therefore is to exert the lager force than does the gravitational earth. the particle to be separated is divided centrifugally into a number of fractions by increasing the applied centrifugal field. pellet and supernatant are separated. which makes it useful in analysis in the locating in boundaries in sedimentation velocity measurements. better for the deproteinisation of physiological fluids for amino acid analysis. Another type of ultracentrifuge is analytical type. In differentiated centrifugation. plasma membranes. subcellular organelles. nucleic acids. shape. ULTRACENTRIFUGATION This technique is used to investigate the different parameter of the molecule such as molecular mass. However. the resulting supernatant then being centrifuged at higher speed to separate medium sized particle and so on.000 g). Therefore.B. The technique can be divided into two types: 1 preparative centrifugation: for separation of the whole cells. Differential centrifugation This method is based upon the difference in the sedimentation rate of particles of different size and density.000 g. Particle separation by the rate zonal technique is based on differences in the size. pellet is washed several time and again centrifugation is done. In this process gravity plays most important role. Principles: Rate depends on the centrifugal field G directed radially outward (angular velocity) G = ωr 2 ω =2π/60 revolution min-1 ultracentrifuge have maximum speed of 600. separation of similar particle by the rate zonal technique is based mainly upon differences in their sizes and .

11) where v is the rate of sedimentation. Fs depends on the volume fraction of the solids present. 921 cm s-2. Here the radius and effective liquid thickness are both small allowing a high angular velocity and hence high centrifugal force.2). sub cellular organelles.1 Kg of wet deposit whereas . lysosome. η is the kinematic viscosity. centrifuges are long and thin enabling rapid acceleration and deceleration. 12% and 20% solids volume fraction respectively. peroxisomes.5. Where discontinuous or semicontinuous removal of precipitate occurs. small models can be used at acceleration factors up to 50. Sedimentation of material in a centrifugal field may be described by (2. ribosomal subunits. is minimised. semicontinuous or continuous removal of solids. Centrifugation is the generally preferred method for the collection of enzyme-containing solids as it does not present a great hazard to most enzymes so long as foam production. For large-scale use. d is the particle diameter. This type of design cannot be scaled-up safely. There are two type of density gradient centrifugation. 0. --1. Fs is a correction factor for particle interaction during hindered settling and θ is a shape factor (=1 for spherical particles). Laboratory centrifuges using tubes in swing-out or angle head rotors have high angular velocity ω and radius of rotation (r) but small capacity (V) and substantial sedimentation distance (D). continuous centrifuges of various types are employed (Figure 2. The rate zonal technique and the isopycnic (equal density) Isopycnic centrifugation depends solely upon the buoyant density of the particles and not its shape or size and is independent of time the size of the particle affecting only the rate at which it reaches its isopycnic position in the gradient.12) The efficiency of the process is seen to depend on the solids volume fraction.05 for 1%. 1Svedberg = 10-13 second Centrifugation separates on the basis of the particle size and density difference between the liquid and solid phases. rs is the particle density. minimizing the down-time required for the removal of the sedimented solids. a rotor of radius 25 cm spinning at 1 rev s-1 has an acceleration factor of approximately 1 G).1 and 0. Low acceleration factors of about 1 500 g may be used for harvesting cells whereas much higher acceleration factors are needed to collect enzyme efficiently. the effective clarifying surface (V/D) and the acceleration factor (ω2r/g.can not be separated easily like mitochondria. rl is the solution density is the angular velocity in radians s1. r is the radius of rotation. The maximum throughput of a centrifuge for efficient use is given by (2.000 g. Only material which reaches a surface during the flow through continuous centrifuges will be removed from the centrifuge feedstock. This residence time will equal the volumetric throughput (Ф) divide by the volume of the centrifuge (V). The technique is useful in separating the particle of same size but differing in density. the continuous removal of supernatant and the discontinuous. the efficiency depending on the residence time within the centrifuge and the distance necessary for sedimentation (D). approximately equaling 1. with consequent enzymic inactivation. These allow the continuous addition of feedstock. where g is the gravitational constant. primarily because the mechanical stress on the centrifuge head increases with the square of the radius. The technique has been used for the separation of enzymes hormones and RNA and DNA hybrids. 3%. 0. The product of these factors (ω2rV/gD) is called the sigma factor Σ and is used to compare centrifuges and to assist scale-up. which must increase with increasing capacity. the precipitate is flushed out by automatic discharge systems which cause its dilution with water or medium and may be a problem if the precipitate is required for further treatment. accumulating 0.

Filtration of particles that are easily compressed leads to filter blockage and the failure of Equation 2. Problems associated with the build-up of the filter cake may also be avoided . contain multiple coned discs in a stack which are spun and on which the precipitate collects. They also may cause loss of enzyme activity from the solution due to physical hold-up in the filter cake. binds to unwanted negatively charged cell constituents. in rotary vacuum filters or centrifugal filters (Figure 2.000 g. Under these circumstances a filter aid. acts as a filter aid and may be incinerated to dispose of hazardous 2.3). It is often difficult for a process development manager to decide whether to attempt to recover enzyme trapped in this way. of course.000 g) so they are suitable only for the collection of comparatively large particles. k is a proportionality constant dependent on the size and nature of the particles. the viscosity of the liquid phase and the maximum allowable pressures.13) Where. A different design which is rather similar in principle is the solid bowl scroll centrifuge in which an Archimedes' screw collects the precipitate so that fluid and solids leave at opposite ends of the apparatus.Multichamber discstack centrifuges. Most flocculants have very definite optimum concentrations above which further addition may be counter-effective. Clearly from Equation 2. such as celite. The capacity and radius of such devices are large and the thickness of liquid is very small. the efficient precipitation of small particles of cell debris can be difficult. by using a centripetal pressurising pump within the centrifuge bowl which forces the sludge out through a valve. Filter aids are generally used only where the liquid phase is required as they cause substantial problems in the recovery of solids.3 to describe the system. however. is mixed with the feedstock to improve the mechanical stability of the filter cake. Flocculation is achieved by adding small amounts of high-molecular-weight charged materials which bridge oppositely-charged particles to produce a loose aggregate which may be readily removed by centrifugation or filtration. This material (Whatman CDR.7 Filtration of enzyme after separation Filtration separates simply on the basis of particle size. The angular velocity. sometimes near-impossible. continuously. The capacities of these centrifuges are only moderate. Coagulation is caused by the removal of electrostatic charges (e. Although many types of centrifuge are available.000 g. It is important to note that the choice of flocculants is determined by the pH and ionic strength of the solution and the nature of the particles. it is essential that the agents used must not inhibit the target enzymes. by pH change) and allowing particles to adhere to each other. is restricted giving a maximum acceleration factor of about 2.2. Its efficiency is limited by the shape and compressibility of the particles. For very small particles k depends on the fourth power of their diameter. due to the large effective surface area. The volumetric throughput of a filter is proportional to the pressure (P) and filter area (AF) and inversely proportional to the filter cake thickness (DF) and the dynamic viscosity (2. Flocculation and coagulation are cheap and effective aids to precipitating or otherwise harvesting whole cells. They may be operated either semi-continuously or. Large-scale simple filtration employs filter cloths and filter aids in a plate and frame press configuration. designed to accumulate up to 5 Kg of deposit. A comparatively recent introduction designed for the removal of cell debris is a moderately hydrophobic product in which cellulose is lightly derivatised with diethylaminoethyl functional groups. This can be done either by coagulating or flocculating particles.large models. cell debris or soluble proteins but. Some flocculants can be rapidly ruined by shear.g. originally designed (by Westfalia and Alpha-Laval) for cream separation. cell debris remover) is inexpensive (essential as it is not reusable). These can only be used at low acceleration (about 3. the efficiency of centrifugation can be improved if the particle diameter (d) is increased. are restricted to 16.

The suspension is sucked through a filter cloth on a rotating drum. less dense. denser. Separation may be achieved in a few minutes. Such centrifugal filters have a large radius and effective liquid depth. many two phase aqueous systems have been found. minimizing the harmful action of endogenous proteases. The systems have also been used successfully for . Aqueous three phase systems are also known.5% potassium phosphate buffer). 10% polyethylene glycol 4000/12. A great variety of separations have been achieved. This is avoided in cross-flow filtration where the flow sweeps the membrane surface clean. They provide the opportunity for the rapid separation of biological materials with little probability of denaturation. Bacillus diameter of about 2µ m). where dextran forms the more hydrophilic. Principles of (a) dead-end filtration and (b) cross-flow filtration. The interfacial tension between the phases is very low (i. This method dispenses with filter aids and uses special symmetric microporous membrane assemblies capable of retaining particles down to 0. or without. ________________________________________ Figure 2. much cheaper hydroxypropyl starch derivatives and salt-containing biphasic systems. However.4. Phases form when limiting concentrations of the polymers are exceeded. Some polymers form the upper hydrophobic phase in the presence of fairly concentrated solutions of phosphates or sulphate (e. The basic design of the rotary vacuum filter. The limiting concentrations depend on the type and molecular weight of the polymers and on the pH.g.by high tangential flow of the feedstock across the surface of the filter. ________________________________________ Figure 2. This has been alleviated by the use of crude unfractionated dextran preparations.g. ________________________________________ A simple and familiar filtration apparatus is the perforate bowl centrifuge or basket centrifuge. however.1 . There have been recent developments that improve their suitability. safety decrees that the angular velocity must be low and so only large particles (e. This produces a filter cake which is removed with a blade.4). efficient mixing under very gentle stirring and rapid partition. Both phases contain mainly water and are enriched in one of the polymers. In this case two phases were formed when agar was mixed with soluble starch or gelatin. A drawback to the useful dextran/polyethylene glycol system is the high cost of the purified Dextran used. by far the most important being the separation of enzymes from broken crude cell material. lower phase and polyethylene glycol the more hydrophobic.g. allowing small droplet size. plant material) can be removed satisfactorily. such as the Disposable Rotary Drum Filter. which may prevent efficient operation. Cell debris is collected on a cloth with. allowing high volumes. ionic strength and temperature of the solution. upper phase. 10% polyethylene glycol 4000/2% dextran T500). in effect a spin drier. These filters are generally rather messy and difficult to contain making them generally unsuitable for use in the production of toxic or recombinant DNA products. the most thoroughly investigated being the aqueous dextran-polyethylene glycol system (e. about 400-fold less than that between water and an immiscible organic solvent). Aqueous biphasic systems are of considerable value to biotechnology. filter aid and can be skimmed off when necessary using a suitable blade. ________________________________________ Chapter-10 Separation in Aqueous biphasic systems The 'incompatibility' of certain polymers in aqueous solution was first noted by Beijerinck in 1296. The filter cake may be rinsed during its rotation.e.3. large interfacial areas. The polymers have a stabilizing influence on most proteins. Since then.1 µm diameter (cf. In dead-end filtration the flow causes the build-up of the filter cake. a process known as crossflow microfiltration (Figure 2.

It is not generally very sensitive to temperature changes. The partition coefficient is exponentially related to the surface area (and hence molecular weight) and surface charge of the particles in addition to the difference in the electrical potential and hydrophobicity of the phases. Such systems are readily amenable to scale-up and may be employed in continuous enzyme extraction processes involving some recycling of the phases. A given protein distributes differently between the phases at different pH's and ionic strength but the presence of phosphate ions affect the partition coefficient in an anomalous fashion because these ions distribute themselves unequally resulting in electrostatic potential (and pH) differences. Such aqueous liquidliquid two-phase systems are finding increasing use in the extractive separation of labile biomolecules such as proteins. offering mild conditions due to the low interfacial tension between the phases (i. generating a new biphasic system. Some polymers form a two-phase system by themselves.the separation of different types of cell membranes and organelles. the purification of enzymes and for extractive bioconversions. --------------------(2. large interfacial areas. A partition coefficient of greater than 3 is required if usable yields are to be achieved by a single extraction process. Separation may be achieved in a few minutes. phosphates or sulfates or at higher temperatures (see below). The influence of pH and salts on protein partition is complex. This means that proteins and larger particles are normally partitioned into one phase whereas smaller molecules are distributed more evenly between phases. solvent with . the purification of enzymes. for example disc-stack centrifuges and counter-current separators. about 400-fold less than that between water and an immiscible organic solvent) allowing small droplet size. A great variety of separations have been achieved. The limiting concentrations depend on the type and molecular weight of the polymers and on the pH. ionic strength and temperature of the solution. The polymers also have a stabilizing influence on most proteins.e. Continuous liquid twophase separation is easier than continuous solid/liquid separation using equipment familiar from immiscible solvent systems.14) Where Ct and Cb represent the concentrations in the top and bottom phases respectively. PEG forming the upper morehydrophobic phase in the presence of fairly concentrated solutions of citrates. ideally the upper phase with cell debris and unwanted enzymes in the lower phase.g. triazine dyes) may be coupled to either polymer in an aqueous biphasic system and thus greatly increase the specificity of the extraction. Both phases contain mainly water (typically 70-90% w/w water) and are enriched in one of the polymers.01-100 whereas the partition coefficients for cells and cell debris are effectively zero. The yield and efficiency of the separation is determined by the relative amounts of material in the two phases and therefore depends on the volume ratio (Vt/Vb). The systems have also been used successfully for the separation of different types of cell membranes. Typical partition coefficients for proteins are 0. Phases form when limiting concentrations of the polymers are exceeded. Affinity ligand (e. cell debris proteins and other material distribute themselves between the two phases in a manner described by the partition coefficient (P) defined as. organelles and actinide ions. the properties of these biphasic systems can be mainly attributed to incompatibility between aqueous pools of low and higher density water. This stage may be used to further purify the enzyme. An enzyme may be extracted from the upper (polyethylene glycol) phase by the addition of salts or further polymer. although aqueous. minimizing the harmful action of endogenous proteases. Cells. This means that systems may be 'tuned' to enrich an enzyme in one phase. A powerful modification of this technique is to combine phase partitioning and affinity partitioning. Although sometimes perceived as due to polymer incompatibilities. by far the most important being the separation of enzymes from broken crude cell material. Each phase may be considered as a different. efficient mixing under very gentle stirring and rapid partition. extractive bioconversions. particularly when phosphate buffers are present.

properties determined by its structuring. PEG usually has a far higher concentration in the upper (low-density) phase of such solutions in spite of its inherent density being greater than water. together with the properties of this PEG phase encourage the belief that it creates a predominantly low-density water environment due to its partially hydrophobic character. This may be explained as the PEG creating a low-density water environment with decreased entropy. in line with the extent of water clustering. A oppositely-ordered compensating effect on the cloud point has been recognized due to binding of the anions to the polymer surface. The partitioning of proteins into the hydrophobic PEG phase shows great sensitivity to the protein's surface hydrophobicity (partition increasing with surface hydrophobicity) and also depends on the PEG size. solubility. Also. This entropy loss is required. Further proof of this may be seen by use of microwave dielectric measurements. gauche. however.22 Å) are close to the O•••O distances (2.24 Å) in water and the next ether (O-C-C-O-C-C-O) (5. trans links across the C-O-C-C. is expected to raise this cloudpoint. C-C-O-C bonds) is the same as the diameter of the spines of the ES water cluster (4. the ether (O-C-C-O) distances (2. At the cloud point. At higher temperatures still.9 Å) of the favored PEG helix (formed by trans. This reduces the entropic cost.< F. Increasing PEG size and concentration both increase the proteins' effective hydration as the PEG is excluded from the proteins' surface.< HPO42. SCN.having negligible effect.7 Å) formed by pentagonal boxes. increasing with PEG molecular mass . but is not great as the water is somewhat ordered already at lower temperatures. which show the water surrounding PEG to be ordered.< SO42. The strongly-held hydration. stronger hydration.g. At low temperatures a solution is formed due to the enthalpy of hydrogen bonding between the PEG and the water more than compensating for the entropy lost in forming the low-density water. whereas that surrounding more hydrophilic polymers is disordered [332].< Cl. at higher PEG size) partitioned proteins are excluded due to the reduced available water content.< CO32. An interesting and revealing phenomenon occurs in PEG solutions as the temperature is raised. and two phases develop.6 Å) distances are close to the next vertex distance on opposite sides of the pentagonal boxes (5. . The stronger hydrogen bonding in D2O. the solution at low temperatures separates into two phases (PEG-rich and PEG-poor) at higher temperature (separating at the cloud-point) and reverts to a single phase at even higher temperatures. These. The relative effect of the ions is the reverse of the Hofmeister series just given with weakly hydrated ions binding best.4 Å). as determined by viscosity. the dissolution of PEG is exothermic (and increasingly exothermic with PEG size). increases from two molecules of water per PEG monomer at very low polymerization (tetramer) to 5 molecules of water per PEG monomer for 45-mer . Li+) raising it. NH4+) tending to lower the cloud point but kosmotropes (e. in turn mainly determined by the methylene groups. showing that the extent of water clustering increases with PEG size.< PO43-). due to the hydrophobicity of the methylene groups.< Br. the water possesses excess energy and cannot be structured by the PEG.having the greatest effect and ionic kosmotropes below Cl. hence.< OH. the greater lowering of the cloud point is in line with greater surface charge density . in line with a shift in the ES CS equilibrium towards the more ordered ES structure. so allowing a solution to form once more. However.< I. Anions have a distinct effect on the cloud point in line with the Hofmeister Series (cloud point lowering: SCN. i. relative to H2O.g.g. the entropy cost is greater as the water is no longer naturally as structured. This tends to raise the cloudpoint at lower salt concentrations as the bound salt increases the polymer net charge and.e. It is interesting and perhaps not simply fortuitous that the diameter (4. greater tendency to avoid low-density water and the greater destruction of the natural structuring of the water. Cations have a lesser but opposite effect to anions with chaotropes (e. when the PEG phase becomes too ordered (e.a Model building shows that optimum hydrogen bonding would tend to distort this PEG helix. O-C-C-O.

preference for PEG-rich phase: I. as a consequence. Ultrafiltration differs from conventional filtration and microfiltration with respect to the size of particles being retained (< 50 nm diameter). particularly the anions (e.> Br. due presumably to their specific chelation to oxygen atoms in the PEG molecules. Ultrafiltration In many cases.> I-). The most popular method. and sold as a solution or as a dried preparation. by increasing the concentration of strongly hydrated (CS-forming) anions.Exceptionally. however. especially when extracellular enzymes are being prepared for sale.> Cl. Anions and cations distribute themselves differently between the phases depending on their affinity for low or higher density water but with the requirements that the phases be electrically neutral and iso-osmotic.000 and usable at pressure up to 2 MPa are available. durability and reliability of membranes .> Cl. The membrane rests on a rigid support at the base of a cylindrical vessel which is equipped with a magnetic stirrer to combat concentration polarization. It uses asymmetric microporous membranes with a relatively dense but thin skin. such as sulfate. The steady improvement in the performance.g. supported by a coarse strong substructure. The reason however is not so much that the O•••O distances (2. or dextran-PEG. There are number types of apparatus available. These are not as compact as capillary systems (area/volume about 25 m1) and are very expensive but are less liable to blockage by stray large particles in the feedstream.and trivalent cations such as Mg2+ and Zn2+ act counter to their normal Hofmeister behavior. For some enzymes rotary evaporation can be considered. Stirred cells represent the simplest configuration of ultrafiltration cell. This configuration of membranes can be scaled up with ease. Various large-scale units are available in which membranes are formed into wide diameter tubes (1 . preservative materials added.> SO42-.79 Å respectively) fit less well with the water cluster spacing but rather that the molecules form almost-linear extended (rather than helical) chains with a pronounced hydrophobic character that have strong intra-molecular attraction. Membranes are usually mounted into modules for convenient manipulation. so producing an interfacial potential difference.> F. negatively charged proteins are partitioned into the upper PEG phase.1 mm diameter and provides large membrane areas within a small unit volume (area/volume about 1000 m-1). Capillary membranes represent a relatively cheap and increasingly popular type of ultrafiltration system which uses micro-tubular membranes 0. Commercial models are available that give ultrafiltration rates of up to 600 L hr-1.9 Preparation of enzymes from clarified solution.2 . A similar Hofmeister Series effect is noticed intensifying the incompatibility between two polymers such as polyethylenimine-PEG. which may aid the partitioning of charged biomolecules. preference for PEG-poor phase: SO42. Preference for the PEG-rich or PEG-poor phase is related to the Hofmeister Series for the structuring ability of the salts. a Note that the polymers formed with either one (-O-C-O-C-O-) or three (-O-C-C-CO-C-C-C-O-) methylene groups between the oxygen atoms are both insoluble in water.2 cm diameter) and the tubes grouped into cartridges. containing pores. some di.> F. The concentration process chosen will be the cheapest which is compatible with the retention of enzyme activity. Membranes possessing molecular weight cut-offs from 1000 to 100. so allowing more sulfate or phosphate ions to partition into their preferred lower phase. Cheaper thin-channel systems are available (area/volume about 500 m-1) which use flat membrane sandwiches in filter press arrangements of various designs chosen to produce laminar flow across the membrane and minimise concentration polarization. enzyme being retained. It is not suitable for large scale use but is useful for preliminary studies and for the concentration of laboratory column eluates. Thus sulfate and phosphate ions prefer the bottom phase and.> Br. the clarified solution is simply concentrated. Cs+ > Na+ > Ba2+ > Ca2+.1. though. followed if necessary by spray drying. is ultrafiltration. whereby water and low molecular weight materials are removed by passage through a membrane under pressure.12 Å and 4. 2.

acids or alkalis. results in little loss of enzyme activity. Ultrafiltration. Increases in the ionic strength of the solution cause a . However. particularly at the laboratory scale. Salting in is the method in which ammonium sulphate is used for dissolving the protein. is one of the best known and used methods of purifying and concentrating enzymes. Problems with membrane blockage and fouling can usually be overcome by treatment of membranes with detergents. with care. some configurations of apparatus. There are method of salting in and salting out. encouraging wide use of the various ultrafiltration configurations. particularly in which solutions are recycled. can produce sufficient shear to damage some enzymes. done efficiently. proteases or. On increasing the ammonium sulphate concentration further proteins starts precipitating this phenomenon is called as salting out. Chapter-10 Concentration and purification of enzyme Concentration by precipitation Precipitation of enzymes is a useful method of concentration and is ideal as an initial step in their purification. The initial cost of membranes remains considerable but modern membranes are durable and cost-effective. All this process must be done in the ice cold solution so that no protein can denature. Salting out of proteins is done by use of ammonium sulphate. Note that protein dissolves least at its isoelectric point.has been a boon to enzyme technologists. It can be used on a large scale and is less affected by the presence of interfering materials than any of the chromatographic methods described later.

and the tendency of proteins to undergo rapid denaturation by these solvents if the temperature is allowed to raise much above 0°C. Ksalt depends on both the enzyme required and the salt used but is largely independent of other factors. otherwise. These act by reducing the dielectric of the medium and consequently reducing the solubility of proteins by favoring protein-protein rather than protein-solvent interactions.11) where S is the enzyme solubility. Organic solvents are not widely used on a large scale because of their cost. Sephadex G-25. These include. B. however. in particular ultrafiltration. cheapness. This may be done by dialysis. Kintercept is independent of the salt used but depends on the pH. temperature and pH are kept constant. enzyme and the other components in the solution. It also reduces the forces holding the salvations shell around the protein molecules. fractionation of protein mixtures by the stepwise increase in the ionic strength can be a very effective way of partly purifying enzymes. ethanol. Heat treatment . special flameproof laboratory areas are used and temperatures maintained below their flashpoints. they may complex undesirably with certain enzymes. Ammonium sulphate is convenient and effective because of its high solubility. polyethyleneimine. Kintercept is the intercept constant. A. the nucleic acids must be removed by precipitation or degraded by the addition of exogenous nucleases. Ksalt is the salting out constant and T is the ionic strength which is proportional to the concentration of a precipitating salt. This equation (2. particularly streptomycin sulphate. for instance. On safety grounds when organic solvents are used. Its large-scale use. the precipitating salt or solvent must be removed. Reproducible results can only be obtained provided the protein concentration. Otherwise. usually positivelycharged materials which form complexes with the negatively-charged phosphate residues of the nucleic acids. such as in the preparation of restriction endonucleases. Some organisms contain sufficient nuclease activity to eliminate this problem but. streptomycin sulphate and protamine sulphate. however.reduction in the repulsive effect of like charges between identical molecules of a protein. Some enzymes do not survive ammonium sulphate precipitation. their flammability. Ammonium sulphate precipitation can be effective in removing nucleic acids but will remove some protein at the same time. temperature. hydrophobic proteins precipitating at lower salt concentrations than hydrophilic proteins. treatment with bovine pancreatic nucleases is probably the most cost-effective method of nucleic acid removal. propan-2-ol and acetone. The practice of using ammonium sulphate precipitation is more straightforward than the theory. All of these are expensive and possibly toxic. and it may release gaseous ammonia. The concentration of the salt needed to precipitate an enzyme will vary with the concentration of the enzyme. Various more specific precipitants have been used. Nucleic acid removal Intracellular enzyme preparations contain nucleic acids which can give rise to increased viscosity interfering with enzyme purification procedures. in order of roughly decreasing effectiveness.11) may also be used to give the minimum salt concentration necessary before enzyme will start to precipitate. where possible contamination of the enzyme product must be avoided. the concentration change necessary to precipitate the enzyme varying according to the magnitude of the salting out constant. Ultrafiltration or by using a desalting column of. lack of toxicity to most enzymes and its stabilizing effect on some enzymes (see Table 2.4). Also. However. particularly at alkaline pH. When these forces are sufficiently reduced. The solubility of an enzyme can be described by the equation (2. it forms dense solutions presenting problems to the collection of the precipitate by centrifugation. Except when enzymes are presented for sale as ammonium sulphate precipitates. the protein will precipitate. the cationic detergent cetyltrimethyl ammonium bromide. They may be necessary. Other salts may be substituted but the more favored alternative is to use organic solvents such as methanol. is limited as it is corrosive except with stainless steel.

Where the enzyme required is relatively heat-stable this allows its easy and rapid purification in terms of enzymic activity. Kd describes that how a substance distributes itself between different type of immiscible phases. affinity and gel exclusion properties of the enzyme. preferred at an earlier stage in the purification where the scale of operation is somewhat greater. therefore. Such equipment must. and hence leaky. restriction endonucleases and therapeutic enzymes). charge versus pH) for all proteins present. Gel exclusion chromatography (also sometimes called 'gel filtration' or just 'gel chromatography' although it does not separate by a filtering mechanism. The method was developed in 1906 by the Russian botanist Mikhail Tswett.e. It is generally left until last as an important final purification step and also as a method of changing the solution buffer before concentration.6µm diameter) which allows their use in very efficient separation processes (high performance liquid chromatography. Ionexchange and affinity chromatographic methods can both rapidly handle large quantities of crude enzyme but ion-exchange materials are generally cheaper and. but at exponentially increasing cost with decreasing bead size. As the sample is applied across the range of pH.g. They are used only for the small-scale production of expensive enzymes. The main problem that has had to be overcome is that of ensuring the matrix has sufficiently large surface area available to molecules as large as proteins (i.e. of course. 1.In many cases. which both depend on the pH. Relatively high pressures are needed to operate such columns necessitating specialized equipment and considerable additional expense.g. Different enzymes have differing susceptibility to heat denaturation and precipitation. larger molecules passing more rapidly through the matrix than smaller molecules) is relatively slow and has the least capacity and resolution. this method produces titration curves (i. they are macroporous) whilst remaining rigid and incompressible under rapid elution conditions. where a high degree of purity is required (e. For enzyme purification there are three principal types of chromatography utilizing the ion-exchange. A large effort has been applied to the development of chromatographic matrices suitable for the separation of proteins. A relatively quick analytical method for obtaining such data utilises a two-dimensional electrophoresis whereby electrophoresis occurs in one direction and a range of pH is produced in the other. 4 . collects fractions and regenerates the column. This method has been particularly successfully applied to the production of glucose isomerase. No interfering activity remains after this treatment and the heat-treated. a rational purification scheme can be devised.g. movement in the electric field determined by the size and sign of a protein's charge. Column manufacturers now supply equipment for monitoring and controlling chromatography systems so that it is possible to have automated apparatus which loads the sample. cells may be immobilised and used directly. where a short incubation at a relatively high temperature is used (e. Although the standard bead diameters of most of these matrices are non-uniform and fairly large (50 – 150 µm). Where sufficient information has been gathered regarding the size and variation of charge with pH of the required enzyme and its major contaminants. many are now supplied as uniform-sized small beads (e. unwanted enzyme activities may be removed by heat treatment. . For such enzymes heattreatment is always considered as an option at an early stage in their purification. Enzyme purification by Chromatography For identification and separation of biochemical compound chromatography is one of the most effective techniques. matrices must generally be hydrophilic and inert. Enzyme preparations that have been clarified and concentrated are now in a suitable state for further purification by chromatography. finishing and sale. In addition. 60 . HPLC). Chromatography involves the principle of partition or distribution coefficient ( Kd ). have fail-safe devices to protect both column and product.25°C for 10 min). usually in that order.

In addition.and anion exchange matrices usually contain the cationic tertiary and quaternary ammonium groups. and the conditions needed to elute proteins are generally severe and may be denaturing. Consequently.g. proteins may be eluted from them under mild conditions. Proteins become bound by exchange with the associated counter-ions. Unadsorbed material may be removed in a variety of manners. Ion-exchange cellulose can be used in both batch and column processes but on a large scale they are used mainly batch wise. but opposite. Adsorption to the exchangers is usually rapid (e. Basket centrifuges are a particularly convenient means of hastening the removal of the initial supernatant and the elution of the adsorbed material. Note that In solutions of pH below their isoelectric point amino acid will be positively charged and bind to cation exchangers whereas in solutions of pH above their isoelectric point amino acid will be negatively charged and can bind to anion exchangers.and -COO. Ion-exchange cellulose and large pore gels are much more generally suitable for enzyme purification and. indeed. Stirring is essential but care must be taken not to generate fine particles (fines). The practical level of substitution of cellulose is limited as derivatisation above one mole per kilogram may lead to dissolution of the cellulose. Therefore pH chosen must be sufficient to maintain a high. Ion-exchange polystyrene resins are eminently suitable for large-scale chromatographic use but have low capacities for proteins due to their small pore size. 2 Affinity chromatography This is a term which is based on specific interaction between the enzyme and the . Ion-exchange materials are generally water insoluble polymers containing cationic or anionic groups. Careful preparation before use and regeneration after use is essential for their effective use. whilst ion-exchange cellulose are widely used for column chromatography on the laboratory scale. This is because the increased speed of large-scale batch wise processing and the avoidance of the deep-bed filtering characteristics of columns outweigh any advantage due to the increase in resolution on columns. anionic or cationic. cellulose derivatives and large-pore gels are available for chromatographic use. A variety of charged groups. dependent upon the pH and their structure and isoelectric point enzyme can be separated. their compressibility causes difficulty when attempts are made to use large scale columns.a. Cation exchange matrices have anionic functional groups such as -SO3-. Ion-exchange chromatography Enzymes possess a net charge in solution. charge on both protein and ion-exchanger and the ionic strength must be sufficient to maintain the solubility of the protein without the salt being able to successfully compete with the protein for ion-exchange sites. in a column and elute using a suitable gradient. Batch wise operations involve stirring the pretreated and equilibrated ionexchanger with the enzyme solution in a suitable cooled vessel. A very wide range of ion-exchange resins. Binding is often strong. Some of the problems with derivatised cellulose may be overcome using more recently introduced materials. The binding is predominantly reversible and its strength is determined by the pH and ionic strength of the solution and the structures of the enzyme and ion-exchanger. Nevertheless such resins are a potential means of concentrating or purifying enzymes. may be introduced. due to the resin hydrophobicity. This is usually done using stepwise increases in ionic strength and/or changes in pH but it is possible to place the exchangers. they do not change volume with pH and ionic strength which allows them to be regenerated without removal from the chromatographic column. less than 30 minutes) but some proteins can take far longer to adsorb completely. Normally the pH is kept constant and enzymes are eluted by increasing the solution ionic strength. many were designed for that task. with general formulae -NHR2+ and -NR3+. More detail description is given in chapter 9. -OPO3. However. Derivatives of cross-linked agarose (Sepharose CL6B) and of the synthetic polymer Trisacryl have high capacities (up to 150 mg protein ml-1) yet are not significantly compressible. plus adsorbed material.

This method has been used successfully but has now been superceded by the employment of a series of water soluble dye as ligand. This dyeaffinity chromatography was allegedly discovered by accident. usually by trial and error. An alternative. This material (Whatman HB1) is designed to interact with proteins by hydrogen bonding. and experience in handling these materials is gained. to calibrate gel exclusion columns. as the ligand. the immobilised ligand is a substrate or competitive inhibitor of the enzyme. certain enzymes being found to bind to the blue-dyed dextran used. as a molecular weight standard. Elution is achieved by changing the pH or ionic strength or by modifying the dielectric constant of the eluent using. though. equally specific approach is to use an antibody (they are specific in binding because they are raised against specific antigen inside an animal) to the enzyme as the ligand. have been found to have surprising degrees of specificity for a wide range of enzymes. enzyme manufacturers will make increased use of these very powerful techniques.immobilised ligand. Particles should retain good flow and porosity properties after attachment of the ligand and should not be capable of the non-specific adsorption of proteins. Proteins are eluted by diluting the ammonium sulphate. > 2M) solution of ammonium sulphate. they often do not perform as well as might be expected due to non-specific binding effects.3 Hydrophobic interaction chromatography (HIC) or affinity elution HIC is found to be very useful when it was noted that certain proteins were unexpectedly retained on affinity columns containing hydrophobic spacer arms. suitable for many enzymes. affinity chromatography achieves a higher purification factor (with a median value in reported purifications of about ten fold) than ion-exchange chromatography (with a median performance of about three fold). This pH gradient decides the final movement of the enzyme in the gel. in spite of it generally being used at a later stage in the purification when there is less purification possible. is to use analogues of coenzymes. Such specific matrices. are very expensive and cannot be generally employed on a large scale. Isoelectric focusing When any enzyme mixture is placed in the gel having solution of different pH then after passing the current a potential difference is established which helps in development of a pH gradient. CHOICE OF MATRIX Careful choice of matrices for affinity chromatography is necessary. In general. ethanediol. because enzyme will stop its movement only after a situation where pH of enzyme will be equal to the pI of the buffer. In its most specific form. Hydrophobic interactions are strong at high solution ionic strength so samples need not be desalted before application to the adsorbent. This introduces more water which competes with protein for the hydrogen bonding sites. More detail account is given in chapter 9. Doubtless as the relative costs of materials are lowered. (See figure below. A recent introduction is cellulose derivatised to introduce even more hydroxyl groups. The selectivity of both of these methods is similar to that of fractional precipitation using ammonium sulphate but their resolution may be somewhat improved by their use in chromatographic columns rather than batch wise. Agarose beads fulfill these criteria and are readily available as ligand supports . Affinity chromatography is not used extensively in the large-scale manufacture of enzymes. A less specific approach. These are much cheaper and. for instance. primarily because of cost. such as NAD+. Samples are applied to the matrix in a concentrated (over 50% saturated. . Electrophoresis This method was developed by Arne Tiselius in 1937 and is based on principle that any charged molecule will move towards the opposite pole. enzyme A 1 forming separate bands in compare to the Enzyme A2). . More detail description is given in chapter 9. Hydrophobic adsorbents now available include octyl or phenyl groups. Additionally. Ideally it should be possible to purify an enzyme from a complex mixture in a single step and. purification factors of up to several thousand-fold has been achieved. indeed.

may not) result in stabilization. polyvinyl alcohol. citrate3increased stabilization ________________________________________ Low molecular weight polyols (e. 2. I-. increased chaotropic effect Cations Al3+. Neutral salts compete with proteins for water and bind to charged groups or dipoles. K+. some are much more effective at their destabilization by binding to them and disrupting the localized structure of water (the chaotropic effect. This derivatisation is sufficient to disguise the proteinaceous nature of the protease and prevent autolysis. Na+. resulting in more rigid structures. Table 2. Ca2+. SO42-. proteins are stabilized by increasing their concentration and the ionic strength of their environment. more commonly. Some hydrophilic polymers (e.1).4. Not all salts are equally effective in stabilizing hydrophobic interactions. sorbitol and mannitol) are also useful for stabilizing enzymes. This may result in the interactions between an enzyme's hydrophobic areas being strengthened causing the enzyme molecules to compress and making them more resistant to thermal unfolding reactions. the controlled use of covalent modification. has given hope that site-specific mutagenesis may lead to enzymes . From this it can be seen why ammonium sulphate and potassium hydrogen phosphate are a powerful enzyme stabilizers whereas sodium thiosulphate and calcium chloride destabilize enzymes. Many specific chemical modifications of amino acid side chains are possible which may (or. Mg2+. This observation. so preventing unfolding.2. aggregation and changes in the covalent structure. Br-. Important lessons about the molecular basis of thermostability have been learned by comparison of enzymes from mesophilic and thermophilic organisms.g. by repressing microbial growth.g. At high concentrations (e. osmotic effect. A frequently found difference is the increase in the proportion of arginine residues at the expense of lysine and histidine residues. In addition ions can offer some protection against oxidation to groups such as thiols by salting-out the dissolved oxygen from solution. salt discourages microbial growth due to its. HPO42-. due to the reduction in the water activity. 20% NaCl).4). and 3. for example Ca2+ stabilises αamylases and Co2+ stabilises glucose isomerases. (CH3)4N+ Anions SCN-. glycerol. ClO4-. Li+. Enzyme immobilization. Consequently. NH4+.11 Maintaining Enzyme Activity The key to maintaining enzyme activity is maintenance of conformation. Many enzymes are specifically stabilized by low concentrations of cations which may or may not form part of the active site. This may be possibly explained by noting that arginine is bidentate and has a higher pKa than lysine or histidine (see Table 2. Cl-. polyvinylpyrrolidone and hydroxypropylcelluloses) stabilise enzymes by a process of compartmentalisation whereby the enzyme-enzyme and enzyme-water interactions are somewhat replaced by less potentially denaturing enzyme-polymer interactions. Glycerol may be used to protect enzymes against denaturation due to ice-crystal formation at sub-zero temperatures. A useful example of this is the derivatisation of lysine side chains in proteases with N-carboxyamino acid anhydrides.g. Three approaches are possible: 1. and by the formation of protective shells which prevent unfolding processes. it forms stronger salt links with bidentate aspartate and glutamate side chains. among others. use of additives. Effect of ions on enzyme stabilization. In general. Table 2. They may also act by stabilizing the hydrophobic effect within the enzymes. These form polyaminoacylated enzymes with various degrees of substitution and length of amide-linked side chains.

The main advantage of adsorption chromatography is in separating isomers. Mass spectrometry. Presence of amide or peptide bond can be calculated by UV-IR spectroscopy. enzymes are further purified by two main common techniques 1. Vmax) than the 'natural' enzyme. Gel filtration. In this chapter we will focus mainly on choice of these techniques and their broad details of the application and principles. SDS –page. Additives of these types must. of course. 2. Amino acid can be determined by Ninhydrin reaction and taking OD at 570 nm. The method was developed in 1906 by the Russian botanist Mikhail Tswett. Enzymes are more stable in the dry state than in solution. Kd describes that how a substance distributes itself between different type of immiscible phases. Calculate the molecular weight by following method Ultracentrifugation. Solid enzyme preparations sometimes consist of freeze-dried protein.Chromatography. be compatible with the final use of the enzyme's product. 3-Molecular-sieve Adsorption Chromatography The stationary phase in adsorption chromatography is silica or alumina particles. benzoic acid esters as preservatives for liquid enzyme preparations.12 HOW TO KNOW THE PROPERTY OF ENZYME 1. 2-Ion-exchange. Direct structure can be determined by the X-ray crystallography. Many specialized technique that use them are: --Basically. 2. Other materials which are added to enzymes before sale may consist of substrates. antibiotics. Chromatography involves the principle of partition or distribution coefficient (Kd). there is several type of chromatographic technique based on following property Adsorption. In the meantime it remains possible to convert lysine residues to arginine-like groups by reaction with activated urea.with significantly improved stability . Example Thin layer chromatography. Chromatography Principles of chromatography For identification and separation of biochemical compound chromatography is one of the most effective techniques. Amino acid sequencing can be done to know the order of amino acid.Gel electrophoresis. Analyte are separated due to their varying degree of adsorption onto the solid surfaces. lactose. Chapter-12 Techniques used in Enzyme characterization 2. More usually they are bulked out with inert materials such as starch. It should be noted that enzymes stabilised by making them more rigid usually show lower activity (i. A. inhibitors of contaminating enzyme activities and chelating agents. which can have very different physisorption characteristics due to steric effects in the molecules. Introduction After salting in and salting out procedure. all type of chromatography consists of two type of phase . carboxymethylcellulose and other poly-electrolytes which protect the enzyme during a cheaper spray-drying stage. thiols to create a reducing environment. Basically.e.

which has led to the development of other divinylbenzene polymers for size exclusion chromatography. Mobile phase: This is chosen according to the nature of the biomolecules. although the surface is often modified by adding organic compounds to resist adsorption. Paper chromatography. Principle Size Exclusion Chromatography differs from conventional chromatography in the behavior of the stationary phase. while in ion-exchange chromatography is the ion-exchanger like Dowex -50 . Size exclusion chromatography was discovered using starch as a packing material. developed for zone electrophoresis. the stationary phase chemically interacts through adsorption. b. there have been many discoveries that have made size exclusion chromatography practical. Mobile phase is poured in the gel and get separated due to attachment with the stationary phase. it led to further interest. Thin layer chromatography. The process developed using Sephadex was used extensively in characterizing polymers and polymer molecular weight distributions. Silica has also been used. Dowex-1 both are anion exchanger. gel. 1. Stationary phase in paper chromatography is the paper. Today. allowing packing materials to be easily customized for different applications.Stationary phase: the phase that can not move and is prepared by dissolving some solid . was applied to chromatography. When using nonpolar organic solvents. Soon. In most forms of chromatography. both gel filtration and gel permeation chromatography are generally referred to as size exclusion chromatography. adsorption chromatography are of following type a. d) High performance liquid chromatography (HPLC) Based on adsorption of solute in the matrix. researchers prefer modified divinylbenzene and polyacrylamide as packing materials. Sephadex is a cross-linked styrene divinylbenzene copolymer. Mobile phase is used to isolate the biomolecules because most of the biomolecules are present in the solution. Two immiscible phases are formed after the distribution of compound in the matrix and is denoted by Kd= concentration of compound in phase A / concentration of compound in phase B. Size exclusion chromatography/ gel filtration (permeation) chromatography History Since the discovery in the 1940s that certain porous material can retain molecules of certain sizes and let others elute. . The amount of cross-linking (controlled by the availability of divinylbenzene) determines pore size. This may be solid. the processes are still used extensively in polymer chemistry but have gained new uses in biochemistry. This initial use of size exclusion was limited to aqueous solvents and called gel filtration chromatography (at Tiselius’s suggestion). liquid. liquid or other material that can be packed inside the column and may provide the support for movement the mobile phase. Although starch was not a very durable material. or solid liquid mixture. this innovation made size exclusion chromatography realistic. a material called Sephadex. c) Affinity chromatography. or various other actions. Column chromatography is of following type a) GPC or gel permeation chromatography b) Ion –Exchange chromatography. The basis of all type of chromatography is the partition or distribution coefficient (Kd). DEAE and sephadex. and the cation exchanger is the CMC carboxymethyl cellulose. COLUMN CHROMATOGRAPHY In the column chromatography column is filled with the stationary phase attached to suitable matrix and mobile phase passed through the column either with the gravity or by applied pressure. charge.

agarose. For maximum resolution.Agarose is a linear polymer of Dgalactose. so poorer resolution results. Since gel have pores after polymerization and it can be used for the separation of the molecule of fixed size. both inside and outside the gel particle. They made strong beads. Here separation of molecule is done on the basis of their molecular size and shape with the help of gel that have pores formed during the gel formation. and polyacrylamide. whereas if the analyte is sufficiently small to gain complete access to the inner mobile phase. the General principle is simple. The space within the gel is filled with liquid occupies most of the gel volume. The most commonly used material is organic gel and they form different type of pores on polymerizations. Vs. For given type of gel Kd depends on the molecular size of the analyte. N’methylene-bis acrylamide. So Kd =1. It forms a gel held together with crosslink of H-bonds. Term gel filtration or exclusion or permeation chromatography is used to describe the separation of molecule of different molecular size utilizing gel material. The pore size is usually much larger than sephadex. DNA. The gel is a three dimensional network whose structure is usually random. using molecular sieve. The gels currently in use are of three types: ---dextran. Therefore.In size exclusion chromatography. According to availability of the mobile phase kd varies between 0 to 1. The product is called as Sephacryl S-300. N’. They are used in the aqueous solution. They will pass through the column at slower rate. and are uncharged. For two substances of different relative molecular mass and kd value for example Kd’ and kd’’ is the difference in their elution volumes. Smaller molecules will be distributed between the mobile phases inside and outside the molecular sieve. do not bind or react with the material that is being analyzed. the stationary phase simply acts as a sieve to filter molecules based on size.Dextran is a polysaccharide composed of glucose residues. It is commercially available in the trade name “Sephadex”. but still they can pass though the spaces around the pore called as void space and appear in the effluent first. It is marketed in the name of Bio-Gel –P. the alkyl dextran is called as N. They can not be used for molecules that have size more than 600. superfine beads are used. The larger size molecule are separated out or excluded from the pores (see figure). Mixed gel of . This is the reason why they can be used for the separation of the large macromolecular substance like protein. denotes the distribution of an analyte (that we have to separate) in a column of a gel is determined solely by the total mobile phase.Kd’’) V1------------------------------------1 The resolution depends on the beads. The coarser beads are unable to hold the fluid. The gels acts as molecular sieves consist of cross linked polymers that are generally inert.methylene bis acrylamide. If the analyte is large then kd =0. There are various materials that are porous in nature. Note that in the column there is always an inert substance. The gel is filled in the column that is equilibrium with the mobile phase. can be derived from the equation and shown to be: VS = (K’d. Kd. Hence they appear last in the effluent. 000. This is the reason why molecules are eluted from the column in order of decreasing size or if the shape is relatively constant.Polyacrylamide gels are produced by cross-linking acrylamide and N.

and mean radii. precipitation. Common bulk purification methods are centrifugation. For chromatography beads of a given porosity. Gel permeation chromatography is normally considered to be a medium. molecules below a certain size for beads of a given porosity will completely penetrate the pore structure and will elute with the "included volume" (Vi). Gel permeation chromatography is a common and often excellent polishing step during any protein purification. gel permeation chromatography provides an estimate of mass for the oligomeric holoprotein. filtration.the fractionation range. Thus. By comparing the elution volumes of protein standards of known molecular mass. Use of gel permeation chromatography as part of a purification scheme Most protein purification schemes involve several steps.g. The radii of the beads are more important for determining the capacity of the column to separate molecules of similar size. it would not be used as a first step in purification except in unusual circumstances (e. Note. gel permeation chromatography is found to separate globular proteins according to mass. etc. the size of this sphere is directly related to the mass of the globular protein. Vi. which gives subunit molecular masses for an oligomeric protein. The pore size of the bead will determine whether a protein of s Moreover. as opposed to fiber proteins). and the elution volumes (Ve) of the proteins of interest. high abundance of the target protein in the starting material). Bulk purification steps are generally useful for samples of low purity and large volume. In general. both of which are typical of the starting material to be used in a protein purification. acid. Polishing purification steps are generally useful for samples of higher quality and smaller volume. average radii. and ion-exchange chromatography. Void volume. Only molecules with masses (hydrodynamic radii) that allow them to penetrate the pore structure of the beads only partially fall within the fractionation range and are separated according to their molecular size (approximately. or polyethylene glycol. solvent. Access to included volume (volume that hydrates the inside of the bed) depends on the size of the sphere (the hydrodynamic radius) that each protein occupies.polyacrylamide and agarose is known as Ultra-Gel.to high resolution chromatographic (polishing) technique. In contrast to denaturing gel electrophoresis. such as with salt. such as a cellular extract. however. these molecules are not fractionated either.The use of a gel permeation column as an analytical tool requires the determination of Vo. These excluded molecules elute in the "void volume" (Vo) of the column and are not fractionated. Common polishing purification methods are ion exchange. Mechanism of separation by size in gel permeation chromatography For our discussion of the theory of gel permeation chromatography. that large deviations from spherical shape can cause a protein to migrate anomalously during gel permeation chromatography that is with regard to migration versus mass. as described above). typical of partially purified fractions obtained from bulk purification steps. In contrast. we will make the assumption that many proteins have a roughly spherical shape (often called globular proteins. affinity. These can be characterized as either bulk or polishing steps. Table:1 MATERIAL: POLYMER TRADE NAME FRACTIONATION RANGE . the molecular mass of an unknown protein can be estimated. The combination of denaturing gel electrophoresis and gel permeation chromatography is thus exceedingly powerful for elucidating basic features of protein structures. and gel permeation chromatography. however. molecules above a certain size will be completely excluded from the pore structure of the beads.. since proteins have roughly constant density (mass per volume). the porosity of the beads determines the size range of molecules that can be effectively separated. Thus. Beads can be prepared with defined degrees of porosity.

0-5 1.0--100.0 4--150 5---600 5---250 10---1500 20—8000 2. it is collected in fractions until all the components of the original sample have passed through the column.1---1. . Due to the differential partitioning of molecules into the pore structure of the beads (as described above). As additional buffer is applied to the top of the column and allowed to flow through the column. the protein sample migrates through the column. a protein sample (often a partially purified fraction from a previous purification step.1. As buffer is eluted from the column.0 Overview of a typical gel permeation chromatography experiment In a typical gel permeation experiment.DEXTRAN < 0. AGAROSE 10-4000 60---20.0—6 BIO-GEL A 15m 40--- P2 P6 P100 5. see below) is applied onto the top of the column packing (beads) and allowed to flow into them.000 3.000 100---50.000 70—40. POLYACRYLAMIDE 0.7 1.5—3.000 A 5m BIO-GEL A 50 SEPHAROSE 4B SEPHYCRYL S G SEPHADEX G 10 G 25 G 50 G 100 200 200 S 300 S 400 2 B 6B 10---5000 15.8 1. the protein molecules are separated.

One can obtain average molecular weights without it. Vo. Repeat for as many points as you proportional to the concentration of polymer: The constant of proportionality is dn/dc. You cannot measure concentrations in an isorefractive solvent (i. the right ordinate.Note that all the large M’s come out at nearly the same volume. ionic strength. not M. So they all elute together at the void volume. Ve trend Obtain DRIA similarly from wish! The DRI response is DRI c ( in g/mL) to select a representative sampling of points.. you are example. One can also obtain the number average molecular weight: Mn = = = = ADVANTAGE OF GEL –CHROMATOGRAPHY In this chromatography the material used is inert one so there is no effect of temp. For example: Mw = = Since. Since there is no adsorption by the inert material so labile material are not affected. size exclusion chromatography . the same specific refractive index increment needed in light scattering. With such a curve. consider point A. Note that the independent variable is plotted on the y axis by convention. the constant of proportionality factors out of the numerator and denominator identically. For indicated by the cross. The elution volume is related in a simple manner to molecular weight. Vo. This is because none of the very large polymers ever enter a pore. one in which dn/dc = 0). pH.e. Application of GPC or gel permeation chromatography Size exclusion chromatography is used primarily in two areas of chemistry: polymer chemistry and biochemistry. However. it is not necessary to actually know dn/dc in simple GPC. They can be used for very labile material like enzyme. In polymer chemistry. Starting at VeA read up until and then read left to get MA from the left y-axis. you hit the M vs. It is customary to plot log(M).

but in both polymer chemistry and biochemistry. the mobile phase--determined by their partition coefficient--measured as an Rf . aldehyde etc. because size exclusion chromatography does not destroy or alter the sample.3. For example. giving the polymer’s producer information about the length of the chains produced. it is usually a mixture of Solutes partition between H2O in the stationary phase and the solvent of the mobile phase. In purification of Macromolecule: GPC can be used to separate the viruses. particularly polyesters and polyamides (1999). The parameter K = Ve---Vo / Vs. Mobile Phase is mixture of organic solvents (alcohols. antibodies. it can be compared with the molecules that it eluded closest to determine an approximate molecular weight. adjust the composition). Antonio Moroni and Trevor Havard discuss the use of solvents in the analysis of engineering thermoplastics. ketone. Anke van Rijk and colleagues used size exclusion chromatography to isolate a small protein called alpha-A-crystalline to study the insertion of hamster alpha-Acrystalline into mice (1999). H2O run across the cellulose paper due to capillary action. proteins. such as o-chloronaphthalene. For example. O’Donohue and E.3. size exclusion chromatography allows the researchers to isolate the biomacromolecule based on its relative size without destroying the sample.1. or a protein of unknown size. In addition. This extension or enlargement causes them to be eluded at later times than expected.1. In biochemistry.can separate polymers with different numbers of monomer units. making these molecules seem larger than they actually are. Biochemical separations may seem to make this more of a separation technique.3-hexafluoro-2-propanol may not be a good solvent for size exclusion chromatography as polar portions of macromolecules (such as polyesters and polyamides) may be solvated and extended. Their experimentation leads to the suggestion that 1. The mobile phase solvent will be less polar than H2O. hormones. One can change the characteristics of separation by changing the polarity of the mobile phase (i. is analyzed along with molecules of known size or molecular weight. Meehan discuss the use of different solvents in size exclusion chromatography and molecular weight detectors (light scattering and viscometry) at high temperatures to characterize polymers. size and molecular weight estimations can be made. drawn by capillary action Solutes move as spots with a rate depending upon how much time they spend in the stationary phase vs. the fractions of polymer can be further tested. in a genetics study.e. where Ve= elution volume. The bound H2O is the Stationary Phase. that can be processed only at temperatures above 135 °C (1999). enzyme. There are two modes of chromatography1: Ascending and 2-Descending. Vt is the total volume of the column PAPER CHROMATOGRAPHY Cellulose has many hydroxyl groups which are polar and bind H2O. It yields a straight line except for very small and very large molecule. If a polymer chain of unknown size. Vs= volume of stationary phase or = Vt---Vo. Vo= void volume. size exclusion chromatography is useful in separating highmolecular-weight molecules from other molecules.The solvent moves through the paper. In study of protein –binding study Estimation of the molecular weight: Gel 1 gel 2 Log M THE plot is between K verses log M (molecular weight). Paper Chromatography is the most common form of cellulose chromatography in which solute is "spotted" on "dry" paper (still contains H2O) encircle by the pencil at a line mark and chromatograph is "developed by dipping one end in the mobile phase. J. then when the compound in question eludes. S.) and possibly water. nucleic acid and polysaccharide.

Other weakly acidic cation exchangers are phenolic hydroxyl group and carboxylic group. The ratio of two gives Rf value. strong cation exchangers: Sulfonic Acid (or derivatives) RSO3. weak anion exchangers: Diethyl-amino-ethyl (DEAE). CMCellulose.H+(s) + Mx+(aq) -SO3. DEAE-Sephadex. CM-Sepharose Choosing an Ion Exchanger Charge -. These may be bonded to a variety of supports: e. Cationic exchanger posses negative charge. For cation-exchange with a sulfonic acid group the reaction is: -SO3.depends upon the charge of the molecules to be separated. DEAE-Sepharose.cation or anion exchanger -. phosphoryl. while anionic exchanger posses positive charge. DEAE-cellulose. the exchanger is said to be in the acid form.value. First –the substances to be separated are bound to the exchanger.Mx+]s [H+]aq Keq = -----------------------------------------[-SO3.H+]s [Mx+]aq Different cations have different values of Keq and are therefore retained on the column for different lengths of time. and a programmable pump that can change the pH of the mobile phase during the separation. The name ion exchange is given because of exchanging ions for ion in aqueous solution. The principle of ion exchange chromatography is that charged molecule adsorb to ion exchangers reversibly so that molecule can be bound or eluted by changing the ionic environment. The negative charge ion exchangers bind to positive charge molecule. Ion –exchange chromatography This type of chromatography is used for many biological materials like amino cid and proteins. Weak Exchanger is useful for labile . The column packing for ion chromatography consist of ion-exchange resins bonded to inert polymeric particles (typically 10 µm diameter). Separation by ion exchange is usually involving two step. This Rf value is fixed and is generally less than one.weak cation exchangers: carboxy methyl (CM).e. The time at which a given cation elutes from the column can be controlled by adjusting the pH ([H+]aq).Mx+(s) + H+(aq) where Mx+ is a cation of charge x. i. If an H+ is bound to group.b. There are two type of ion exchanger 1) cationic exchanger 2) Anion exchanger.strong anion exchangers: quaternary ammonium salts R-N (CH3)+ Weak Ion Exchangers: based upon weak acids or bases which are charged only over a limited pH range a. The equilibrium constant for this reaction is: [-SO3. CM-Sephadex. using the conditions that make them more stable and tight binding. Ion exchange separation is carried out mainly in column packed with an ion –exchangers. which have ionisable groups. tertiary amines b.g. Most ion-chromatography instruments use two mobile phase reservoirs containing buffers of different pH. For cation separation the cation-exchange resin is usually a sulfonic or carboxylic acid. The sulphonic acid groups are called as a strongly acidic cation exchanger. It can exchange one H+ for one Na+ or two H+ for one Ca++. This will also depend upon pH. A typical group used in ion exchange is the sulphonic group. they have either negative or positive charge. Strong Ion Exchangers: based upon strong acids or bases and are charged over a wide range of pH a. SO3 -. (s) indicates the solid or stationary phase. and (aq) indicates the aqueous or mobile phase. and for anion separation the anion-exchange resin is usually a quaternary ammonium group. the tighter it binds to the exchanger and less readily it is displaced by other ion. The more highly charged molecule to be exchanged. Maximum distance covered by the solvent is noted and the distance covered by the solute is noted.

r is the distance between the groups. and D is the dielectric constant of the solvent which is increased with higher ionic strength thus weakening the force between the solute and the ion exchanger. Finer the mesh the slower will be the flow rate. must be weakened. For example to separate the sugar mixed with the polar and non- . (Most common method): F = q1 q2 / D r2 q1and q2 are the charges on two groups. Gel chromatography is an effective means of removing ion from solutions of macromolecules. Small mesh sizes improve the resolution but decrease flow rate. the binding strength.molecules usually adsorb tightly in the column so. The available capacity is the capacity under particular conditions like pH. The sephadex and bio-gel exchanger offers a particular advantage for macromolecules that are unstable in low ionic strength. this interaction. Eluting Ion Exchange Columns -. and anionic buffer is used with the cationic exchanger. The cellulose ion exchanger proved to be best for the macromolecule like protein and the nucleic acid. agarose. Macromolecular separation always needs larger pore size while micro molecule can be separated by the small pore.by mixing two buffers The basic principle of ion exchange chromatography is that the affinity of substance for the exchanger depends on both the electrical properties of the material and the relative affinity of other charged substances in the solvent. A related resin called a mixed bed resin is used to prepare deionized water. Hence. Large molecules may be unable to penetrate the pore so the capacity will decrease with increase in molecular weight. Because the cross links in these materials maintain the insolubility of the matrix even cross link in these materials maintain the insolubility of the matrix even if the matrix is highly polar.change pH -. It is measured in term of milliequivalent of exchangeable group permiligram of dry weight. Another way to look at this is that other ions in the buffer compete for the ion exchanger binding site. The extent to which an ion exchanger is charged depends upon the pH.molecules such as proteins. The total capacity of ion exchanger is measure of its ability to take up exchangeable ion. cellulose and copolymer of styrene and vinyl benzene. In deciding the eluting condition. For selection of buffer following rule is always adopted. peptides etc. ionic strength. For materials that have either single charge the choice of exchanger is not difficult. it is important to consider how eluting fluid will affect the assay for the material. also can change the charge of a weak ion exchanger .changes the charges on the molecules being separated. amino acids. But in case of material that have both negative and positive charge the choice depends upon the charge that is stable. As we know above the isoelectric point if the pH is stable one then an ion exchanger can be used. For best ionic resolution ionic condition should be same as the eluting condition. If stable below isoelectric point then a cation exchanger can be used. One important use of on exchange is in desalting. Simply because. For example if spectral analysis is to be done then eluting fluid should not absorb in the required wavelength. depends on the ionic factor.These changes can be made stepwise by changing the buffer reservoir (step gradient) or as gradient -. Finer mesh means an increased surface to volume ratio and therefore increased capacity and decrease time for exchange to occur for given volume of exchanger. It is because of larger available capacity. The cationic buffer is used with the anionic exchanger. thus altering the charge of the material.The matrix is used of various materials like dextran. The porosity of the matrix is very important factor because of charged group are present both outside and inside of the matrix. Since pore also act like molecular sieve. Strong Exchanger is used for more stable molecules such as nucleotides. which increases the zone spreading and decrease resolution. Ion exchanger has been found to be an effective way to separate weakly polar substances by using the exchanger as the matrix for partition chromatography. a weak exchanger can be used. Ion exchanger come in variety of particle size called as mesh size. If the substance is labile. bound material can be eluted by changing pH.

Commonly use a more polar organic solvent like acetonitrile. The pressure used affects the resolution of the bands. or mixtures of these with H2O. or glass beads . EtOH. N2) and Stationary Phase is a liquid coating on an inert solid support. polyethylene glycol) but the column is run at higher temperature where the coating melts.100 m long) Packed column -. At present time this procedure is applied principally with the ion-exchange and adsorption chromatography of small molecule. Gas / Liquid Chromatography In gas chromatography mobile Phase is usually a gas -. the flow rate of a column drops as the particle size decreases and this allow sufficient time for significant diffusional spreading to occur. Highperformance liquid chromatography (HPLC) columns are stainless steel tubes.polar solvent. The use of very fine particle and very high pressure to maintain adequate flow rate is called as HPLC or high performance or high pressure liquid chromatography. However.g. This hydrocarbon chains is bound to an inert matrix to provide hydrophobicity. This decreases the resolution. with a typical column having a diameter of 3—6mm and a length of 10-20 cm operating at 50-200 pounds per square inches [ 5—20 kg/ cm2 ] . This is called as reverse phase chromatography. At atmospheric pressure diffusion spreading causes the bands to overlap. Detectors present are of various types. Most powerful detector is a mass spectrometer (GC/Mass Spec. However. Usually the resolving power of a column increases with the length of the column and the number of theoretical plates per unit. there may be insufficient time for molecules in the mobile phase equilibrate and the bands are also broadened. Diffusional spreading can be reduced if the transit time of the mobile phase in the column made small. Short. GC Can provide very high resolution by making very long columns. Also it requires the less amount of test material. Ar. Use of shorter hydrocarbon chains less densely packed is called Hydrophobic Interaction Chromatography HIGH PRESSURE LIQUID CHROMATOGRAPHY [HPLC] Conventional liquid chromatography uses plastic or glass columns that can range from a few centimeters to several meters. There is open tube or capillary operation and other a coat the inside surface of a long thin tube (30 . The polar solvent binds to the matrix forming the polar stationary phase and sugar partition between this phase and the mobile weakly phase. For e. peptide.) which give mass spectrum that can be used to identify and quantitate samples as them come off the column. Reversed Phase Chromatography In reverse phase chromatography stationary Phase is apolar (hydrophobic) and is reversed with respect to cellulose chromatography.usually inert (He. teflon powder. which are placed before an analytical column to trap junk and extend the lifetime of the Stationary Phases. a sample of 0.01 to 0. HPLC is known for its rapid separation with extraordinary resolution of peaks. the solution is applied to the column.1 ml. Note that sample must be somewhat volatile and stable at higher temperature. with the longer columns finding use for preparative-scale separations. DMSO. It is because of its small fraction of the void volume. propanol. small carbohydrate and t-RNA. .g. fast analytical columns. This can be done by establishing a pressure difference across the top and bottom of the column to force the liquid through the bed. if pressure (and hence flow rate is very high. can be used. ethylene glycol. The number of plates increases as the available surface area per unit length of the column becomes greater – in other words as the matrix particle become smaller.larger diameter column is packed with an inert support -commonly diatomaceous earth. The most common lengths are 10-100 cm. typically of 10-30 cm in length and 3-5 mm inner diameter. Sample usually injected as a liquid which is then heated to vaporize it. Coating may be solid at room temp (e. and guard columns. Hydrophobicity can be varied by changing the hydrocarbon chain length or by aromatic groups. Other phase is mobile Phase that depends upon hydrophobicity of stationary phase.

Reverse-phase partition chromatography uses a relatively nonpolar stationary phase and a polar mobile phase. methylene chloride. The stationary phase is a bonded siloxane with a polar functional group. or chloroform. and phenyl groups. acetonitrile. with the smaller sizes. such as n-hexane. Normal-phase partition chromatography uses a polar stationary phase and a nonpolar organic solvent. water. Reverse-phase chromatography is the most common form of liquid chromatography. 3-5 µm. or mixtures of these solvents. as the mobile phase. primarily due to the wide range on analyte that can dissolve in the mobile phase. being used in analytical columns. The most common functional groups in order of increasing polarity are: cyano: -C2H4CN diol: -C3H6OCH2CHOHCH2OH amino: -C3H6NH2 dimethylamino: -C3H6N(CH3)2 Chromatographic Separation principle Commercial name Phase Type of support Adsorption Partisil C8 Corasil Pellumina Partisil Micro Pak Al Bondapak C18 ULTRA pak TSK ODS Octylsilane Silica Alumina Silica Alumina Porous Pellicular Pellicular Microporous Microporous Pellicular Porous Ion exchange Partisil-SAX MicroPak-NH2 Strong base Weak base Porous Porous Exclusion Styragel Superpose Bio-glass Nature of stationary . Analyte separate as they travel through the column due to the differences in their partitioning between the mobile phase and the stationary phase. and the larger particles being used in preparative-scale HPLC.Partition Chromatography In partition chromatography the stationary phase is bonded to inert particles of 3-10 µm of diameter. The most common bonded phases are n-octyldecyl (C18) and n-decyl (C8) chains. such as methanol.

hydroxyl. may be used. in a single process. if a ligand were found that attached to the two unwanted molecules. the ligand must also remain active toward the target molecule. Ligand attachment requires that the matrix be activated and then react with the ligand to fix them onto the matrix. bind to the desired molecule within a solution to be analyzed . allowing the desired molecule to elute while the other two were retained in the column. Choosing the correct ligand is the first hurdle. leaving the desired product in the column. Affinity chromatography operates on the principle that ligand (as stated in the Table). must also often stand up to decontamination when purifying pharmaceutical compounds. During this process. Biological systems have millions of ligand (also known as receptors). Specificity based on three aspects of affinity—the matrix. All other compounds in the solution will elude. and most can be affixed to the matrix and used to isolate the desired molecule. and thio groups. Matrix materials simply hold the active ligand and provide a pore structure to increase the surface area to which the molecules can bind. and the attachment of the ligand to the matrix—is the hallmark of this process and the reason for its success. With new materials and new demands. carbonyl. but the only ligand that binds the molecule also trapped two additional. and it has become essential with the growing demand of the biotech in the 1980s and 1990s. are easily activated and can serve as the sites to which the ligand attach. it utilizes the property of biological affinity of the substances to be separated. that have many hydroxyl groups that can be activated. For example. Then. the ligand. Substituent groups within the matrix. the ligand will interact only with the desired molecule and form a permanent bond. you could run a normal affinity column and collect these three molecules. The desired molecule is then removed from the column by using a wash (typically changing the pH) that lowers the dissociation constant and allows recovery of a nearly pure sample. even from complex mixtures. Decontamination is typically performed by rinsing the column with sodium hydroxide or urea. in addition to requiring activation. but suitable support materials were not available until much more recently. which uses ligand to remove everything but the target molecule from the solution. if you were looking for a molecule from a cell. such as amino. different molecules. that ligand could be used in the size exclusion chromatography and affinity column. The matrix. the technique became extremely useful in the 1960s. then a technique called negative affinity. attached to a matrix made up of an inert substance. or all will be for naught. Different matrix materials are stable in different pH ranges.Biotech research has used affinity chromatography because of its ability to separate one desired species from a host of other biological molecules. adding the third aspect of selection for affinity . As the name suggests. Matrix materials are often polysaccharides.Fractogel TSK Glass Polystyrene-divenyl benzene Agarose Polyvinylchloride Rigid solid Semi-rigid gel Soft-gel Semi-rigid gel Affinity chromatography Principle Affinity chromatography is almost exclusively used for the purification of biological molecules such as proteins and other macromolecules. Ideally. such as agarose. The ligand must bind strongly with the molecule that is to be recovered. The technique has been known for almost a century. If the ligand chosen can bind to more than one molecule in the sample in question. As a consequence. it is capable of giving absolute purification.

antigen.chromatography. a) Used mainly for small ligand. or an allosteric activator. thiol gp. pea nut can be used for separation of lactose. and does not denature after application of extreme pH and ionic strength. e) Epoxy –activated Agarose. Before using. Table9. and polyacrylamide.This is useful for linkage of sugars and carbohydrate or any material containing OH gp. The ligand should be coupled without altering its binding properties. then an ML complex will be formed. 6-AMINO-HEXENOIC ACID & 1. 2. CNBR activated agarose CNBr is negatively charged so they can react strongly with the amino group. The technique was originally developed for the purification of enzymes. Co-enzyme. Maltose Can be separated by the jack bean. 1. exhibit minimum absorption. Coupling of N-nucleophile to CNBr. 1. The choice of ligand depends on the specificity of the substance. b) They can solve the steric problem c) Only a Spacer can be attached between matrix and ligand. amino gp. f) Thiopropyl-Agarose. 4. For example for an enzyme ligand must be the substrate. a reversible inhibitor. 3. Matrix should have broad range of thermal and chemical stability. and membrane receptors and even to whole cells and cell fragments. nucleic acid and most protein to agarose. it should be treated with the cysteine. The most useful material is the Agarose. The technique can be shown by the following equation: if we assume M is the macromolecule and L ligand is attached to the matrix. 2 Affinity chromatography Substance Use Lectin Polysaccharides and glycoprotein present on the RBC membrane.Can be Used or sulpher containing protein.6 DIAMINO HEXANE. but it has been extended to nucleotides. This results in isourea linkage that carries potential charge and thus can act as an ion-exchanger. Con A Sepharose They selectively can binds to the N-acetyl glucosamine residue. since they have broad range of thermal tolerance. It is extremely useful for coupling enzyme. It should not absorb any chemical or substance to be purified itself. maintain good flow property after coupling. inhibitors. METHOD OF LIGAND IMMOBILIZATION Linking or coupling of the ligand to matrix material is called as immobilization. immunoglobulin. A ligand must bind tightly. D-glucosamine Can be separated by clam . Helix pomatia lectin Used for separation of the pure T-cell Lactose Caster bean. Carbomyl diimidazole activated agarose. During the elution the matrix should not be destroyed. 2. M + L ML For the success of the experiment following steps must be kept in mind. 3. d) Spacer can be attached by reacting functional group C00H (by using Agarose) and NH2 (group of 6AHA). Therefore they can be used for the lymphocyte separation. nucleic acids. antibody.

Loukas and colleagues studied the existence of one type of suspected opiate receptor in the brain (1994). Early applications were in the separation of biomacromolecules from other biological compounds. Detection is done by the several methods. Urease. indicating that the molecule is more tightly bound to the ligand.5 cm from edge by means of micropipette or microsyringe. For e.g. DNA polymerase. androgen receptor Imino-diacetic acid Zn+2. ion-exchange.25 mm thick slurry is prepared in water is applied to glass with the help of plate spreader. In addition to biological separation. 5’ ADP. The distribution coefficient is represented by the Kd. the broader the chromatographic peak. but they are predominantly in the field of biochemistry. The longer a molecule stays on the column. The movement of analyte is expressed by the retardation factor. such as molecules from an entire cell. exclusion chromatography. This is also helpful in the activation of the plate. The layer is formed must be thin Mobile phase is passed through the stationary phase under capillary force. fluorescent dye (this can be Distance moved by the solute from origin / Distance moved by the solvent from . Ribosome. Thin layer chromatography Principle in this type of chromatography the stationary phase is generally the glass plastic or metal foil plate. Distribution process is based on the fact of adsorption. affinity chromatography can be used to determine dissociation constants of ligand and molecules.5 cm it is left for one hour. sugar. Rf= origin. For example. catecholamine Heparin-Agarose Blood protein. partition. S. CaSO4 is mixed to facilitate adhesion of the adsorbent to the plate. The TLC plate is dipped in the developing phase to depth of about 1. Sample is applied to the plate 2—2. Protein A Can be used for separation of IgG (using Fc region) Poly A Can be separated of m-RNA Boronate polyacrylamide RNA. researchers have immobilized large molecules on the matrix and used them to separate the small molecules that bind to them. Applications The applications of affinity chromatography have been numerous.NADP+ Can be separated by using 2. The plate is dried at 100—1200C. Cu +2 Can be separated Octyl –agarose Protein Can be separated Thio-propul Sepharose Can be separated by clam Protein. In addition to using small ligand to separate large molecules. K’ can be calculated by = 1—Rf/ Rf Process of preparation of the thin layer plate A thin layer of about . and Papain. These studies may one day lead to a better understanding of how drugs such as opium affect our brains. The mobile liquid phase passed through the thin layer plate. This quantitative information can be used in further studies of the ligand or molecule. and the analyte is distributed well in the layer. They separated the ( -opioid binding protein by using an opioid receptor antagonist that was specific to the ( -opioid binding protein. This continues to be the primary and extremely important field for affinity chromatography.

which relies heavily on instrumental analysis. It took two more decades before Jorgen son and Lukacs developed the first truly useful CE instrument and showed that it had exceptional resolution (1981). Electrophoretic technique Electrophoresis. For radiolabel led compound autoradiography can be used. migration due to an electric field. For compound having different functional group—adsorption chromatography. Two factor that affect the TLC has. Equally if not more important. allowing for easy densitometry studies. Low polar compound---liquid chromatography. Hjerten. In the 1960s. the technology on which this technique depends. Applications of these modern forms are extensive. vi. such as high-quality capillary silica tubing and extremely sensitive detectors. A wider range of sorbant can be used and also due to easy detection of spot and separation of chromatogram. Movement of compound can be observed by the specific Rf values. HPTLC and HPPLC Thin-layer chromatography (TLC) gets the high-performance (HP) treatment through several optimization steps leading to improved efficiency and automation. For investigating the unsaturated compound I2 vapour can be used. Capillary electrophoresis was pioneered by S. iii. which interferes with charged species migration. H2SO4 for organic acid. HPTLC shows better optical properties than standard TLC.However. natural products analysis (including a variety of botanicals and herbals). and in environmental analysis for determining such things as pesticides in drinking water. Selection of chromatographic system i. Theory. The layers are made smaller (0. CE uses the phenomenon of electroosmotic flow to separate analytes. CE .25 mm). Rhodamine B is used for the detection of the lipid. in food analysis. High surface to volume ratio and the weight ratio. had yet to mature. Better resolution as well as a 10-fold improvement in detection limits are key to HPTLC’s increasing popularity. Arne Tiselius rediscovered this technique in 1937 and used it to separate human serum proteins. Water soluble but strong ionic compound---ion-exchange chromatography. In subsequent years. Ligand having specific binding property—Affinity chromatography. development focused on new gel materials that minimized convection. polyacrylamide gels were developed. HPTLC can also be used with centrifugation to force the sample outward in what is known as rotational planar chromatography (RPC). Br2 vapour is used for olefins detection. who built tube electrophoresis units as early as 1959. ii. ADVANTAGE OF TLC OVER OTHER CHROMATOGRAPHY TECHNIQUE IT has great resolving power and greater speed of separation. SbCl2 for steroid and tarpenoid. H2SO4 and KMN03 for hydrocarbon. especially to the speed of the run. By using a forced-flow mobile phase and a sealing chamber.02 mm instead of 0. iv. v. and the grain size is more uniform. These adaptations are collectively responsible for what is known as modern TLC. If necessary 2D gels electrophoresis can be used in the TLC plate this can be used by placing the plate in the buffer at right angle to first separation. Michaelis (1909). which ushered in the widespread use of gels in biological studies. they have smaller mean grain sizes of the coating particles (down to 7 µm instead of 12–30 µm).absorbed by the UV light. Volatile compound ----gas liquid chromatography. Commonly used separating agent is the Ninhydrin for detection of the amino acid. vii. Compound differ in molecular size—exclusion chromatography. gaining the added benefits that pressure provides. Water soluble but weakly ionic---reverse phase liquid chromatography. researchers can transform HPTLC into high-pressure planar liquid chromatography (HPPLC). including routine use in the pharmaceutical industry and clinical analysis. was first discussed by L.

protein. The column itself becomes negative if the cathode is on the product side and the anode is on the sample side. However. they will elute last because of their struggle to reach the anode. Terabe and colleagues developed a variant of CE that allows determination of nonpolar molecules (1984. Agar can be used more for analysis of DNA. Force can be represented by the following equation: F= qv/ velocity q-= charge and V is the potential difference. The substances separated at the low temperature to save it from heating effect. creating a dense +ve area. 1985). The function of buffer is to maintain the constant ionization pattern of the molecule. Many important biomolecules can be separated by this technique like: amino acid. For this purpose vertical slab gel can be used. Generally pore size of the 1% Agar has pore size larger than proteins and nucleic acid. This cross linking attaches new anti conventional properties. N’ bis acrylamide. Hydrophobic molecules will enter the micelle. But in the early 1980s. A reversed-phase HPLC packing material is used to retain the analytes at different rates on the liquid stationary phase. a. peptide. THERE is some necessary component for the polymerizations of the acrylamide like the N. Generally the agarose concentration is used in the 1% to 3% . precluding the identification and analysis of polar compounds. These cations pull the rest of the fluid with them. This area is then attracted by the cathode and starts to flow toward the product end of the column. but they will also exit in a fashion similar to liquid partition chromatography. While. When the column is negative. Therefore. Agarose Gel Electrophoresis Agarose is prepared from natural compound isolated from Gracilaria and Geladium. Traditional CE uses polar solvents. And so electrophoresis is called as polyacrylamide gel electrophoresis or PAGE. Agarose has property to dissolve in the hot water making liquid solution while it solidifies in the cooled medium and forms a rigid gel. Electrophoretic mobility (μ) is defined as ratio of velocity to field strength. b. The gel is prepared from the material like agarose or polyacrylamide and the separation of compound is done in the buffer. Micellar electrokinetic capillary chromatography (MEKC) uses surfactants or polymers to form micelles—small spheres with hydrophobic centers and ionic shields around the outside—that travel against the liquid flow toward the anode (the charges could be switched with different surfactants) but are swept with the electroosmotic flow toward the cathode.6. the separation of molecules is based on their polarity as polar molecules are slowed by entering the micelle. The gel is formed due to inter and intra-H bonding. The main problems occurs during electrophoresis is the phenomenon of electro endosmosis. Electrodes are placed in both reservoirs. Polyacrylamide Gel Electrophoresis After the polymerization of acrylamide it forms a polymer compound called as polyacrylamide. It describes the migration of a charged particle under the influence of electric field. 1% Agar is used for the immuno-elctrophoresis or flat bed electrophoresis. small concentration of agarose gives larger pore size.instruments consist of a capillary column between two reservoirs of buffer solution (the solution used as the solvent). ionisable group. High concentration of agarose gives usually smaller pore size. which places a potential across the column. The pore size is decided by the initial concentration of the agarose.anhydro galactose. Note that bis acrylamide is . nucleotide. The basic repeat unit is the galactose and 3. cations migrate toward the column wall. This can be avoided by the substituting some other functional group like sulphate.

Initiation of the polyacrylamide is difficult so some more compound is added like ammonium persulphate. This is done to sharpen the protein band.8 and resolving gel has pH about 8. For protein separation SDS –PAGE can be used the usual percentage lies between the 3 to 30 % of acrylamide.N’N’’ tetramethylenediamine. and for tracking the protein bromo-phenol blue is used. Native (buffer) gel In this method SDS is not used for the separation of the protein subunit prior to loading. Generally 5% at the top. After the protein is reaches the bottom. The negatively charged protein is attracted toward the anode. a calibration curve can be constructed. e.7) and according to the different electrophoretic nobilities and the sieving effect of the gel. This can allow separation of the protein in the range of 100. For protein of molecular weight more than 150. Staining of gel is done for 2-3 hrs. It allows the free movement of the protein so that one type of protein either same in the molecular weight or charge can stalk at one position.000 to 10. After this process the protein is separated in the gel having smaller pore size. and the TEMED or N. The distance moved by the protein of unknown Mr is then measured and its log Mr and hence Mr can be determined from calibration curve.> PROTEIN-SDS > GLYCINATE So protein SDS band lies in between the Cl. while 15% gel is used at the . The method is generally used for the enzyme –substrate reaction and total protein content. A protein generally have single band in the protein unless it has two same subunit. 000. Here protein is separated according to the native charge of protein at pH of the gel (normally pH 8. S2O8 + eSO4 2+ SO4 – IF this free radical is represented as R’.5% gel is used.8. With β mercaptoethanol and SDS.N. Molecular weight of protein Mr can be determined by the comparing its mobility with those of a number of protein used as standard. which have lower mobility than chloride ion of the loading buffer and the stacking gel Cl. the gel is removed and placed in stain more generally the Coomassie blue. c. The protein first of all loaded in the stalking gel. And destaining requires overnight. Stalking gel generally uses lower percentage of the gel that provides the larger pore size. And thus protein is analyzed in qualitative way. Problem arises during the electrophoresis is the settling of sample and exact tracking of the protein position in the gel. protein can be denatured into the simple protein. PH of the stacking gel is generally 6. For settling problem sucrose solution is used. Free radical generation can be done by the photo polymerization. Gradient gel The protein is separated according to the gradient formed by the different concentration of the gel. 000 7. Generally 15 % polyacrylamide gel is used in the separating gel. All these process can be done by the vertical slabs. Actually addition of TEMED IS necessary as it acts like the catalyst. On average it has seen that one SDS can binds to the two amino acid.essentially two acrylamide linked by the methylene group. But note that glycinate ion have a lower electrophoretic mobility than protein –SDS complex. d. Since protein separation done in the two steps: first it is separated in the stalking gel. glycine is added. In this master mix. Oxygen can be used for the removal of the free radicals. The gel is then placed in the destain solution. and size exclusion chromatography on it is resolved by the resolving gel.and glycinate ion. SDS-PAGE SDS is an anionic detergent (CH3—(CH2)10 –CH2OSO3-Na+). Low percentage gel can be used for the separation of the DNA. It helps in the better separation of the protein. By plotting a graph of distance moved against log Mr for each of standard proteins.

Note: separation of protein is called as western blotting. Agarose gel of 0. This is based on forming a pH gradient. The ion-exchanger is fixed in the column and set to a particular temperature and pH. This prevents the protein from being washed out while it is stained.1 μg of protein. a standard IP of known protein can be determined and with the help of this calibration curve. Here riboflavin is used for initiation of the polymerization of the gel. In another step the protein is run in presence of SDS. means it can detects the 0. Chromatofocussing gives a good resolution of quit complex mixture of protein provided that there is close difference in their isoelectric point. f. while separation of protein is called as southern blotting. It is difficult to observe in the gel. Coomassie blue is highly sensitive. IEF is highly sensitive technique and used for studying heterogeneity of the protein. Just beyond the isoelectric point the protein is bind to the positive charge of the ionexchange column. The difference of pH lies between 3-4 unit from upper and bottom. it gives poor resolution of compound having same isoelectric point. It gives pink red bands. Protein can be further analyzed and protein can be purified. where the silver is deposited to give black band. IP of unknown protein can be determined. It also helps in isolating the extra protein in the expression.3% will separated double stranded DNA molecules of about 5 and 60 . Iso-electric focusing: IEF gel: It utilizes the horizontal gels and separates the protein according to the isoelectric point of the protein. So the convenient way is to use agarose to analyze the DNA. i. Generally the technique is used in the translational product of the gene or mRNA. Silver stain is more sensitive. It is 100 time more sensitive than CBB. As we know that protein is the ampholytes. Glycoprotein can be detected by the stain called as PAS (periodic acid stiff stain). 8M urea and non-ionic detergent. Staining is done in the 0. g. Quantitative analysis of the protein can be done by the method called as scanning densitometry. computerized 2D is done. In the first dimension –isoelectric focusing is done in polyacrylamide gel in narrow tubes in the presence of ampholytes. DETECTION. For determining the IP of the protein. AGAROSE GEL ELECTROPHORESIS OF DNA As we know that DNA is larger than proteins and therefore they are unable to enter the polyacrylamide gel. Chromatofocussing Is suitable for the protein separation. Ag+ ion is reduced to metallic silver on the protein. the charge on the protein decreases slowly and at one point protein stops where IP is equal to the charge.bottom. This acid –methanol mixture acts as a denaturant to precipitate or fix the protein in the gel. As they proceed further. 2D-PAGE This technique utilizes the technique of both IEF and the SDS-PAGE. it can be separated easily. On adding protein at top of column the protein moves at its isoelectric point. CBB or Coomassie blue R -250 is most commonly used for the detection of the protein in the gel. It can be used immediately after the electrophoresis. Denatured protein is separated in the gel according to the isoelectric point.1% (w/v) CBB in methanol: water: glacial acetic acid. Now a days to reduce so much of the labor. Protein having pH lower than iso-electric point will be positively charged. and will initially migrate towards the cathode. The main advantage is that – very similar molecular weight can be resolved. Protein band can be cut out of protein and sequence by the gas phase sequencer. ESTIMATION AND RECOVERY OF PROTEIN GELS. Note that – single stranded DNA is always expressed in nt (nucleotide) while double stranded DNA is expressed always in the base pair or kilo base pair.

Enzymes may be immobilized on solid carriers by various techniques. It must be noted that all oxygen should be removed prior to use of the gel by degassing it in the vacuum. the comb is introduced in the gel. magnetite. and is initiated by the addition of ammonium persulphate and the base N. and (c) the properties and limitations of the support system (Bickerstaff. Also open DNA moves slower in compare to the closed circular DNA. 10% to 20 % acrylamide are used in techniques such as SDS gel electrophoresis. matrix) and decide on the method of immobilization.8% gels which are suitable for separation for the DNA molecule in the range of 0. or entrapment (Katchalski-Katzir. In this well. The gel is boiled and poured in the tank and left for the solidifying.N. The gel is sealed surrounding the gel tank which is later on opened before placing them in the electrophoresis tank. Ethidium bromide is introduced in between the DNA double stranded DNA. Generally. When one wants to do enzyme immobilization. N’ –methylene bis acrylamide (bis-acrylamide). one must select the carrier (support. Bromophenol blue is generally used and also ethidium bromide. such as carrier-binding. It is generally believed that the best support for one enzyme may not work as well for another enzyme. ie. etc.N’. Polyacrylamide polymerization is the free –radical catalysis. Preparation of the gel Gel is prepared by dissolving agarose in the water according to the need. The smaller fragment of the DNA moves faster in the gel in compare to the larger fragments. kappa-carrageenan. polyurethane. Actually the bis –acrylamide is two acrylamide joined by the methylene. 1993). This requires the presence of smaller amount of the N. Carriers or support may be selected on the basis of the following considerations: . There is sufficient variety to accommodate almost all available industrial enzymes. and that each enzyme ought to be evaluated to determine the optimal carrier matrix system and conditions. Most of the lab uses 0. The molecular weight of the DNA can be known from the calibration curve prepared from the known standard of the DNA. So they are also called as PAGE. 1997). 1993).kb size. but it needs riboflavin which generated the free radical. whereas 2% gels can be used for the separation of the sample between 0. Induction of the polymerization can be done by a method called as photo-polymerization. Electrophoresis tank is filled with the buffer. the decision will depend on (a) the various characteristics of the enzymes. Along with the sample some dye and glycerol is also loaded so that they can’t float in the buffer. so that well can form in the gel. In SDS gel.N’. Interestingly. Immobilization requires carriers to bind and to support the enzymes. Polyacrylamide gel Polymerization of the acrylamide results in the formation of the polyacrylamide. the operational conditions. Chapter -13 Enzyme Immobilization Introduction The word "immobilized enzyme" was coined by Katchalski-Katzir in 1971 (KatchalskiKatzir.1 and 3 kb. Some of these include alginate. The volume is calculated by measuring the length and breadth of the gel tank. cross-linking. DNA material is loaded.TETRAMETHYLENE DIAMINE (TEMED). there are very few detailed studies to compare the efficacy of various immobilization methods or immobilization supports. (b) requirements of the specific application. TEMED catalyses the decomposition of the persulphate ion to give a free radical (a molecule with unpaired electron). Acrylamide gel can be made with a content between 3 to 30 %.5 kb ---10 kb. A variety of carriers have been tested with different enzymes. Aim of Enzyme Immobilization Enzyme immobilization is aimed to restrict the freedom of movement of an enzyme. In the mean time.

or within. they should be stabilised against denaturation and utilised in an efficient manner. density. other molecules. However due to denaturation. including the reactants. available functional groups for modification. 3. one phase containing the enzyme and the other phase containing the product. some other material. The term 'immobilisation' does not necessarily mean that the enzyme cannot move freely within its particular phase. Beside this industrial scale chemical preparation. Their high initial cost. health and safety for process workers and end product users. mechanical stability of support material when subject to pressure or water flow. Reaction: Immobilized enzymes must not show any diffusion limitations on mass transfer of cofactors. they retain some activity after the reaction which cannot be economically recovered for re-use and is generally wasted. [b] Chemical properties chemical property like hydrophilicity. [f] Economic Availability and cost of carrier materials. pressure drop are the main requirements of the immobilization. [d] Resistance Immobilized enzyme must be resistance against bacterial or fungal attack. compressibility of carriers. reagents and technical skills is required. they do lose activity with time. These are usually inert polymeric or inorganic matrices. This activity residue remains to contaminate the product and its removal may involve extra purification costs. Alginate is not compatible with phosphate buffer. When they are used in a soluble form. permeability. In order to eliminate this wastage. effective working life. medical or pharmaceutical applications. simple and economic methods must be used which enable the separation of the enzyme from the reaction product. Beads. inertness to enzyme(s). may be used to immobilise the enzymes by making them insoluble. Compatibility with certain buffers e. acrylamide is toxic. organic solvents. This is known as immobilisation and may be achieved by fixing the enzyme to. [e] Safety Immobilized enzyme must be safe from toxicity of component reagents. The easiest way of achieving this is by separating the enzyme and product during the reaction using a two-phase system. and give an improved productivity. disruption by chemicals. pore volume. Several hundred enzymes are commercially available that are very costly. The enzyme is imprisoned within its phase allowing its re-use or continuous use but preventing it from contaminating the product.g. sheets. also known as substrates (not to be confused with the enzymes' reactants). If possible. although this is often the case. fibers). Carriers must be "safe" if the end product is to be used for food. An important factor determining the use of enzymes in a technological process is their expense. Immobilized enzymes must not be biodegradability for example in waste treatment polyurethane is not naturally occurring and is not easily degraded. special equipment. continuous processing. [c] Stability Immobilized enzyme must have stability on storage. feasibility for scale-up. should only be incidental to their use. and re-usability must not be very costly. shape or form (eg. temperature. Therefore. therefore. porosity. are able to move freely between the two phases. pH. substrates or products. and enzymes such as proteases. available surface area. For example. . substrate(s) or cofactor(s).[a] Physical properties Strength. chemicals. 2. As enzymes are catalytic molecules. flow rate. although some are much cheaper and many are much more expensive. its use may be undesirable for cleanup. Limitations of Immobilized Enzyme 1. they are not directly used up by the processes in which they are used. ability to be regenerated or reused. A wide variety of insoluble materials. Side reactions. residual enzyme activity on storage.

porosity. Immobilised enzyme systems. (a) enzyme non-covalently adsorbed to an insoluble particle. ideally they should be cheap enough to discard. due to steric difficulties.g. The most important benefit derived from immobilisation is the easy separation of the enzyme from the products of the catalysed reaction. or a reductive surface environment (e.1. The ideal support is cheap. but conditions are usually available where these properties are little changed or even enhanced. membrane confinement Figure 6. and discourage microbial growth and non-specific adsorption. is greatly increased as it may be more fully used at higher substrate concentrations for longer periods than the free enzyme.g. This prevents the enzyme contaminating the product. determine the maximum binding capacity. manganese dioxide. the hydrophobic-hydrophilic balance) on both the enzyme and support. particularly if the enzyme is noticeably toxic or antigenic. The manufacture of high-valued products on a small scale may allow the use of relatively expensive supports and immobilisation techniques whereas these would not be economical in the large-scale production of low added-value materials. 2. It will increase the enzyme specificity (kcat/Km) whilst reducing product inhibition.Immobilisation of enzymes often incurs an additional expense and is only undertaken if there is a sound economic or process advantage in the use of the immobilised. Some matrices possess other properties which are useful for particular purposes such as ferromagnetism (e. which catalytically removes the inactivating hydrogen peroxide produced by most oxidases). magnetic iron oxide. with a considerable saving in enzyme. It also allows continuous processes to be practicable.g. The nature of the support will also have a considerable affect on an enzyme's expressed activity and apparent kinetics. A substantial saving in costs occurs where the carrier may be regenerated after the useful lifetime of the immobilised enzyme. density. Immobilisation often affects the stability and activity of the enzyme. shape. enabling transfer of the biocatalyst by means of magnetic fields). for enzymes inactivated by oxidation).1): I. titania. Of particular relevance to their use in industrial processes is their cost relative to the overall process costs. (b) enzyme covalently attached to an insoluble particle. covalent binding III. Methods of immobilizations There are four principal methods available for immobilising enzymes (Figure 3.e. operational stability and particle size distribution of the supporting matrix will influence the reactor configuration in which the immobilised biocatalyst may be used. (d) enzyme confined within a semipermeable membrane. enzymes. (c) enzyme entrapped within an insoluble particle by a cross-linked polymer. 3. The actual capacity will be affected by the number of potential coupling sites in the enzyme molecules and the electrostatic charge distribution and surface polarity (i. ________________________________________ Carrier matrices Carrier matrices for enzyme immobilisation by adsorption and covalent binding must be chosen with care. pore size distribution. so immobilised. The surface density of binding sites together with the volumetric surface area sterically available to the enzyme. labour and overhead costs. inert. Clearly most supports possess only some of these . minimising downstream processing costs and possible effluent handling problems. shift the pH optimum to the desired value for the process. Insoluble immobilised enzymes are of little use. The productivity of an enzyme. rather than free (soluble). The form. where any of the reactants are also insoluble. however. adsorption II. entrapment IV. a catalytic surface (e. Advantages of Immobilizations 1. physically strong and stable.

Adsorption of enzymes In adsorption.1). ionic and H-bonding interaction. Possible steric hindrance by the carrier material. No chemical changes to carriers or enzyme. and the physical characteristics of both the enzyme and the support. may be used economically due to the ease with which they may be regenerated when their bound enzyme has come to the end of its active life. porous carbon. The driving force causing this binding is usually due to a combination of hydrophobic effects and the formation of several salt links per enzyme molecule. Although the physical links between the enzyme molecules and the support are often very strong. although more expensive than these other supports. cofactor or contaminants to the carrier may result in diffusion limitations and mass transfer problems. but a thorough understanding of the properties of immobilised enzymes does allow suitable engineering of the system to approach these optimal qualities. Little or no damage to the enzyme. Adsorption utilizes existing surface interactions between enzyme and carrier. Normally. temperature. Adsorption of enzymes on to insoluble supports is a very simple method of wide applicability and capable of high enzyme loading (about one gram per gram of matrix). the enzyme is bound to the carrier material via reversible surface interactions. 2. The particular choice of adsorbent depends principally upon minimising leakage of the enzyme during use. . surface area. This may result in reduced catalytic activity. ________________________________________ Figure 6. contaminant binding. followed. pH. etc. The forces are generally weak. Binding of protons to the support may change the pH of the microenvironment and change the reaction rate. and does not require chemical activation or modification. collision or abrasion can cause desorption. cheap and immobilization can be done quickly. a process which may simply involve washing off the used enzyme with concentrated salt solutions and re-suspending the ion exchanger in a solution of active enzyme. Desorption may occur on changing environmental conditions (such as pH. Figure 6. Schematic diagram showing the effect of soluble enzyme concentration on the activity of enzyme immobilised by adsorption to a suitable matrix. they may be reduced by many factors including the introduction of the substrate. after a sufficient incubation period. porosity. Simple. but there are sufficiently large to allow reasonable binding. hydrous metal oxides. 4. clays. The forces involved are electrostatic. agitation by stirring.features. Overloading of carrier possible. Simply mixing the enzyme with a suitable adsorbent. by washing off loosely bound and unbound enzyme will produce the immobilised enzyme in a directly usable form (Figure 3. under appropriate conditions of pH and ionic strength. one does not see any damage to the enzyme by the carriers. Some physical factors such as flow rate. The carriers with adsorption properties are selected on the basis of knowledge of their compatibility with the enzyme. 3.2 showing the carrier that adsorbs the Enzyme Some advantages of adsorption include 1. ionic strength. Ion-exchange matrices.2). The amount adsorbed depends on the incubation time. Some disadvantages of adsorption include Desorption or leakage of enzyme from support.2. Care must be taken that the binding forces are not weakened during use by inappropriate changes in pH or ionic strength. ionic strength) or on conformational changes arising from substrate/cofactor binding. Non-specific binding by substrate. such as van der Waals forces. and possibly hydrophobic forces. Easily reversible. glasses and polymeric aromatic resins. Examples of suitable adsorbents are ion-exchange matrices (Table 3. This may in turn change the kinetic properties of the reactions.

in addition to the stability of the covalent link.0 100 34 ________________________________________ Covalent coupling This involves the formation of a covalent bond between the enzyme and the carrier material.e.2 Relative usefulness of enzyme residues for covalent coupling Residue Content Exposure Reactivity Stability of couple Use Aspartate + ++ + + + Arginine + ++ ± Cysteine ± ++ Cystine + ± ± Glutamate + ++ + + + Histidine ± ++ + + + Lysine ++ ++ ++ ++ ++ Methionine ± Serine ++ + ± + ± Threonine ++ ± ± + ± Tryptophan ± - .cellulose (carboxymethyl). Tests must be run to ensure the formation of covalent bonds will not inactivate the enzyme. Only small amounts of enzymes may be immobilised by this method (about 0. They also appear to be only very rarely involved in the active sites of enzymes. ________________________________________ Table 6. found in enzymes. for covalent link formation depends upon their availability and reactivity (nucleophilicity). once formed (Table 3. hydroxyl (OH) group from Ser.5 0 100 pH 4.7 100 75 pH 7. CM. Those on the enzymes are usually amino acid residues such as amino (NH2) group from Lys or Arg. and sulfhydryl (SH) group from Cys. The reactivity of the protein side-chain nucleophiles is determined by their state of protonation (i. Functional groups that affects the covalent coupling The relative usefulness of various groups. Lysine residues are found to be the most generally useful groups for covalent bonding of enzymes to insoluble supports due to their widespread surface exposure and high reactivity. which helps to maintain enzyme activity in a hydrophilic milieu. and very little leakage of enzyme from the support occurs.2). While many supports exist. Glu. The strength of binding is very strong.> -NH2 > -COO.02 gram per gram of matrix) although in exceptional cases as much as 0.1 Preparation of immobilised invertase by adsorption (Woodward 1985) Support type % bound at DEAE-Sephadex anion exchanger CM-Sephadex cation exchanger pH 2. especially in slightly alkaline solutions.________________________________________ Table 6. Immobilisation of enzymes by their covalent coupling to insoluble matrices is an extensively researched technique. however. For example. Through chemical modifications. Thr.> -OH >> -NH3+where the charges may be estimated from a knowledge of the pKa values of the ionising groups (Table 1. charged status) and roughly follows the relationship -S. functional groups on the carriers can be altered to different forms to accommodate different kinds of covalent bonds to be formed with the enzyme.> -SH > -O. The bond is normally formed between functional groups on the carrier and the enzyme.3 gram per gram of matrix has been reported. an important factor for enzyme immobilization appears to be hydrophilicity. and DEAE-cellulose (diethylaminoethyl). This increases the range of immobilization methods that can be used for a given carrier.1) and the pH of the solution. carboxyl (COOH) group from Asp. chemical modification of -OH group can give rise to AEcellulose (aminoethyl).

production of ready-activated Sepharose and the investigation of alternative methods. if rather expensive. mild and often successful method of wide applicability. This reacts with primary amino groups (i. and lysine groups through amide formation with acyl chlorides or anhydrides). by cross-linking the enzyme plus a non-catalytic diluent protein within a porous sheet (e. (d) Glutaraldehyde [6. Sepharose is a commercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack. It usually consists of an equilibrium mixture of monomer and oligomers.3d). Chemically it is an agarose (poly{β1. for use in biosensors.3c) are very useful bifunctional reagents as they allow the coupling of amines to carboxylic acids.4-(3. The reactive glass may be linked to enzymes by a number of methods including the use thiophosgene. The hydroxyl groups of this polysaccharide combine with cyanogen bromide to give the reactive cyclic imido-carbonate.~ ++ - _+ + ++ ++ - + + + ++ + + + . This is a simple.3b). cyanogen bromide [6. Figure 3. ________________________________________ . (e) The use of trialkoxysilane to derivatise glass.g. often involving chloroformates. (c) Carbodiimide [6. Glutaraldehyde is another bifunctional reagent which may be used to cross-link enzymes or link them to supports (Figure 13. lens tissue paper or nylon net fabric). the attachment of tyrosine groups through diazo-linkages.3] (b) Chloroformates may be used to produce similar intermediates to those produced by cyanogen bromide but without its inherent toxicity.3-D-galactose-α-1. Carbodiimides (Figure 13.3e).e. activated by cyanogen bromide. ________________________________________ A.3. to produce similar intermediates (Figure 3.Tyrosine + + C terminus ++ N terminus ++ Carbohydrate .11.3] . Conditions are chosen to minimise the formation of the inert substituted urea. Conditions are chosen to minimise the formation of the inert carbamate.6-anhydro)-L-galactose}) gel. (b) Ethyl chloroformate [6. The use of trialkoxysilanes allows even such apparently inert materials as glass to be coupled to enzymes (Figure 13.~ ++ ± - The most commonly used method for immobilising enzymes on the research scale (i.3] (c) Carbodiimides may be used to attach amino groups on the enzyme to carboxylate groups on the support or carboxylate groups on the enzyme to amino groups on the support. using less than a gram of enzyme) involves Sepharose. Careful control of the reaction conditions and choice of carbodiimide allow a great degree of selectivity in this reaction.3] (d) Glutaraldehyde is used to cross-link enzymes or link them to supports. There are numerous other methods available for the covalent attachment of enzymes (e. The product of the condensation of enzyme and glutaraldehyde may be stabilised against dissociation by reduction with sodium borohydride.3a). as shown.e.5. (e) 3-aminopropyltriethoxysilane Figure 6. It is particularly useful for producing immobilised enzyme membranes. mainly lysine residues) on the enzyme under mildly basic conditions (pH 9 . (a) Activation of Sepharose by cyanogen bromide.g. The high toxicity of cyanogen bromide has led to the commercial.~ ++ Others .

________________________________________ Figure 6. The activity of the immobilised enzyme is then simply restored by washing the immobilised enzyme to remove these molecules. the substrates. This product may then be copolymerised and cross-linked with acrylamide (CH2=CH-CO-NH2) and bisacrylamide (H2N-CO-CH=CHCH=CH-CO-NH2) to form a gel. in most configurations. (E) with its active site unchanged and ready to accept the substrate molecule (S). where calcium alginate is widely used. animal and plant cells. Amounts in excess of 1 g of enzyme per gram of gel or fiber may be entrapped. all of which depend for their utility on the semipermeable nature of the membrane. (c) Enzyme bound in a non-productive mode due to the inaccessibility of the active site. The entrapment process may be a purely physical caging or involve covalent binding. effectively displacing the enzyme away from the steric influence of the surface. but restricted in movement by the lattice structure of a gel.4. ________________________________________ Entrapment and Encapsulation In both of these methods. Encapsulation generally refers to larger capsules that allow for co-immobilization of different combinations of enzymes for selected applications.4). As an example of this latter method. Both (c) and (d) may be reduced by use of 'spacer' groups between the enzyme and support.e. followed by extrusion through a spinneret into a solution of an aqueous precipitant.. these are very easy to use for a wide variety of enzymes (including regenerating . (a) Immobilised enzyme. Nonproductive modes are best prevented by the use of large molecules reversibly bound in or near the active site. the enzyme remains free in solution. native) form of the enzyme. Although costly.It is clearly important that the immobilised enzyme retains as much catalytic activity as possible after reaction. product or a competitive inhibitor ensures that the active site remains unreacted during the covalent coupling and reduces the occurrence of binding in unproductive conformations. Distortion can be prevented by use of molecules which can sit in the active site during the coupling process. be ensured by reducing the amount of enzyme bound in non-catalytic conformations (Figure 3. Membrane confinement Membrane confinement of enzymes may be achieved by a number of quite different methods. while allowing free movement of substrates. as shown in (b). for example. This can. or by the use of a freely reversible method for the coupling which encourages binding to the most energetically stable (i. The porosity of the gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzyme. cofactors and products. Hollow fiber membrane units are available commercially with large surface areas relative to their contained volumes (> 20 m2 l-1) and permeable only to substances of molecular weight substantially less than the enzymes. red blood cells can be used as encapsulation capsules. Notably. making up an emulsion of the enzyme plus cellulose acetate in methylene chloride. Entrapment is the method of choice for the immobilisation of microbial. Enzymes may be entrapped in cellulose acetate fibers by. the difficulty which large molecules have in approaching the catalytic sites of entrapped enzymes precludes the use of entrapped enzymes with high molecular weight substrates. This must confine the enzyme whilst allowing free passage for the reaction products and. Entrapment of enzymes Entrapment of enzymes within gels or fibers is a convenient method for use in processes involving low molecular weight substrates and products. The simplest of these methods is achieved by placing the enzyme on one side of the semipermeable membrane whilst the reactant and product stream is present on the other side. on the expressed activity of an immobilised enzyme. (d) Distortion of the active site produces an inactive immobilised enzyme. However. The effect of covalent coupling. the enzymes' surface lysine residues may be derivitized by reaction with acryloyl chloride (CH2=CH-CO-Cl) to give the acryloyl amides. Immobilisation of the enzyme in the presence of saturating concentrations of substrate. in part.

Table 6. the entrapment method may not work well with cellulose substrate because the substrate itself is large and will not readily go into the entrapment matrix to reach the enzyme. Methods compatible with the enzyme. For example. Characteristics Adsorption Covalent binding Entrapment Membrane confinement Preparation Simple Difficult Difficult Simple Cost Low High Moderate High Binding force Variable Strong Weak Strong Enzyme leakage Yes No Yes No Applicability Wide Selective Wide Very wide Running Problems High Low High High Matrix effects Yes Yes Yes No Large diffusional barriers No No Yes Yes Microbial protection No No Yes Yes ________________________________________ ________________________________________ There are several methods of immobilization (Bickerstaff. Crosslinking This method joins the enzyme to each other to form a large. This allows for easier recovery of the enzymes for repeat use. surrounding the soluble enzyme. The micro-capsules and liposomes are washed free of non-confined enzyme and transferred back to aqueous solution before use. Liposomes are concentric spheres of lipid membranes. paper and pulp and specialty chemicals.6-diaminohexane. Chapter-14 Introduction to Industrial biotechnology Industrial biotechnology applies the techniques of modern molecular biology to improve the efficiency and reduce the environmental impacts of processes in industries like food production.coenzyme systems.6) shell around the aqueous droplets which traps the enzyme.3 presents a comparison of the more important general characteristics of these methods.g. 3-D structure without any support. The resultant reaction forms a thin polymeric (Nylon-6. encapsulation allows for ease of handling and storage of enzymes. substrate or cofactor should be selected. altered pH-activity profile. textiles. As an example of the former. the enzyme is dissolved in an aqueous solution of 1. 1997). ________________________________________ Table 6. Just as biotechnology is transforming the pharmaceutical industry. may also be used within such reactors. This can be done through chemical (covalent bonding) or physical means (flocculation). Encapsulation often improves the operational stability and sometimes efficiency of enzyme catalysis under industrial conditions. some observers predict the same impact in the industrial sector. grain cleaning. chloroform.3 Generalised comparison of different enzyme immobilisation techniques. This is then dispersed in a solution of hexanedioic acid in the immiscible solvent. Encapsulation of Enzymes Enzymes. Tests must be done to ensure the active site remains free and available for catalytic activity. and also allows their easy separation from the medium. encapsulated within small membrane-bound droplets or liposomes. In addition. e. Sometimes other properties of the enzyme may be altered by immobilization. They are formed by the addition of phospholipids to enzyme solutions. They are also easier to scale up. as listed below. Today . see Chapter 8) without the additional research and development costs associated with other immobilisation methods. The improved stability and ease of separation of immobilized enzymes make them suitable to be used in a variety of bioreactors.

considerably smaller and the plant may be prefabricated cheaply off-site Immobilised enzymes . Both improved economics as well as the manufacture of novel products not possible or practical by traditional chemical approaches have been achieved. for instance. Even today.up to 90% of the enzymes used in large scale. signal cells to turn on and off and perform other complex functions. The manufacturing process uses renewable resources as a raw material feedstock. the mild conditions in which they work and their high biodegradability. Less than 1 percent of the microorganisms in the world have been cultured and characterized. Most proteases. for commercial applications result from the exploitation of rDNA methods in the manufacturing process or for the improvement of the catalysts themselves. the enzyme catalase. no enzyme can withstand temperatures above 100°C for long. Enzymes are proteins produced by all living organisms. The plant size needed for continuous processes is two orders of magnitude smaller than that required for batch processes using free enzymes. metals and metal oxides. Other animals use enzymes to break cellulose into sugar or break up proteins. Due to their efficiency. enzymes help digest food. Enzyme processes are therefore potentially energysaving and save investing in special equipment resistant to heat. enzymes are very well suited to a wide range of industrial applications. bases. The opportunity to identify unique bioactive molecules is vast. such as enzymes. much of the science is focused on the development of new technological approaches that will allow the future solution of problems of fundamental understanding as well as practical application. Contrary to inorganic catalysts such as acids. oil field chemicals and specialty chemical synthesis reactions. In other words.Being formed to work in living cells. can break down several types of protein. including paper manufacturing. sometimes need to run at very high or very low temperatures or high or low pHs. Most enzymes function optimally at a temperature of 30-70°C and at pH values which are near pH 7. special enzymes have been developed that work at higher temperatures. Most enzymes are manufactured in fermentation systems like human therapeutic proteins. Enzymes are very efficient catalysts. The capital costs are. each enzyme can break down or synthesize one particular compound. For certain technical applications. In humans. enzymes can work at atmospheric pressure and in mild conditions in terms of temperature and acidity. they limit their action to specific bonds in the compounds with which they react. For example. However. enzymes are very specific. The progress made in applying the techniques of genetic engineering in the development of industrial-scale processes that produce or utilize enzymes has been simply amazing. the specific action of enzymes allows high yields to be obtained with a minimum of unwanted byproducts. Scientists can locate enzymes in the natural environment with special functional characteristics that have significant commercial value. which is found abundantly in the liver and red blood cells. therefore. pressure or corrosion. Industrial biotechnology companies develop biocatalysts. is so efficient that in one minute one enzyme molecule can catalyze the breakdown of five million molecules of hydrogen peroxide into water and oxygen.Nature provides rich resources for innovative solutions for many industries. textile processing. The challenge is to find organisms that can survive and even thrive in these environments. but in each protein molecule only certain bonds will be cleaved depending on which enzyme is used. specific action. Chemical processes. Companies involved in industrial biotechnology are constantly striving to discover and develop high-value enzymes and bioactive compounds that will enhance current industrial processes. In some cases. In industrial processes. Using biotechnology. the desired enzyme can then be manufactured in commercial quantities. Application of Immobilised-Enzyme Processes Introduction Immobilise -enzyme systems are used where they offer cost advantages to users on the basis of total manufacturing costs. to be used in chemical synthesis.

0.3. Batches of 97 DE glucose syrup at the final commercial concentration (71% (w/w)) must be kept warm to prevent crystallisation or diluted to concentrations that are microbiologically insecure.4 Aspartame a Dihydropyrimidinase 1-High-fructose corn syrups (HFCS) With the development of glucoamylase in the 1940s and 1950s it became a straightforward matter to produce high DE glucose syrups. on a weight basis. The use of this phosphohexose isomerase may be ruled out as a commercial enzyme because of the cost of the ATP needed to activate the glucose and because two other enzymes (hexokinase and fructose-6-phosphatase) would be needed to complete the conversion.1. ________________________________________ Table 7.5.3). However.g.1. the majority being converted to various hydroxy acids).23 Lactose-free milk and whey Lipase 3.1. giving tiny yields and many by-products (e.2.1.3. Fructose is 30% sweeter than sucrose.5.1.5. can produce such glucose isomerases: The commercial enzymes are produced by Actinoplanes missouriensis.12 L-Alanine Cyanidase 3.1.1.1.1. on a weight basis. these xylose isomerases were found to isomerise α-D-glucopyranose to α-D –fructofuranose.2. At about this time.2.1.22 Raffinose-free solutions Thermolysin 3. and twice as soluble as glucose at low temperatures so a 50% conversion of glucose to fructose overcomes both problems giving a stable syrup that is as sweet as a sucrose solution of the same concentration (see Table 14. and is comparatively insoluble.2. The isomerisation is possible by chemical means but not economical.1 M glucose 'isomerised' with 1. as they have specificities for glucose and fructose which are not much different from that for xylose and ways are being found to avoid the necessity of xylose as inducer.5 High -fructose corn syrup Histidine ammonia-lyase 4. the existence of such an enzyme was not suspected. mainly bacteria.2.3.22 M KOH at 5°C under nitrogen for 3.1 Some of the more important industrial uses of immobilised enzymes Enzyme EC number Product Aminoacylase 3. enzymes were found that catalyse the conversion of D-xylose to an equilibrium mixture of D-xylulose and D-xylose in bacteria.2.I.5.offer greatly increased productivity on an enzyme weight basis and also often provide process advantages (see Chapter 6) Currently used immobilised-enzyme processes are given in Table 7.x Formic acid (from waste cyanide) Glucoamylase 3. 2-GLUCOSE ISOMERASE Glucose is normally isomerised to fructose during glycolysis but both sugars are phosphorylated. When supplied with cobalt ions.1.3 Cocoa butter substitutes Nitrile hydratase 4. which have the additional benefit of substantially stabilising the .3 D-Glucose Glucose isomerase 5.2.14 L-Amino acids Aspartate ammonia-lyase 4.and L-amino acids Invertase 3. these have shortcomings as objects of commerce: D-glucose has only about 70% of the sweetness of sucrose.5. Now it is known that several genera of microbes.1.1 L-Aspartic acid Aspartate 4-decarboxylase 4.24.11 Penicillins Raffinase 3.3 Urocanic acid Hydantoinasea 3.1. until the late 1950s. Bacillus coagulans and various Streptomyces species. Only an isomerase that would use underivatised glucose as its substrate would be commercially useful but.2 D. They are remarkably friendly enzymes in that they are resistant to thermal denaturation and will act at very high substrate concentrations.26 Invert sugar Lactase 3.x Acrylamide Penicillin amidases 3.5 months gives a 5% yield of fructose but only 7% of the glucose remains unchanged.1. these should perhaps now no longer be considered as xylose isomerases.

The pH is adjusted to 7. and this presents a problem. plus in some cases a protein diluent.enzymes at higher operational temperatures. At this level the substrate stream is normally made 3 mM with respect to Mg2+. left from previous processing. causing inhibition. immobilised glucose isomerase was used in a batch process. Immobilisation is generally by cross-linking with glutaraldehyde. This proved to be costly as the relative reactivity of fructose during the long residence times gave rise to significant by -product production.7 0. They are used with high substrate concentration (35-45% dry solids. after cell lysis or homogenisation.5-8. ________________________________________ Table 7. labour and energy costs. The Ca2+ concentration of the glucose feedstock is usually about 25 m. Originally. Nowadays most isomerisation is performed in PBRs (Table 14. Some of the improvement that may be seen for PBR productivity is due to the substantial development of this process. The vast majority of glucose isomerases are retained within the cells that produce them but need not be separated and purified before use. Although differerent immobilisation methods have been used for enzymes from differerent organisms. Excess Mg2+ is uneconomic as it adds to the purification as well as the isomerisation costs. All glucose isomerases are used in immobilised forms. but the immobilisation methods now used fix the cobalt ions so that none needs to be added to the substrate streams. half-life (h) 30 300 1500 Active life. half-lives 0. Comparison of glucose isomerisation methods Parameter Batch (soluble GI) Batch (immobilised Gl) Continuous (PBR) Reactor volume (m3) 1100 1100 15 Enzyme consumption (tonnes) 180 11 2 Activity. difficulties were encountered in the removal of the added Mg2+ and Co2+ and the recovery of the catalyst. Also. £ tonne-1 5 5 1 Conversion cost. a Treatment with activated carbon. The need for Co2+ has not been eliminated altogether.2). At higher concentrations of calcium a Mg2+ : Ca2+ ratio of 12 is recommended.7 2 3 Residence time (h) 20 20 0. Ca2+ competes successfully for the Mg2+ binding site on the enzyme. ________________________________________ .8 6.2 < 0.1 Product refining Filtration C-treatmenta Cation exchange Anion exchange C-treatment Cation exchange Anion exchange C -treatment Capital. the principles of use are very similar.6 Colour formation (A420) 0.2. £ tonne-1 500 30 5 All processes start with 45% (w/w) glucose syrup DE 97 and produce 10000 tonnes per month of 42% fructose dry syrup. 93-97% glucose) at 5560°C.0 using sodium carbonate and magnesium sulphate is added to maintain enzyme activity (Mg2+ and Co2+ are cofactors).5 Co2+ (tonnes) 2 1 0 Mg2+ (tonnes) 40 40 7 Temperature (°C) 65 65 60 pH 6.8 7.

10).5 and it is purified by ion-exchange chromatography and treatment with activated carbon. ________________________________________ Figure 7. The fractionation process.. the pH of the syrup is lowered to 4 . is only economic if run continuously.51 times the initial productivity of one column. In effect. Diagram showing the production rate of a seven-column PBR facility on start -up. It may be seen that the final average production rate is higher when the PBRs are individually operated for shorter periods but this 29% increase in productivity is achieved at a cost of 50% more enzyme. 55% fructose is required. most manufacturers adjust flow rates so as to produce 42-46% (w/w) fructose (leaving 47-51 % (w/w) glucose). the feed flow rate is adjusted according to the enzyme activity. reactor is being refilled with fresh biocatalyst. The final average productivity is 3. Insoluble material is removed by filtration.23 times the initial productivity of one column. At equilibrium at 60°C about 51 % of the glucose in the reaction mixture is converted to fructose. Immobilised glucoamylase is used in some plants to hydrolyse oligosaccharides in . Several reactors containing enzyme preparations of different ages are needed to maintain overall uniform production by the plant (Figure 14.Precautions It is essential for efficient use of immobilised glucose isomerase that the substrate solution is adequately purified so that it is free of insoluble material and other impurities that might inactivate the enzyme by chemical (inhibitory) or physical (pore-blocking) means.e. However. .5% initial activity) before replacement.-. The half-life of most enzyme preparations is between 50 and 100 days at 55°C.10. The final average productivity is 2. At any time a maximum of six PBRs are operating in parallel. This may be removed by vacuum de-aeration of the substrate at the isomerisation temperature or by the addition of low concentrations (< 50 ppm) of sulphite. after three half -lives). whilst the seventh. and soluble materials are removed by ion exchange resins and activated carbon beads. A shorter PBR operating time also results in a briefer start-up period and a more uniform productivity. The columns are brought into use one at a time. ——— PBR activities allowed to decay through three half-lives (to 12. assuming exponential decay of reactor activity. it is normally concentrated by evaporation to about 70% dry solids. This done.PBR activities allowed to decay through two half-lives (to 25% initial activity) before replacement. For use in the better colas. this means that glucose produced by acid hydrolysis cannot be used. exhausted. This is produced by using vast chromatographic columns of zeolites or the calcium salts of cation exchange resins to adsorb and separate the fructose from the other components.For many purposes a 42% fructose syrup is perfectly satisfactory for use but it does not match the exacting criteria of the quality soft drink manufacturers as a replacement for sucrose in acidic soft drinks. To maintain a constant fructose content in the product. an enzyme bed volume of about 4 m3 is needed. To produce 100 tonnes (dry substance) of 42% HFCS per day. Activity decreases. sometimes after treatment with flocculants. The glucose-rich 'raffinate' stream may be recycled but if this is done undesirable oligosaccharides build up in the system. Typically a batch of enzyme is discarded when the activity has fallen to an eighth of the initial value (i. there still remains the possibility of inhibition due to oxidised by-products caused by molecular oxygen. ________________________________________ After isomerisation... The fructose stream (90% (w/w) fructose. although basically very simple. because of the excessive time taken for equilibrium to be attained and the presence of oligosaccharides in the substrate stream. In its lifetime 1 kg of immobilised glucose isomerase (exemplified by Novo's Sweetzyme T) will produce 10 -11 tonnes of 42% fructose syrup (dry substance). 9% glucose) is blended with 42% fructose syrups to give the 55% fructose (42% glucose) product required.. Then. following a first-order decay equation. as its low quality necessitates extensive and costly purification. due to the more rapid replacement of the biocatalyst in the PBRs.

This process results in a 3% increase in productivity and a significant reduction in the costs of the disposal of waste molasses. Enzyme. it would be totally unacceptable to use an enzyme preparation containing invertase to remove this material during sucrose production. Immobilised raffinase may also be used to remove the raffinose and stachyose from soybean milk. stirring is stopped and the juice pumped off the settled bed of enzyme.Clearly the need for a second large fructose enrichment plant in addition to the glucose isomerase plant is undesirable and attention is being paid to means of producing 55% fructose syrups using only the enzyme. glucose isomerase and sucrose phosphorylase (EC 2.1). Both these alternatives present a more than considerable challenge to enzyme technology! The present world market for HFCS is over 5 million tonnes of which about 60% is for 55% fructose syrup with most of the remainder for 42% fructose syrup.the raffinate. such microbes may be the basis of totally novel processes. dried and used directly as the immobilised-enzyme preparation. The thermodynamics of the system favour fructose production at higher temperatures and 55% fructose syrups could be produced directly if the enzyme reactors were operated at around 95°C. A mould. This will not be easy but is achievable if the commercial pull (i. so yeast invertase was used instead. here the substrate concentration is comparatively low (around 20% dry solids) so the formation of isomaltose by the enzyme is insignificant. was unavailable. raffinoseutilizer. Another ambition of the corn syrup industry is to produce sucrose from starch. is replaced by new enzyme added with the next batch of juice. In the period 1941 -1946 the acid. Plainly. The galactose released is destroyed in the alkaline conditions of the first stages of juice purification and does not cause any further problems while the sucrose is recovered. Yeast cells were autolysed and the autolysate clarified by adjustment to pH 4. money available) is sufficient: phosphorylase starch (Gn) + orthophosphate starch (Gn-1) + α-glucose-1-phosphate [7.4.7.7). When the removal of raffinose is complete.4. The use of miscible organic co -solvents may also produce the desired effect. 3-Use of immobilised raffinase The development of a raffinase (α-D-galactosidase) suitable for commercial use is another triumph of enzyme technology. Provided they are able to release sucrose without hydrolysis when the stress is released. Mortierella vinacea var. It is stirred with the sugar beet juice in batch stirred tank reactors. followed by filtration through a bed of calcium sulphate and .The high-fructose syrups can be used to replace sucrose where sucrose is used in solution but they are inadequate to replace crystalline sucrose. These sugars are responsible for the flatulence that may be caused when soybean milk is used as a milk substitute in special diets.2] sucrose phosphorylase -glucose-1 -phosphate + fructose sucrose + orthophosphate [7. This market is still expanding and ensures that HFCS production is the major application for immobilised-enzyme technology.1] glucose isomerase glucose fructose [7.1. previously used in the manufacture of Golden Syrup. This is grown in a particulate form and the particles harvested. but the thermodynamics do not favour the conversion so means must be found of removing sucrose from the system as soon as it is formed. lost by physical attrition. fills the requirements. This can be done using a combination of the enzymes phosphorylase (EC 2.1. It has been necessary to find an organism capable of producing an -galactosidase but not an invertase.e.3] A further possible approach to producing sucrose from glucose is to supply glucose at high concentrations to microbes whose response to osmotic stress is to accumulate sucrose intracellularly. 4-Use of immobilised Invertase Invertase was probably the first enzyme to be used on a large scale in an immobilised form (by Tate & Lyle).

allowing their specific hydrolysis to D-carbamoyl amino acids which can be converted to the D-amino acids by chemical treatment with nitrous acid. The enzyme is immobilised by adsorption to anion exchange resins (e.6]) at pH 8. It is impossible to produce inverted syrups of equivalent quality by acid hydrolysis. The scale used was large. in part. the product did not have the subtlety of flavour of the acid-hydrolysed material and the immobilised enzyme process was abandoned when the acid became available once again. Recently. A layer of the bone char containing invertase was included in the bed of bone char already used for decolourising the syrup. 5-Production of amino acids Another early application of an immobilised enzyme was the use of the aminoacylase from Aspergillus oryzae to resolve racemic mixtures of amino acids.5]) followed by enzymic hydrolysis with hydantoinase and a carbamoylase (reaction scheme [14. the limiting factors being microbial contamination or loss of decolourising power rather than loss of enzymic activity. DEAE-Sephadex) and has an operational half-life of about 65 days at 50°C in PBRs with residence times of about 30 min.7] fumaric acid L-aspartic acid A crude immobilised aspartate ammonia-lyase (50000 U g-1) may be prepared by entrapping Escherichia coli cells in a κ-carageenan gel crosslinked with glutaraldehyde and hexamethylenediamine. not surprisingly. These products are easily separated by differential crystallisation and the N-acyl-D-amino acids racemised chemically (or enzymically) and reprocessed. however. it has been relaunched using BrimacTM. where the invertase -char mix is stabilised by cross-linking and has a half-life of 90 days in use (pH 5. D -Amino acids are important constituents in antibiotics and insecticides. Enzymic inversion avoids the high-colour. They may be produced in a manner similar to the L-amino acids but using hydantoinases of differing specificity. aspartate ammonia-lyase -OOCCH=CHCOO. 50°C).5] [7. Figure [7.5. to the success of HFCS as a high-quality low-colour sweetener.4] Chemically synthesised racemic N-acyl-DL-amino acids are hydrolysed at pH 8. The revival is due. The immobilised enzyme has proved a more economical process than the use of free enzyme mainly due to the more efficient use of the substrate and reductions in the cost of enzyme and labour. The process is operated in a PBR at pH .g.+ NH4+ -OOCCH2CH(NH3+)COO[7.5. relatively low conversion and batch variability problems of acid hydrolysis. It may be produced from fumaric acid by the use of the aspartate ammonia-lyase (aspartase) from Escherichia coli.adsorption into bone char. The preparation was very stable. Novel and natural L-amino acids can be produced by the chemical conversion of aldehydes through DL-amino nitrites to racemic DL-hydantoins (reaction scheme [14. The process was cost-effective but. Both enzymes may be obtained from Arthrobacter species. Although free invertase may be used (with residence times of about a day). They remaining L-hydantoin may be simply racemised by base and the process repeated. A productivity of 16 tonnes of inverted syrup (dry weight) may be achieved using one litre of the granular enzyme.6] L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of the low -calorific sweetener aspartame. the use of immobilised enzymes in a PBR (with residence time of about 15 min) makes the process competitive. [7. the bed of invertase-char being 2 ft (60 cm) deep in a bed of char 20 ft (610 cm) deep. high salt-ash. the cost of 95% inversion (at 50% (w/w)) being no more than the final evaporation costs (to 75% (w/w)).5 to give the free L-amino acids plus the unhydrolysed N-acyl-D-amino acids. The Pseudomonas striata enzyme is specific for Dhydantoins. The reactors may be re-activated in situ by simply adding more enzyme.

pH 7. ampicillin. pH optimum 6.000 Ukg1penicillin G. producing about two tonnes of 6-aminopenicillanic acid kg-1of immobolised enzyme. Such specific hydrolysis may be simply achieved by use of penicillin amidases (also called penicillin acylases). A crude immobilised-enzyme preparation consisting of heat -treated cells entrapped in a polyacrylamide gel has been used to effect this conversion. 400 U g-1). Product inhibition by galactose and unwanted oligosaccharide formation are both noticeable under the diffusion-controlled conditions usually prevalent. pH optimum 3. with a reported operational halflife of 680 days at 37°C. It represents one of the earliest successful processes involving immobilised enzymes and is generally used in batch or semicontinuous STR processes (40. [7. Both problems may be reduced by an increase in the effectiveness factor and a reduction in the degree of hydrolysis or initial lactose concentration. whilst causing no hydrolysis of the intrinsically more labile but pharmacologically essential β-lactam ring. The organism cannot be used directly as it has urocanate hydratase activity. whereas fungal enzymes are more useful with acid whey. as the fats and proteins in the milk emulsion tend to coat the biocatalysts. The control of microbial contamination within the bioreactors is the most critical practical problem in these processes. 2 h) where it may be reused over 100 times. Immobilised lactases are particularly affected by two inherent short-comings. where it has an active life of over 100 days. coli and has been immobilised on a number of supports including cyanogen bromide-activated Sephadex G200. They are used in PBRs.5 using ammonium fumarate as the substrate. which removes the urocanic acid. Due to the different pH optima of fungal and yeast lactases.g. . pencillin-V-amidase being much more specific than pencillinG-amidase.p. 500 U g-1. 90 U g-1). Penicillin amidase may be obtained from E. oryzae.8] .7 Production of antibiotics Benzylpenicillins and phenoxymethylpenicillins (penicillins G and V. 35°C. 30 min) inactivates this unwanted activity but has little effect on the histidine ammonialyase. are difficult to attain. the yeast enzymes are useful at the neutral pH of both milk and sweet whey.8. This both reduces their apparent activity and increases the probability of microbial colonisation. The amide link may be hydrolysed conventionally but the conditions necessary for its specific hydrolysis.8. but the relatively high cost of the enzyme is an added incentive for its use in an immobilised state. pH optimum 4. Yeast lactase has been immobilised by incorporation into cellulose triacetate fibres during wet spinning.8. but such conditions also lead to a reduction in the economic return. in Italy. To some extent. respectively) are produced by fermentation and are the basic precursors of a wide range of semisynthetic antibiotics.0 -1.4 -6. Urocanic acid is a sun-screening agent which may be produced from L-histidine by the histidine ammonia -lyase (histidase) from Achromobacter liquidum . A. showing a half-life of 180 days at 37°C. 6. However.Immobilised lactases are important mainly in the treatment of whey.5.5 mm diameter porous silica (35 nm mean pore diameter) using glutaraldehyde and γ-aminopropyltriethoxysilane (Asperigillus niger. Different enzyme preparations are generally used for the hydrolysis of the penicillins G and V.A. a process developed by Snamprogetti S. The reasons for its utility has been given earlier . this may be overcome by the use of regular sanitation with basic detergent and a dilute protease solution. a brief heat treatment (70°C. The fibres are cut up and used in a batchwise STR process at 5°C (Kluyveromyces lactis.5.0 -3.Use of immobilised lactase Lactase is one of relatively few enzymes that have been used both free and immobilised in large-scale processes. Fungal lactases have been immobilised on 0. e. It has also been used in PBRs.

With discoveries of monoclonal antibodies different specific detection become easier with the help of different assay system like ELISA. The major application in the clinical field is the determination of the concentration of the substrate secreted in the body fluids with the help of enzymes and comparing it with the normal fluid concentration. These problems may be overcome by the use of immobilised nitrile hydratase (often erroneously called a nitrilase).5 in a semibatchwise process. It may be produced by the addition of water to acrylonitrile. amylase. Now a day up to 25% of the enzyme is used today for the clinical diagnosis.+ NH4+ [7. as it contains only very low amidase activity which otherwise would produce unwanted acrylic acid from the acrylamide. reflecting the magnitude of the potential . however.10] Many other potential and proven antibiotics have been synthesised in this manner. Due to enhancing in knowledge of enzyme it is possible to use it for rationale drug design to inhibit specific enzyme. Using 1% (w/v) immobilised-enzyme concentration (about 50.02% (w/w) acrylic acid. CH2=CHCN + H2O CH2=CHCONH2 [14.11] This process may be achieved by the use of a reduced copper catalyst (Cu+).9] The penicillin-G-amidases may be used 'in reverse' to synthesise penicillin and cephalosporin antibiotics by non -equilibrium kinetically controlled reactions. It is used at 10°C and pH 8. The closely related enzymes cyanidase and cyanide hydratase are used to remove cyanide from industrial waste and in the detoxification of feeds and foodstuffs containing amygdalin (see equation [7. Development of medical applications for enzymes have been at least as extensive as those for industrial applications. The enzyme from Rhodococcus has been used by the Nitto Chemical Industry Co.12] HCN + H2O HCONH2 [7.13] Chapter-15 Application of Enzyme in clinical diagnosis and Industries Introduction Most important aspects of enzyme activities is the clinical diagnosis.only hydrolytic enzyme were used such as lipase.000 U l-1) the process takes about a day. containing negligible substrate and less than 0. phosphatase. the yield is poor. Trypsin. Product concentrations of up to 20% (w/v) acrylamide have been achieved.[7. unwanted polymerisation or conversion to acrylic acid (CH2=CHCOOH) may occur at the relatively high temperatures involved (80 -140°C) and the catalyst is difficult to regenerate. keeping the substrate acrylonitrile concentration below 3% (w/v). Preparation of acrylamide Acrylamide is an important monomer needed for the production of a range of economically useful polymeric materials. This is due to increased knowledge in the metabolic pathway and the role of enzyme. Ltd. Ampicillin has been produced by the use of penicillin-G-amidase immobilised by adsorption to DEAE -cellulose in a packed bed column: [7. Before 1940 . HCN + 2H2O HCOO.12]). Immobilised nitrile hydratase is simply prepared by entrapping the intact cells in a cross-linked 10% (w/v) polyacrylamide/dimethylaminoethylmethacrylate gel and granulating the product. Acrylamide production using this method is about 4000 tonnes per year. using a variety of synthetic β-lactams and activated carboxylic acids. and pepsin and in diagnosis only 5% enzyme were in the use.0-8.

. semen . due to increase cell turnover number. lactate dehydrogenase is used mostly.1 . aminotransferase are monitored for several months to follow progress of disease. ALT or alanine aminotransferase or serum glutamate pyruvate transaminase(SPGT) . Ortnithin carbamoyltransferase is almost exclusive to liver. the most important thing is the availability of enzyme in the blood urine or in the tissue. aspartate aminotransferase. 1. Other body fluids that can be used is the body pleura. the most successful applications are extracellular: purely topical uses. The first enzyme that was used in the diagnosis of the heart disease was the aspartate aminotransferase. amylase . Other enzyme is the lactate dehydrogenase. In the disease state a tissue become inflamed and swelling occur and it become nacrotic. and lactooylglutathione lyse. In the case of cirrhosis or hepatitis. isocitrate dehydrogense 5’ nucleotide and glutathione transferase. For example glutamate S –transferases has a half lie of 90 minute. peritoneum. stomoach. In that state it may secrete large amount of the enzymes from the dead cells for example release of glutamate dehydrogenase from the dead mitochondrial cells. cerebrospinal canal. Then it has been observed that they were rapidly degraded and removed from the blood plasma. and neoplasia. Other most studied enzyme in the blood serum is the LDH lactate dehydrogenase. and γ. glucose-6 phosphate. due to tissue damage. acid phosphatases in the prostate and acetylcholineestarase in erythrocyte. deudonium.Clinical Enzymology of liver disease. pericardium. ceruloplasmine. Sometime these enzyme also secreted in the hepatitis so to decide which is the disease better to measure the ratio. At present. Obstructive jaundice can be determined by the measurement of aminotransferases and alkaline phosphatase. The variety of enzymes and their potential therapeutic applications are considerable.dehydrogenase and aspartate aminotransferase. the removal of toxic substances and the treatment of lifethreatening disorders within the blood circulation. and alkaline phosphates. nacrosis of the heart tissue. To know amount of or fate of serum enzyme labeled DH injected in the rabbit. creatine kinase. vagina. The enzyme most used in the diagnosis is the (ST or SGOT) aspartate amino transferase. γ-glutamyltransferase used extensively in the liver specially in the case of alcoholism. The most common disease of the liver is the hepatitis. synovia. cellular proliferation.glutamyl transerase. In forensic science main enzyme that is used is the adenosine deaminase. Activities of the enzyme in the serum may increases due to several reason. but now creatine kinase. 2. creatine kinase. phosphoglucomutase amino peptidase. There are some tissue specific enzyme that may be detected from them. The ratio is higher in the case of obstructive jaundice than in viral hepatitis.In Heart disease The main disease of the heart is the myocardial infarction and.rewards: for example. α-amylase α-amylase is an endoamylase and hydrolyze amylopectine. cirrosis. For example.with the elctrophoresis technique we can determine many more enzyme in the serum like alkaline phosphatase. Most of the clinical assay is done with the help of serum. RBC and WBC. tumors infection. Determination of enzyme activities For the determination of enzyme activities for clinical diagnosis. Its highest concentration . carbonate dehydrates and phosphatase esterase. For example. The ratio of glutamate dehydrogenase / aminotranserase changes after one week of the viral hepatitis.Note that the stay of the release enzyme is very sort. and lactate dehydrogenase. A selection of those enzymes which have realised this potential to become important therapeutic agents is shown in Table 8. Enzymes are released during the nacrosis into the plasma. Serum alkaline phosphatase is mainly important in the liver disease. . alkaline phosphatase. The three enzymes that is assayed are creatine kinase. adenylate kinase. pancreatic enzymes have been in use since the nineteenth century for the treatment of digestive disorders. Urine can also be used.

Creatine Kinase and fructose bisphosphate aldolase. Also this enzyme is detected during the duodenal ulcers. Table 8. Alkaline phosphates This enzyme is detected in the obstructive jaundice. amylase activity also increases during the mumps. The action of the asparaginase does not affect the functioning of normal cells which are able to synthesize enough for their own requirements. cardiac muscle and brain. This enzyme is normally found in the serum. Enzyme deficiencies Phenylketonuria is detected by presence of phenylalanine in the urine. when salivary glands become inflamed. It is also useful in the detection of bone disease like ostomalacia. This enzyme concentration also increases during the pregnancy. which restricts their ability to synthesise the normally non-essential amino acid L-asparagine. In Treatment of cancer A major potential therapeutic application of enzymes is in the treatment of cancer. The enzyme is administered intravenously. During the pancreatic carcinoma it is found in higher concentration in the pancreas. rickets and hyperparathyroidism. albinism . It is only effective in reducing asparagine levels within the bloodstream.is found in the intestine and salivary gland. Therefore.g. Some inborn error of metabolism is also detected are relatively harmless e. In normal person its concentration is detected in low concentration in the urine and the serum but it is increases many fold during the pencreatitis. This half-life may be increased 20-fold by use of polyethylene glycol-modified asparaginase. alkaptonuria. Asparaginase has proved to be particularly promising for the treatment of acute lymphocytic leukemia. Pains occur severely in the upper duodenum. This enzyme is found in highest concentration in the prostrate carcinoma. A 60% incidence of complete remission has been reported in a study of almost 6000 cases of acute lymphocytic leukemia. showing a halflife of about a day (in a dog). Its concentration (creatine kinase) raised during brain stroke. Its action depends upon the fact that tumour cells are deficient in aspartate-ammonia ligase activity.1 showing List of defective enzyme and their diseases Alkaptonuria Phenylketonuria Maple syrup disease Galactosomia uridyltransferasae Glycogen storage Fructosuria Gaucher disease Tay Sachs Wilson disease Acetalasaemia Xerderma pigmentosa homogenistate 1 oxidase phenylalanine 4 monooxygenase oxo acid decorboxylase galactose-1 phosphate glucose 6 phosphate fructokinase glucocerbrosidase β-NAcetykll –D-hexosaminidase . they are forced to extract it from body fluids. but reduce the free exogenous concentration and so induces a state of fatal starvation in the susceptible tumour cells. Acid phosphatase. Creatine kinase predominantly occurs in the skeletal muscle.

g. The main inhibitor is used allopurinol. Inhibition of HIV protease. serum creatine and non-enzymic method are still being used in clinical chemistry. Figure below showing structure of HIV protease. The second method is glucose oxidase is more commonly used. Other competitive inhibitors of HMGCo-A is namely mevinolin.P-type ATPase catalase DNA binding protein helicases Enzyme inhibitors and drug design Inhibition of hydroxymethylglutaryl Co-A reductase (HMGCo-A).1. hexokinase couple with glucose 6-phosphate dehydrogenase.1 L-Asparagine H2O L-aspartate + NH3 Leukaemia . The HIV1 protease catalyses this protein sequence.5.g. Figure 8. It resemble pepsin. glucose oxidase coupled with peroxidase. ________________________________________ Table 8. There are number of enzymatic advantage method estimate the metabolite in presence of many other substances an inhibitor present in serum may completely inhibit enzyme activity.The replication of HIV-1 is the cause of AIDS. Many inhibitors have been designed that are analogous like rennin.2 Some important therapeutic enzymes Enzyme EC number Reaction Use Asparaginase 3. and plasma glycerides enzyme methods are now used almost exclusively e. during hypercholesterolemia. Inhibition of xanthin oxidase. and other retroviral protease. compactin and monacol K which inhibits the enzyme. Blood glucose In the investigation of diabetes there are three enzymatic method that are available. Gout disease is characterize by uric acid that is end product of purine breakdown in humans derived from breakdown of nucleic acid. HIV protease is an aspartyl protease having a sequence Asp-thr-Gly at its active site. For the estimation of certain metabolite e. 1 HIV protease Use of enzyme in determination of concentration of metabolite of clinical importance. glucose.

they are antigenic and can elicit an immune response which may cause severe and life-threatening allergic reactions. Other methods have also been shown to be successful. in spite of their high cost. they often cause increased immunological response and additionally may cause blood clots.35 Hyaluronate hydrolysis Heart attack Lysozyme 3.c streptococcal cysteine proteinase d urate oxidase. In contrast to the industrial use of enzymes.2.6 Penicillin penicilloate Penicillin allergy Streptokinasec 3.3 Urate + O2 allantoin Gout Urokinasee 3. This has proved easier than the immunological problem to combat.1. pyrogens and other harmful materials within a therapeutic enzyme preparation is totally forbidden.SO32. has been shown to retain its anti-tumour effect whilst possessing no immunogenicity. as examples.4.2. this encourages the use of animal enzymes.31 Plasminogen plasmin Blood clots a Hyaluronoglucosaminidase.1. Unfortunately a number of factors severely reduce this potential utility: a) They are too large to be distributed simply within the body's cells.4. relative to those of microbial origin.3.1.Collagenase 3. by disguising the enzyme as an apparently nonproteinaceous molecule by covalent modification.4 RNA hydrolysis Antiviral β-Lactamase 3. Their effective lifetime within the circulation may be only a matter of minutes. particularly those involving entrapment of the enzyme within artificial liposome’s.24. Asparaginase.21.4 Protein hydrolysis Inflammation Uricased 1. although these methods are efficacious at extending the circulatory lifetime of the enzymes.+ SCNCyanide poisoning Ribonuclease 3.+ CN. However.e plasminogen activator ________________________________________ Disadvantages of Using Enzyme as Therapeutic Agents As enzymes are specific biological catalysts.1. synthetic microspheres and red blood cell ghosts. by disguise using covalent modification.3 Collagen hydrolysis Skin ulcers Glutaminase 3.7.4.on continued use.21. A number of methods are being developed in order to overcome this by targeting enzymes.1 S2O32. Clearly the presence of toxins. particularly . b thiosulphate sulfurtransferase. they should make the most desirable therapeutic agents for the treatment of metabolic diseases.1. therapeutically useful enzymes are required in relatively tiny amounts but at a very high degree of purity and .22.5. b) Being generally foreign proteins to the body. Effectively.26.8.4.17 Bacterial cell wall hydrolysis Antibiotic Rhodanaseb 2. in some cases.5. It has proved possible to circumvent this problem.2. enzymes with covalently attached external α-galactose residues are targeted at hepatocytes and enzymes covalently coupled to target-specific monoclonal antibodies are being used to avoid non-specific side-reactions.10 Plasminogen plasmin Blood clots Trypsin 3. modified by covalent attachment of polyethylene glycol. This is the major reason why enzymes have not yet been successful applied to the large number of human genetic diseases.2 L-Glutamine H2O L-glutamate + NH3 Leukaemia Hyaluronidasea 3.

INDUSTRIAL ENZYME PROCESS AT HIGH TEMPERATURE 8. e.food processing Alkaline proteases 40-60 Detergents Lipases 30-70 Detergents. brewing baking detergents Gluco amylase 50-60 Maltodextrin hydrolysis alpha amylase (fungal) 50-60 Maltose Pullulanase 50-60 high glucose syrups Xylose isomerase 45-55 High fructose syrups Pectinase 20-50 clarification of juices /wines Cellulose 45-55 cellulose hydrolysis Lactase 30-50 lactose hydrolysis . heavy metal leaching and bioconversion of crude oils. these extremozymes are extremely valuable tools for study of protein stability Thermozymes are used in the molecular biology experiments for example taq polymerase and in the detergents (e. T. food processing Enzyme used in Industries Thermozymes Thermozymes are thermostable enzymes that function optimally at 60 0C and 125 0C. amylases. isooctane. cyclooctane. see Table 4. alpha amylase. 50000 for the enzyme alone. This leads to production of acids. The cost of the enzyme is about Rs. . The costs of such enzymes may be quite high but still comparable to those of competing therapeutic agents or treatments. or cholesterol or can utilize sulphure compounds have been isolated. food processing Acid protease 30-50 food processing fungal protease 40-60 baking .4) is prepared from human urine (some genetically engineered preparations are being developed) and used to dissolve blood clots. The favoured kinetic properties of these enzymes are low Km and high Vmax in order to be maximally efficient even at very low enzyme and substrate concentrations.g.) proteases and starch processing (e. Strain Y-40 degrades hydrocarbons and belongs to Candida. with the cost of treatment in a case of lung embolism being about Rs. Therapeutic enzyme preparations are generally offered for sale as lyophilised pure preparations with only biocompatible buffering salts and mannitol diluent added. It can grow well in the presence of solvents such as n-octane. waste treatment and pulp and paper manufacture.g.g. Obligate extremophiles such as thiobacillus thiooxidans and thiobacillus ferrooxidanse occur in highly acidic environments. Thermostable enzymes such as DNA polymerase. Organic solvent tolerant bacteria that exhibit the ability to degrade crude oil. Thermozymes are more stable because they are active against denaturing condition Hyperthermophiles have good potential for use in novel biotechnological processes including oil coal and waste gas desulphurization. In spite of this.g. urokinase (a serine protease.3 Enzyme operating temperature 0C Major applications alpha amylase (bacterial) 90-100 starch hydroysis.brewing.(generally) specificity. glucsoe isomerases) various lipase and proteases oxidoreductase are used in the diagnostics. 5000 mg-1. polyaromatic hydrocarbons. Pseudomonas putida can grow in more than 50% toluene. ferroxidans reduces sulphure compounds as well as ferrous ion to ferric. Thus the sources of such enzymes are chosen with care to avoid any possibility of unwanted contamination by incompatible material and to enable ready purification. the market for the enzyme is worth about Rs 350M year-1. xylanases proteases and lipase are required in basic research and biotechnology. As an example. or kerosene at a concentration of 50 % (v/v) the first strain that isolated for organic solvent tolerant bacterium e.

details of the enzymes used and the ways in which they are used. Their action. Most are used by industry to produce improved or novel products. smell. to produce or process foodstuffs. Most such enzymic conversions benefit from the use of immobilised enzymes or biphasic systems The use of enzymes in detergents The use of enzymes in detergent formulations is now common in developed countries. The use of enzymes in the non-food (chemicals and pharmaceuticals) sector is relatively straightforward. are sold directly to the public. xylanases are produced by the alkalophiles Aeromonas strain 212 that can hydrolyze the beta 1. NaCI) and sugars as preservative. grass. Many extremophiles are used for craft industry for leather tanning (B. fibres of carboxymethyl cellulose or similar protective colloid. Although the cost of enzymes for use at the research scale is often very high. licheniformis) with the help of various alkalophiles. often after a preliminary period of soaking. T. Although a wide range of lipases is known. In spite of the fact that the detergent industry is the largest single market for enzymes at 25 . Products are generally separated and purified and. Many of the most useful.g. when substituting malt with unmlated barley to increase the amount of free amino acids. but least-understood. colour and texture. This was combatted by developing dust-free granulates (about 0. sweat. etc. textile desizing and detergent formulation. Neutral metalloproteases from B. The large-scale use of enzymes in solution Several enzymes. however apparently straightforward. have rarely been published.-4 linkage in the xylan polysaccharides in the crop plant. uses of free enzymes are in the food industry. This core is coated with inert waxy materials made from paraffin oil or polyethylene glycol plus various . subtilis and B. they are not prone to the subtleties available to food products. together with endogenous enzymes. where there is a clear large-scale need for an enzyme its relative cost reduces dramatically with increased production.30% of total sales. Early attempts to use proteases foundered because of producers and users developing hypersensitivity. is complicated due to the effect that small amounts of by-products or associated reaction products have on such subjective effects as taste. milk. amyloliquifaciens can be used in the brewing industry. Alkalophiles such as bacillus strains N-4 are used for cellulose digestion in the waste water treatment to digest cellulose. using modern bleaching and brightening agents.Currently these bacteria are utilized in the Biomining used in minerals leaching for example. However. with over half of all detergents presently available containing enzymes. containing inorganic salts (e. to bypass long and involved chemical synthetic pathways or for use in the separation and purification of isomeric mixtures. especially those used in starch processing. are now traded as commodity products on the world's markets. far more effectively than non-enzyme detergents. In general. notably those in detergents. which are only rarely substantially refined. Relatively few enzymes. therefore. The use of enzymes allows lower temperatures to be employed and shorter periods of agitation are needed. high-fructose syrup manufacture. ferroxidans used to isolate sulphure from the coal. the difference between looking clean and being clean may be difficult to discern. starches and lipids. clothes that have been starched must be freed of the starch. enzyme detergents remove protein from clothes soiled with blood. Dirt comes in many forms and includes proteins. Detergent enzymes must be cost-effective and safe to use.5 mm in diameter) in which the enzyme is incorporated into an inner core. bound with reinforcing. Using detergents in water at high temperatures and with vigorous mixing. it is only very recently that lipases suitable for use in detergent preparations have been described. They show good activity and stability at pH 9. In addition. At present only proteases and amylases are commonly used. Here they are used. it is possible to remove most types of dirt but the cost of heating the water is high and lengthy mixing or beating will shorten the life of clothing and other materials. meat tenderizers and garden composting agents.

respectively. Preparations containing both Termamyl and Alcalase are produced. Savinase and Esperase may be used at up to pH 11 and 12. water softener) 2. It may be replaced by sodium carbonate plus extra protease. used for removing stubborn stains. licheniformis. from an alkalophilic strain of B. loosens dirt) 1. It follows that the ability to withstand the conditions of use is a more important criterion than extreme cheapness.5 Sodium carboxymethyl cellulose (dirt-suspending agent) 1.0 Soap (sodium alkane carboxylates) 3. thermolysin) would lose their metal cofactors due to complexing with the water softening agents or hydroxyl ions. Novo Industri A/S produce and supply three proteases. from B. ________________________________________ Table 15. papain) would be oxidised by the bleaching agents. In addition to the granulated forms intended for use in detergent powders.6 Sodium metasilicate (binder. GistBrocades produce and supply Maxatase. They convert their substrates into small.0 Bacillus protease (3% active) 0. oxidants such as sodium perborate which generate hydrogen peroxide. Once released from its granulated form the enzyme must withstand anionic and non-ionic detergents. loosens dirt)a 38. subtilis).5. Alcalase.4 . optical brighteners and various lessreactive materials (Table 15. liquid preparations in solution in water and slurries of the enzyme in a non-ionic surfactant are available for formulating in liquid 'spotting' concentrates. only 0. giving preferred cleavage on the carboxyl side of hydrophobic amino acid residues but capable of hydrolysing most peptide links.4 Compositions of an enzyme detergent Constituent Composition (%) Sodium tripolyphosphate (water softener. all at pH values between 8. The -amylase supplied for detergent use is Termamyl.0 Sodium alkane sulphonate (surfactant) 25. from an alkalophilic strain of a B.g. the enzymes must retain activity up to 60°C. Although one effect of incorporating enzymes is that lower washing temperatures may be employed with consequent savings in energy consumption. licheniformis.g. The enzymes are supplied in forms (as described above) suitable for formulation by .5. which later disperse in the wash. licheniformis which is also used in the production of glucose syrups. -Amylase is particularly useful in dish-washing and de-starching detergents. This combination of materials both prevents dust formation and protects the enzymes against damage by other detergent components during storage.8% crude enzyme by weight (about 1% by cost). It should be noted that all the proteolytic enzymes described are fairly nonspecific serine endoproteases.hydrophilic binders.0 Sodium sulphate (filler. Only serine protease. licheniformis and Savinase. Termamyl being sufficiently resistant to proteolysis to retain activity for long enough to fulfil its function. Enzymes are used in surprisingly small amounts in most detergent preparations.3 Foam-controlling agents Trace Perfume Trace Water to 100% a A recent trend is to reduce this phosphate content for environmental reasons. ________________________________________ The enzymes used are all produced using species of Bacillus. soaps. the enzyme from B. Esperase. amyloliquefaciens (often mistakenly attributed to B. mainly by just two companies.8 Fluorescent brighteners 0.0 Sodium perborate tetrahydrate (oxidising agent)25. may be used in detergent formulations: thiol proteases (e.4).0. readily soluble fragments which can be removed easily from fabrics. Alcalase and Maxatase (both mainly subtilisin) are recommended for use at 10-65°C and pH 7-10. and metalloproteases (e. from B.0 and 10.

A third wash includes hypochlorite as bleach which would inactivate the enzymes rapidly. particularly. Proteolysis generally increases the solubility of proteins at their isoelectric points. the levels of peroxide at this stage being insufficient to inactivate the enzymes. Added peroxidases may aid the bleaching efficacy of this peroxide. from which water is run to waste. lipoxygenase and glycerol oxidase as means of generating hydrogen peroxide in situ. Industrial laundering requires effectiveness at minimum cost so heated water will be re-used if possible. then to a second wash. Home detergents will probably include both an amylase and a protease and a lengthy warm-water soaking time will be recommended. Apart from the pre-soak stage.and β- . 15. The cellulase also aids the removal of soil particles from the wash by hydrolyzing associated cellulose fibres. by then. and they may be highly specific in their choice of cleavable peptide links or quite non-specific. The water from these stages is discarded to the sewer. its hydrophobic N-terminal region associating with the lipophilic regions of the otherwise insoluble . Rennet (mainly chymosin). in the lowering of the brightness of colours. Thus white cotton uniforms from an abattoir can be segregated for washing. usually microbial. resulting in a change in the 'feel' of the fabric and. There are. These ancient discoveries have led to the development of various food applications for a wide range of available proteases from many sources. 15. small fibres are raised from the surface of cotton thread. Domestic users are familiar with powdered preparations but liquid preparations for home use are increasingly available. then to a bleaching stage and rinsing. This is essentially a batch process: hospital laundries may employ continuous washing machines. opportunities for enzymes such as glucose oxidase. The κ-casein acts by stabilizing the colloidal nature of the milk. There are opportunities to extend the use of enzymes in detergents both geographically and numerically. perhaps. the process operates countercurrently. During use. into the first wash at 60°C and pH 11. the hypochlorite concentration is insufficient to harm the enzyme. only protease being required.13 Rennet in cheese making The action of rennet in cheese making is an example of the hydrolysis of a specific peptide linkage. between phenylalanine and methionine residues (-Phe105Met106-) in the κ-casein protein present in milk . Treatment with cellulase removes the small fibres without apparently damaging the major fibres and restores the fabric to its 'as new' condition. Enzymes are used in the pre-wash and in the first wash. obtained from the fourth stomach (abomasum) of unweaned calves has been used traditionally in the production of cheese. They have not found widespread use in developing countries which are often hot and dusty. Similarly. The recent availability of a suitable lipase may increase the quantities of enzymes employed very significantly. Household laundering present’s problems quite different from those of industrial laundering: the household wash consists of a great variety of fabrics soiled with a range of materials and the user requires convenience and effectiveness with less consideration of the cost. Large laundries can separate their 'wash' into categories and thus minimise the usage of water and maximise the effectiveness of the detergents.5. The water from this stage is used again for the pre-wash but. Proteases may be used at various pH values.12 Applications of proteases in the food industry Certain proteases have been used in food processing for centuries and any record of the discovery of their activity has been lost in the mists of time. making frequent washing of clothes necessary.detergent manufacturers. papain from the leaves and unripe fruit of the pawpaw (Carica papaya) has been used to tenderize meats. which transfer less-initially-dirty linen from a pre-rinse initial stage. at 71°C and pH 11. containing hydrogen peroxide. A recent development in detergent enzymes has been the introduction of an alkaline-stable fungal cellulase preparation for use in washing cotton fabrics. A pre-wash soaking for 10-20 min at pH up to 11 and 30-40°C is followed by a main wash for 10-20 min at pH 11 and 60-65°C. at 32°C and pH 8.

the enzyme has been secreted in an active form only from the latter. Application of this knowledge allows the tailoring of the flavour of protein hydrolysates. usually non-specifically. increasing the amount of free butyric acid. consisting of mainly chymosin with a small but variable proportion of pepsin. 2 Outline method for the preparation of cheese. removes this protective effect. the bitterness being less intense with smaller peptides and disappearing altogether with larger peptides. using enzymes such as subtilisin.H2N-aa-aa-aa-COO. The development of unwanted bitterness in ripening cheese is an example of the role of proteases in flavour production in foodstuffs. peracids). resulting in the release of a hydrophilic glycosylated and phosphorylated oligopeptide (caseino macropeptide) and the hydrophobic para-κ-casein. reduces its thermostability by about 10°C and renders it more comparable with calf rennet. is a relatively expensive enzyme and various attempts have been made to find cheaper alternatives from microbial sources These have ultimately proved to be successful and microbial rennets are used for about 70% of US cheese and 33% of cheese production world-wide. Non-terminal hydrophobic amino acid residues in medium-sized oligopeptides give bitter flavours.1). further hydrolysis. the extent of hydrolysis (degree of hydrolysis) is described in DH units where: (15.1) Commercially. which convert methionine residues to their sulfoxides. H2O2.+ H2N-aa-aa-COO. At the high pH needed for effective use of subtilisin. as indeed occurs naturally in the production of soy sauce. little activity remains.5 DH greatly increases its 'whipping expansion'. DH values of up to 30 are produced using protein preparations of 8-12% (w/w).g. Partial hydrolysis of soya protein. The enzymes are formulated so that the value of the enzyme : substrate ratio used is 2-4% (w/w). Chymosin is a relatively unstable enzyme and once it has done its major job. to around 3.2] where aa is an amino acid residue. When proteases are used to depolymerise proteins.casein molecules. protons are released during the proteolysis and must be neutralised: subtilisin (pH 8. appetising flavours akin to that of monosodium glutamate. However. Correctly applied proteolysis of inexpensive materials such as soya protein can increase the range and value of their usage. which are then compressed and turned into cheese (Figure 4. Calf rennet. to around 6 DH . The action of endogenous proteases in meat after slaughter is complex but 'hanging' meat allows flavour to develop.5) H2N-aa-aa-aa-aa-aa-COO. ________________________________________ The major problem that had to be overcome in the development of the microbial rennets was temperature lability. This is a rare example of enzyme technology being used to destabilise an enzyme Attempts have been made to clone chymosin into Escherichia coli and Saccharomyces cerevisiae but. so far. Lipases from Mucor miehei or Aspergillus niger are sometimes used to give stronger flavours in Italian cheeses by a modest lipolysis. the enzyme from Mucor miehei retains activity during the maturation stages of cheese-making and produces bitter off-flavours. ________________________________________ Figure 15. in addition to tenderising it. Hydrolysis of the labile peptide linkage between these two domains. The coagulation process depends upon the presence of Ca2+ and is very temperature dependent (Q10 = 11) and so can be controlled easily. allowing coagulation of the milk to form curds.+ H+ [15. whilst its negatively charged C-terminal region associates with the water and prevents the casein micelles from growing too large. It has been found that peptides with terminal acidic amino acid residues give meaty. The presence of proteases during the ripening of cheeses is not totally undesirable and a protease from Bacillus amyloliquefaciens may be used to promote flavour production in cheddar cheese. They are added to the milk (30 U l-1) before the addition of the rennet. Treatment of the enzyme with oxidising agents (e.

which has significant effects on the physical properties of the starch but does not interfere with its hydrolysis. liver and kidneys that otherwise could be sold and. On slaughter. except in products such black puddings. they are not generally acceptable in foodstuffs because of their colour. physiological and even psychological (i. Subtilisin is added and hydrolysis is allowed to proceed batchwise. 15. being reasonably heat stable. The haem-containing precipitate is recycled and the light-brown supernatant is processed through activated carbon beads to remove any residual haem. may be spray-dried and is used in cured meats. for instance from potato.5 ppm of the inactive enzyme needs to be injected. Meat of older animals remains tough but can be tenderised by injecting inactive papain into the jugular vein of the live animals shortly before slaughter. Proteases are used to recover protein from parts of animals (and fish) would otherwise go to waste after butchering. Acid . when the hydrophobic haem molecules precipitate. 15.16 The use of enzymes in starch hydrolysis Starch is the commonest storage carbohydrate in plants. Weakgluten flour is required for biscuits in order that the dough can be spread thinly and retain decorative impressions. to around 18 DH. dough may be prepared more quickly if its gluten is partially hydrolysed. Chemical differences are less marked. with neutralization of the released protons. The purified hydrolysate. The major difference is the ratio of amylose to amylopectin. it has found disfavour as it destroys the animals heart.e. 15. The enzyme is inactivated by a brief heat treatment at 85°C and the product is centrifuged. A heat-labile fungal protease is used so that it is inactivated early in the subsequent baking. This is a very effective process as only 2 . no residual activity allowed into meat products. phenomenon of 'mouth feel' in soft drinks.15 Baking industry Proteases are also used in the baking industry. The gluten in the flour derived from these must be extensively degraded if such flour is to be used efficiently for making biscuits or for preventing shrinkage of commercial pie pastry away from their aluminium dishes. sausages and luncheon meats. obtained in 60% yield. the resultant reducing conditions cause free thiols to accumulate in the muscle. but valuable.improves its emulsifying capacity. In the past this has been obtained from European domestic wheat but this is being replaced by high-gluten varieties of wheat.g. If their flavours are correct. Excessive degradation is avoided to prevent the formation of bitter peptides. To recover this.2% approximately). Recently.14 Meat tenderization Meat tenderisation is the softening of the meat for uses in the food by the endogenous proteases in the muscle after slaughter is a complex process which varies with the nutritional. corn starch from waxy maize contains only 2% amylose but that from amylomaize is about 80% amylose. The meat slurry produced is used in canned meats and soups. soya protein hydrolysates may be added to cured meats. Some starches. bones are mashed incubated at 60°C with neutral or alkaline proteases for up to 4 h. Injection of the active enzyme would rapidly kill the animal in an unacceptably painful manner so the inactive oxidised disulfide form of the enzyme is used. activating the papain and so tenderising the meat. The protein is of a high quality nutritionally and is de-haemed using subtilisin. however. e. contain covalently bound phosphate in small amounts (0. Starch from all plant sources occurs in the form of granules which differ markedly in size and physical characteristics from species to species. by microbes and by higher organisms so there is a great diversity of enzymes able to catalyse its hydrolysis. It is used by the plants themselves. frightened or not) state of the animal at the time of slaughter. Large quantities of blood are available but. Hydrolysed proteins may develop properties that contribute to the elusive. its action is difficult to control and persists into the cooking process. About 5% of the meat can be removed mechanically from bone. Red cells are collected and haemolysed in water. Where appropriate.

It is now largely replaced by enzymic processes. gave rise to high colour and saltash content (after neutralisation). as it required the use of corrosion resistant materials. corn is the world's most abundant source and provides most of the substrate used in the preparation of starch hydrolysates. Although starches from diverse plants may be utilised. expression: (15.6glycosidic branch points which constitute about 4 . starch is usually the major component of reaction mixtures. liquefaction. a. DE indicates the extent to which the starch has been cleaved. The starch and glucose syrup industry uses the expression dextrose equivalent or DE. ________________________________________ Of the two components of starch. where: (15 . During starch hydrolysis. but not identical. and Processes in which it is necessary to eliminate starch. This is due to the residues involved in α-1.5 Enzymes used in starch hydrolysis . 3 The use of enzymes in processing starch. involving the dissolution of the nano-gram-sized starch granules to form a viscous suspension. ________________________________________ Table 15. Gelatinization is achieved by heating starch with water. involving the production of glucose and maltose by further hydrolysis. such as glucose syrup production. see equation (15.hydrolysis of starch has had widespread use in the past.6% of the glucose present. and Saccharification. Gelatinized starch is readily liquefied by partial hydrolysis with enzymes or acids and saccharified by further acidic or enzymic hydrolysis. Typical conditions are given. Most hydrolytic enzymes are specific for α-1. this is usually determined analytically by use of the closely related. such as the processing of sugar cane juice. Acid hydrolysis appears to be a totally random process which is not influenced by the presence of α-1. Further hydrolysis using acid is not satisfactory because of undesirably coloured and flavoured breakdown products. DE represents the percentage hydrolysis of the glycosidic linkages present. In the former processes.3): gelatinization. Enzymes of various types are used in these processes. Very considerable amounts of 42 DE syrups are produced using acid and are used in many applications in confectionery. Some of the most impressive recent exercises in the development of new enzymes have concerned debranching enzymes. similar in definition to the DH units of proteolysis. There are three stages in the conversion of starch (Figure 15. with concomitant loss in viscosity. pure maltose has a DE of about 50 (depending upon the analytical methods used.4-glucosidic links yet the -1. Pure glucose has a DE of 100. whereas in the latter processes. and occurs necessarily and naturally when starchy foods are cooked. It is necessary to hydrolyse starch in a wide variety of processes which may be condensed into two basic classes: Processes in which the starch hydrolysate is to be used by microbes or man.3) In practice. small amounts of starch which contaminate nonstarchy materials are removed.6glucosidic links must also be cleaved for complete hydrolysis of amylopectin to glucose. Acid hydrolysis of starch has long been used to produce 'glucose syrups' and even crystalline glucose (dextrose monohydrate). needed more energy for heating and was relatively difficult to control. ________________________________________ Figure 15.4)) and starch has a DE of effectively zero. to describe its products. amylopectine presents the great challenge to hydrolytic enzyme systems.4) Thus. involving the partial hydrolysis of the starch.6-glucosidic linkages.

from nonreducing ends. Similar processes may be used with B. containing 20-80 ppm Ca2+ (which stabilizes and activates the enzyme) and the enzyme is added (via a metering pump). than the B. . G3.3 A.4-oligosaccharide links are cleaved to give α-dextrins and predominantly maltose.4 oligosaccharide links are cleaved to give α-dextrins and predominantly maltose and G3 oligosaccharides Saccharifying α-amylase 3.2. G3. which is resistant to hydrolysis by glucoamylase and α-amylases. the products of bacterial and fungal -amylases are in the configuration and the products of β-amylases are in the β-configuration. These tanks contain baffles to discourage backmixing.4-linked glucose residues. Different approaches for starch liquefaction Various manufacturers use different approaches to starch liquefaction using αamylases but the principles are the same. One reason for the confusion in the nomenclature is the use of the anomeric form of the released reducing group in the product rather than that of the bond being hydrolysed.2 Malted barley Only α-1.1. in the presence of starch.1 Bacillus amyloliquefaciens Only α-1. combined with the significant shear forces.4-oligosaccharide links are cleaved to give α-dextrins and predominantly maltose (G2).5.0-6. subtilis (amylosacchariticus) Only α-1.5-0.1 B.1. Hydrolysis to the required DE is completed in holding tanks at 90-100°C for 1 to 2 h.2. G4 and up to 50% (w/w) glucose β-Amylase 3.Enzyme EC number Source Action α-Amylase 3. G4 and G5 oligosaccharides Aspergillus oryzae. Commercial enzymes used for the industrial hydrolysis of starch are produced by Bacillus amyloliquefaciens (supplied by various manufacturers) and by B. The partly gelatinized starch is passed into a series of holding tubes maintained at 100105°C and held for 5 min to complete the gelatinization process. G6 and G7 oligosaccharides B.4-α-D-glucan glucanohydrolases) are endohydrolases which cleave 1. the slurry of starch plus enzyme is pumped continuously through a jet cooker.2.1.1.4 and α1. licheniformis Only α1. although all these enzymes cleave between α-1. to give α-glucose Pullulanase 3. begins the hydrolysis. The αamylase is usually supplied at high activities so that the enzyme dose is 0.4-oligosaccharide links are cleaved to give α-dextrins with maltose. at pH 6. which is heated to 105°C using live steam. The maximum DE obtainable using bacterial α-amylases is around 40 but prolonged treatment leads to the formation of maltulose (4-α-D-glucopyranosyl-Dfructose). The residence time in the jet cooker is very brief. niger α-1.4-links are cleaved. When Termamyl is used. DE values of 8-12 are used in most commercial processes where further saccharification is to occur.2.5).41 B.6-links are cleaved to give straight-chain maltodextrins ________________________________________ The nomenclature of the enzymes used commercially for starch hydrolysis The nomenclature of the enzymes used commercially for starch hydrolysis is somewhat confusing and the EC numbers sometimes lump together enzymes with subtly different activities (Table 15. niger Only α-1. They differ principally in their tolerance of high temperatures. acidopullulyticus Only α-1.6 kg tonne-1 (about 1500 U kg-1 dry matter) of starch. amyloliquefaciens α-amylase. A. from the nonreducing ends. α-amylase may be subclassified as liquefying or saccharifying amylases but even this classification is inadequate to encompass all the enzymes that are used in commercial starch hydrolysis. Termamyl retaining more activity at up to 110°C. The α-amylases (1.6-α-D-glucosidic branch points.2. For example. Gelatinization occurs very rapidly and the enzymic activity.1.6-links are cleaved. licheniformis (supplied by Novo Industri A/S as Termamyl).4-α-D-glycosidic bonds and can bypass but cannot hydrolyze 1. to give limit dextrins and β-maltose Glucoamylase 3. The principal requirement for liquefaction to this extent is to reduce the viscosity of the gelatinised starch to ease subsequent processing. G3. Granular starch is slurried at 30-40% (w/w) with cold water.

They reduce the time between cutting and threshing of the wheat.5 and operates most effectively at 60°C.3-α-linked glucans.0. however.2% maltose and 0.5 .4-α-glucosidase (1. This has the drawback that a final 'cooking' stage must be introduced when the required DE has been attained in order to gelatinize the recalcitrant starch grains present in some types of starch which would otherwise cause cloudiness in solutions of the final product. this may be replaced later by some acid-stable -amylase which is normally present in the glucoamylase preparations.97% glucose. -----------400 U kg-1 A. Saccharification is normally conducted in vast stirred tanks. saccharification is most often conducted as a batch process. using various enzyme solutions.65 . 1. ________________________________________ Figure 15. ———200 U kg-1 Aspergillus niger glucoamylase. of course. It follows that the maximum DE attainable is 96 . to avoid retrogradation (the formation of intractable insoluble aggregates of amylose. which releases β-D-glucose from 1. Fungal α-amylase also finds use in the baking industry. niger glucoamylase. carefully liquefied starch at 8 -12 DE can be hydrolysed completely to produce a final glucoamylase reaction mixture with DE of 100 but.3. which previously was sufficient to allow a limited sprouting so increasing the amounts of endogenous enzymes. ________________________________________ The saccharification process takes about 72 h to complete but may. ••••••••• 200 U kg-1 A.2% (w/w) isomaltose (α-D-glucopyranosyl-(1.6)-D-glucose). denaturing rapidly during baking.amyloliquefaciens α-amylase but the maximum temperature of 95°C must not be exceeded.tonne-1 starch (200 U kg-1). The cost of concentrating the product by evaporation decreases that a substrate concentration of 30% is used. The cooling must b rapid. The % glucose formed from 30% (w/w) 12 DE maltodextrin. Saccharification The liquefied starch is usually saccharified but comparatively small amounts are spray-dried for sale as 'maltodextrins' to the food industry mainly for use as bulking agents and in baby food. It often needs to be added to bread-making flours to promote adequate gas production and starch modification during fermentation. 1 . so liquefied starch must be cooled and its pH adjusted before addition of the glucoamylase.80 litre enzyme preparation. The fungal enzymes are used rather than those from bacteria as their action is easier to control due to their relative heat lability. this can be achieved only at comparatively low substrate concentrations. The latter should give the most economical use of the enzyme.98 with syrup composition 95 . The alternatives are to meter the enzyme at a fixed ratio or to add the whole dose of enzyme at the commencement of the filling stage. At present these are produced using the exoamylase.4-α-.4-α-D-glucan glucohydrolase. The glucoamylase most often used is produced by Aspergillus niger strains. commonly called glucoamylase but also called amyloglucosidase and γ-amylase).6-α.0 . Any remaining bacterial -amylase will be inactivated when the pH is lowered. directly for the production of high-fructose syrups or for the production of crystalline glucose.and 1. which may take several hours to fill (and empty). glucan 1. When conditions are correct the glucoamylase is added.4. This has become necessary since the introduction of combine harvesters. niger glucoamylase plus 200 U kg-1 Bacillus acidopullulyticus pullulanase. In this case. The liquefied starch at 8 -12 DE is suitable for saccharification to produce syrups with DE values of from 45 to 98 or more. at 60°C and pH 4. in practice. so time will be wasted if the enzyme is added only when the reactors are full. This material is used after concentration. The greatest quantities produced are the syrups with DE values of about 97. This has a pH optimum of 4. usually at the dosage of 0. Whereas liquefaction is usually a continuous process. The relative improvement on the addition of pullulanase is even greater at higher substrate concentrations. 4. In theory. residual enzymic activity may be destroyed by lowering the pH towards the end of the heating period. the process that gives rise to the skin on custard). be .

especially as the combined action of β-amylase and pullulanase give almost quantitative yields of maltose. The pullulanase from Klebsiella aerogenes which has been available commercially to some time is unstable at temperatures over 45°C but the B. to about 90 DE eventually. syrups containing maltose as a major component have been produced by treating barley starch with barley β-amylase.99 DE and 95 . when a maximum DE has been attained. This is not done on a significant scale nowadays . 35 . 2 .3-α-links in tetrasaccharides are in the proportions 300 : 6 : 1). by heating to 85°C for 5 min.6-α and 1. because one molecule of water is taken up for each glycosidic bond hydrolysed (molecule of glucose produced). 10% maltotriose.98 DE) and higher substrate concentrations (30 . when the product is to be used to manufacture high-fructose syrups. which acts as an exo hydrolase on starch dextrins. In addition.6-α-linkages.7% (w/w) glucose) tend not to crystallise. It should be remembered that the dry substance concentration increases by about 11 % during saccharification. rather than 97 . produced by a strain of Bacillus acidopullulyticus.speeded up by the use of more enzyme.17 Production of syrups containing maltose Traditionally. which is a true endohydrolase. there is a saving in the cost of further processing. acidopullulyticus pullulanase is an excellent example of what can be done if sufficient commercial pull exists for a new enzyme. 30 . 1. It is necessary to stop the reaction.5).4-α-linkages (e. It might be expected that β-amylase would be used to produce maltose-rich syrups from corn starch. their breakdown is slow compared with that of 1. for industrial use such an enzyme must be compatible with glucoamylase. and isoamylase (EC.3).30%) may be treated. High conversion syrups (60 . even below 0°C and are relatively non-hygroscopic. The development of a suitable α-D-glucosidase. pH 4. in order to reduce the reversion.4-α.4-β-D-glucan maltohydrolases) are exohydrolases which release maltose from 1.70 DE. They are used for soft candy and in the baking. would be an equally useful step for industrial glucose production. acidopullulyticus enzymes can be used under the same conditions as the Aspergillus glucoamylase (60°C. The practical advantage of using pullulanase together with glucoamylase is that less glucoamylase need be used This does not in itself give any cost advantage but because less glucoamylase is used and fewer branched oligosaccharides accumulate toward the end of the saccharification. brewing and soft drinks industries. Further incubation will result in a fall in the DE. It is not surprising that more details of the screening procedures used are not readily available. Two types of debranching enzymes are available: pullulanase.37% maltose. Screening of new strains of bacteria for a novel enzyme of this type is a major undertaking. The development of the B.6-α-linkages.97% (w/w) glucose. They are used for the production of hard candy and frozen deserts. 15. the rates of hydrolyzing the 1. Novo Industri A/S have recently introduced a suitable pullulanase.40% dry solids rather than 25 .43% glucose.4-α-linked glucans but neither bypass nor hydrolyse 1. β-Amylases (1. 45-60% (w/w) maltose. all by weight) resist crystallisation above 4°C and are sweeter (Table 4. High-maltose syrups (40 .0-4.50 DE. 15% other oligosaccharides.68). the point at which isomaltose production becomes significant occurs at higher DE (Figure 4. Continuous saccharification is possible and practicable if at least six tanks are used in series. caused by the formation of isomaltose as accumulated glucose re-polymerises with the approach of thermodynamic equilibrium (Figure 15. Final step in syrup formation The saccharified syrup is filtered to remove fat and denatured protein released from the starch granules and may then be purified by passage through activated charcoal and ion-exchange resins.3).1. The extra cost of using pullulanase is recouped by savings in evaporation and glucoamylase costs.g. It follows that higher DE values and glucose contents can be achieved when pullulanase is use (98 .3.4).2. Although glucoamylase catalyses the hydrolysis of 1. It is clear that the use of a debranching enzyme would speed the overall saccharification process but.

2 Raffinose 0. solids) Sucrose 1.1 Aspartame 180 a HFCS.3 High-conversion syrup 65 DE 0.3) are produced from liquefied starch of around 11 DE at a concentration of 35% dry solids using fungal -amylase alone.0 and a reaction temperature not exceeding 55°C. less likely crystallise) than pure sucrose syrups. the glucose content of the final product will be higher than that of high-maltose syrups. fungal α-amylases. are not totally compatible. 15. The autolysate was added to the syrup (70% sucrose (w/w)) to be inverted . These may be tailored to customers' specifications by adjusting the activities of the two enzymes used but inevitably.e. when the required composition is reached.3 Glucose syrup 97 DE 0.2 Hydrolysed sucrose 1. however. fungal α-amylases requiring a pH of not less than 5. Although this enzyme is unusual in that it suffers from substrate inhibition at high sucrose levels ( > 20% (w/w)). high-fructose corn syrup. High-maltose syrups (see Figure 15. 5Traditionally. it is possible to use this in combination with fungal -High-conversion syrups are produced using combinations of fungal α-amylase and glucoamylase. completely or partially.7 Fructose 1.7 Maltose syrup 44 DE 0.3 Lactose 0.3 Galactose 0. Instead fungal β-amylases. not sufficiently temperature stable (although some thermostable β-amylases from species of Clostridium have recently been reported) and are easily inhibited by copper and other heavy metal ions. It is now possible to produce starch hydrolysates with any DE between 1 and 100 and with virtually any composition using combinations of bacterial αamylases.18 Enzymes in the sucrose industry The sucrose industry is a comparatively minor user of enzymes but provides few historically significant and instructive examples of enzyme technology The hydrolysis ('inversion') of sucrose.because presently available β-amylases are relatively expensive. Presently available enzymes. The most familiar 'Golden Syrup' produced by acid hydrolysis of one of the less pure streams from the cane sugar refinery but other types of syrup are produced using yeast (Saccharomyces cerevisiae) invertase. Saccharification occurs over 48 h.0 Glucose 0. The stability of glucoamylase necessitates stopping the reaction. as glucoamylase is employed. to glucose and fructose provides sweet syrups that are more stable (i.7 Maltose 0. by which time the fungal -amylase has lost its activity. this does not prevent its commercial use at even higher concentrations: Figure 15.0 HFCS (55% fructose) 1. Invertase was produced on site by autolysing yeast cells.7 Glucose syrup 11 DE <0. ________________________________________ Table 15.1 Glucose syrup 42 DE 0.6 The relative sweetness of food ingredients Food ingredient Relative sweetness (by weight. glucoamylase and pullulanase.5 HFCS (42% fructose)a 1. characterised by their ability to hydrolyse maltotriose (G3) rather than maltose (G2) are employed often in combination with glucoamylase.1 Hydrolysed lactose 0. by heating. Now that a good pullulanase is available.

The production of hydrolysates of a low molecular weight compound in essentially pure solution seems an obvious opportunity for the use of an immobilised enzyme. An enzyme sufficiently stable for prophylactic use would be required in order to benefit from economies of scale. Raffinose may be hydrolysed to galactose and sucrose by a fungal raffinase. 15. These enzymes are comparatively unstable of low activity against native lignocellulose and subject .5) from Leuconostoc mesenteroides on sucrose and found as a slime on damaged cane and beet tissue. This problem can be overcome by using the most thermostable -amylases (e. yet this is not done on a significant scale. probably because of the extreme simplicity of using the enzyme in solution and the basic conservatism of the sugar industry.19 Glucose from cellulose There is very much more cellulose available. Other problems involving dextran and raffinose required the development of new industrial enzymes. some commercial. pH 5. yet cellulose is not a significant source of pure glucose. Invertase finds another use in the production of confectionery with liquid or soft centres. A typical waste cellulolytic material contains less than half cellulose. The fundamental reason is that starch is produced in relatively pure forms by plants for use as an easily biodegradable energy and carbon store. At this level of enzyme. A Dextran is produced by the action of dextransucrase (EC 2. A combination of enzymes is needed to degrade this mixture.72 h at 50°C and pH 4.together with small amounts of xylene to prevent microbial growth. most of the remainder consisting of roughly equal quantities of lignin and pentosans. Other enzymes are used as aids to sugar production and refining by removing materials which inhibit crystallisation or cause high viscosity. Termamyl at about 5 U kg-1) which are entirely compatible with the high temperatures and pH values that prevail during the initial vacuum evaporation stage of sugar production. sugar cane contains significant amounts of starch. Dextran can produce extreme viscosity it process streams and even bring plant to a stop. In some parts of the world.3 ppm (w/w)) amounts of invertase. This produces plate-like or needle-like crystals which are not readily harvested by equipment designed for the approximately cubic crystals otherwise obtained. These are used (e. These centers are formulated using crystalline sucrose and tiny (about 100 U kg-1. is produced at low temperatures in sugar beet. over a period of days or weeks. Cellulose is structural and is purposefully combined and associated with lignin and pentosans. The enzyme and xylene were removed during the subsequent refining and evaporation. inversion of sucrose is very slow so the centre remains solid long enough for enrobing with chocolate to be completed. Because only small quantities are produced for use.1. Now. some technical. Raffinose. commercially produced invertase concentrates are employed. depending on the water content of the centre preparation.5. 1 h) only in times of crisis as they are not sufficiently resistant to thermal denaturation for long-term use and are inactive at high sucrose concentrations. which becomes viscous. The reasons for this are many. 55°C. Then. than starch. 0. 10 U kg-1 raw juice. as a potential source of glucose. dead trees take several years to decay even in tropical rainforests. Extreme dextran problems arc frequently solved by the use of fungal dextranases produced from Penicillium species. Inversion was complete in 48 . Both dextran and raffinose have the sucrose molecule as part of their structure and both inhibit sucrose crystal growth. Partially inverted syrups were (and still are) produced by blending totally inverted syrups with sucrose syrups. thus slowing filtration processes and making the solution hazy when the sucrose is dissolved. which consists of sucrose with α-galactose attached through its C-1 atom to the 6 position on the glucose residue.4. this enzyme is relatively expensive.g.5. sucrose hydrolysis occurs and the increase in solubility causes the centers to become soft or liquid.g. so as to resist biodegradation. especially when processing has been delayed in hot and humid climates.

Of the Thai. Commercial cellulase preparations from Trichoderma reesei consist of mixtures of the synergistic enzymes: cellulase (EC 3. are tolerant. although many cellulolytic enzymes exist and it is possible to convert pure cellulose to glucose using only enzymes. Granular starch is readily stirred in slurries containing 40% (w/v) solids and is easily solubilised but.21. Endo-1. quite possible that it may be used. They are used for the removal of relatively small concentrations of cellulose complexes which have been found to interfere in the processing of plant material in. which currently commands a price of over twice that of corn starch. Exo-cellobiohydrolase is product inhibited and additionally appears to be inactivated on binding to the surface of crystalline cellulose. Relatively pure cellulose is valuable in its own right. respectively.2.2.4-β-glucosidase (EC 3. and cellulose 1. Additionally. glucan 1. The enzymes might be improved by strain selection from the wild or by mutation but problems caused by the physical nature of cellulose are not so amenable to solution. With the increasing world shortage of pulp it cannot be seen realistically as an alternative source of glucose in the foreseeable future. Exo-1.7% (w/v) in milk and the whey (supernatant) left after the coagulation stage of cheese-making. for example. and exo-1. which is of little or no value as a by-product and is difficult an expensive to remove. to produce ethanol. and only very rarely some individuals suffer from inborn metabolic lactose intolerance or lactase deficiency.1. the young of all mammals clearly are able to digest milk but in most cases this ability reduces after weaning.to both substrate and product inhibition. as a paper pulp and chipboard raw material. the cost of this conversion is excessive.4-β-glucosidase is a product-inhibited enzyme.2.6). It should be noted that cellobiose is a nonfermentable sugar and must be hydrolysed by additional β-glucosidase (EC 3. even when pure.74).1.4-β-glucosidase and exo-cellobiohydrolase. 97%. 15.4-β-glucosidase. Its presence in milk makes it unsuitable for the majority of the world's adult population. particularly in those areas which have traditionally not had a dairy industry.4).4-β-cellobiosidase (EC 3. also called cellobiase for maximum process efficiency (Figure 15. Real lactose tolerance is confined mainly to peoples whose origins lie in Northern Europe or the Indian subcontinent and is due to 'lactase persistence'. Knowledge of enzyme systems capable of degrading lignocellulose is advancing rapidly but it is unlikely that lignocellulose will replace starch as a source of glucose syrups for food use. || represents inhibitory effects. fibrous cellulose forms immovable cakes at 10% solids and remains insoluble in all but the most exotic (and enzyme denaturing) solvents. however. It is. ________________________________________ Proper economic analysis reveals that cheap sources of cellulose prove to be generally more expensive as sources of glucose than apparently more expensive starch. ________________________________________ Figure 15. are reported to be lactose intolerant. It acts synergistically with both exo-1. an endo-1.2. Consequently. whereas 84% and 96% of the US White and Swedish populations. Chinese and Black American populations.1.91).4-β-glucanase is the rate-controlling activity and may consist of a mixture of enzymes acting on cellulose of different degrees of crystallinity. an exo-cellobiohydrolase (see Figure 15. 90% and 73% respectively.6). the utilisation of the glucose by the yeast removing its inhibitory effect on the enzymes.4-D β-glucanase. in a process involving the simultaneous use of both enzymes and fermentative yeasts. 6 Outline of the relationship between the enzyme activities in the hydrolysis of cellulose. both of which may be noticed at birth.20 The use of lactases in the dairy industry Lactose is present at concentrations of about 4.1. . Impure cellulose often contains almost an equal mass of lignin. the brewing and fruit juice industries.

0-4. at high lactose and galactose concentrations. Such treatment also has the additional benefits of reducing the solution viscosity. once the stabilising effect of the pectins on the colloidal haze has been removed. Figure 15.15). Insoluble plant material is easily removed by filtration.1.g. a necessary stage before the polygalacturonase can act fully (the increase in the methanol content of such treated juice is generally less than the natural . 15. with pH optima (pH 4. increasing the volume of juice produced (e. wine. Adding to this problem. These problems may be overcome by hydrolysis of the lactose in whey. after homogenisation. 7 Lactose may be hydrolysed by lactase.5-6. the yield of juice from white grapes can be raised by 15%). subtle but generally beneficial changes in the flavour and.21 Enzymes in the fruit juice. Lactases are now used in the production of ice cream and sweetened flavoured and condensed milks. it may be prepared from the dairy yeast Kluyveromyces fragilis (K. When added to milk or liquid whey (2000 U kg-1) and left for about a day at 5°C about 50% of the lactose is hydrolysed. 20°C. with varying degrees of methyl esterification. 20 U kg-1. respectively) more suited to whey hydrolysis. however. They are associated with other plant polymers and. lactase shows significant transferase ability and produces β-1. marxianus).2.4-anhydrogalacturonic acid polymers. It also improves the 'scoopability' and creaminess of the product.The need for low-lactose milk is particularly important in food-aid programmes as severe tissue dehydration. responsible for the random hydrolysis of 1.1. Commercially.6-linked galactosyl oligosaccharides.0 and 3. with a pH optimum (pH 6. This prevents the use of concentrated whey syrups in many food processes as they have a unpleasant sandy texture and are readily prone to microbiological spoilage.4.5-7. much more soluble and capable of forming concentrated. marxianus var. the product being about four times as sweet (see Table 15.2.11).6). shorter fermentation times. or settling and decantation. These consist primarily of α1. a β-galactosidase.D-galactosiduronic linkages. In addition. 1 month of storage). with the cell debris. pectinesterase (EC 3. brewing and distilling industries One of the major problems in the preparation of fruit juices and wine is cloudiness due primarily to the presence of pectins. diarrhoea and even death may result from feeding lactose containing milk to lactose-intolerant children and adults suffering from protein-calorie malnutrition. in the case of wine-making. These enzymes are subject to varying degrees of product inhibition by galactose. Generally. In all these cases. niger. Commercial pectolytic enzyme preparations are produced from Aspergillus niger and consist of a synergistic mixture of enzymes: polygalacturonase (EC 3. Another problem presented by lactose is its low solubility resulting in crystal formation at concentrations above 11 % (w/v) (4°C). microbiologically secure. the disposal of such waste whey is expensive (often punitively so) due to its high biological oxygen demand. giving a sweeter product which will not crystallise if condensed or frozen. syrups (70% (w/v)).0) suitable for the treatment of milk. The cloudiness that they cause is difficult to remove except by enzymic hydrolysis. lactase usage has not reached its full potential. This method enables otherwise-wasted whey to replace some or all of the skim milk powder used in traditional ice cream recipes. as present enzymes are relatively expensive and can only be used at low temperatures. which releases methanol from the pectyl methyl esters. hydrolysis of the lactose to glucose and galactose would prevent the (severe) digestive problems.0. Smaller amounts of lactase may be added to long-life sterilised milk to produce a relatively inexpensive lactose-reduced product (e.g. or from the fungi Aspergillus oryzae or A.

3-βxylosidase. EC 3. corn or rye). In brewing. saccharification continues throughout the fermentation period. by an elimination reaction releasing oligosaccharides with non-reducing terminal 4-deoxymethyl-α-D-galact-4enuronosyl residues.concentrations and poses no health risk). strictly not a pectinase but its adventitious presence is encouraged in order to reduce hemicellulose levels. after use.g. breakdown of barley β-1. where this is used the wort must be boiled for a much longer period (e. which cleaves the pectin. particularly in the production of high-gravity beer. These require a higher degree of saccharification at lower starch concentrations to reduce the alcohol and total solids contents of the beer. Papain is used in the later post-fermentation stages of beer-making to prevent the occurrence of protein. Cellulases are also occasionally used. for maximum economic return extra enzymes are added to achieve their rapid saccharification. For this reason the saccharification process is stopped. whiskey from barley malt. which catalyses the oxidation of β-glucose to glucono-1. Although malt enzyme may also be used to hydrolyse these adjuncts.2. have become more popular.2. 'light' beers. Scotch malt whisky manufacture uses barley malt exclusively) the enzymes are naturally present but in others (e. barley malt supplies the major proportion of the enzyme needed for saccharification prior to fermentation. xylan 1. The enzymes used in brewing are needed for saccharification of starch (bacterial and fungal α-amylases).1. after about 75% of the starch has been converted into fermentable sugar. 15. A great variety of carbohydrate sources are used world wide to produce distilled alcoholic drinks.g. Recently. by boiling the 'wort'. They are suitable for direct addition to the fruit pulps at levels around 20 U l-1 (net activity). In the distilling industry. In some cases (e.2. EC 3. grain spirits production) the more heat-stable bacterial -amylases may be used in the saccharification.g. and α-Larabinofuranosidase.10). without the necessity of pectin methyl esterase action. EC 3.4. Many of these contain sufficient quantities of fermentable sugar (e. particularly where wheat is used as adjunct but also to help breakdown the barley β-glucans.1. of lower calorific content.22 Glucose oxidase and catalase in the food industry Glucose oxidase is a highly specific enzyme. and hemicellulase (a mixture of hydrolytic enzymes including: xylan endo-1. Often other starch containing material (adjuncts) are used to increase the fermentable sugar and reduce the relative costs of the fermentation. Hydrogen peroxide is an effective bacteriocide and may be removed.1.37.2. Enzymes with improved characteristics of greater heat stability and lower pH optimum are currently being sought.and β-1. rum from molasses and brandy from grapes). amyloliquefaciens α-amylase. from the fungi Aspergillus niger and Penicillium.55). pectin lyase (EC 4.32. It finds uses in the removal of either glucose or oxygen from foodstuffs in order to improve their storage capability.5-lactone (which spontaneously hydrolyses non-enzymically to gluconic acid) using molecular oxygen and releasing hydrogen peroxide. others contain mainly starch and must be saccharified before use (e.and tannin-containing 'chillhaze' otherwise formed on cooling the beer.g.2.linked glucan (βglucanase) and hydrolysis of protein (neutral protease) to increase the (later) fermentation rate. The optimal activity of these enzymes is at a pH between 4 and 5 and generally below 50°C. This may be achieved by the use of glucoamylase and/or fungal -amylase during the fermentation.3.g.4-β-xylosidase.4] . where extra protein is added. It not necessary nor desirable to saccharify the starch totally. by treatment with catalase (derived from the same fungal fermentations as the glucose oxidase) which converts it to water and molecular oxygen: catalase 2H2O2 2H2O + O2 [4. as non-fermentable dextrins are needed to give the drink 'body' and stabilise its foam 'head'. 30 min) to inactivate it prior to fermentation. Due to the extreme heat stability of the B.

the kinetics of the reaction and the chemical and physical properties of an immobilization support including whether it is particulate. in addition to the more obvious costs concerned with building and running the enzyme reactor. labour. and its density. depreciation.e. There are several important factors that determine the choice of reactor for a particular process. or series of vessels. overheads and process development. downstream processing. free or immobilised). Other contributing factors are the form of the enzyme of choice (i. robustness. This must be inclusive of the costs associated with substrate(s). Chapter-16 Enzyme Reactors Introduction An enzyme reactor consists of a vessel. Glucose oxidase and catalase may be used together when net hydrogen peroxide production is to be avoided. Other uses are in the removal of oxygen from the head-space above bottled and canned drinks and reducing non-enzymic browning in wines and mayonnaises. In general. substrate and product. used to perform a desired conversion by enzymic means. the possible need for pH and temperature control.For most large-scale applications the two enzymic activities are not separated. A mixture of the enzymes is used (165 U kg-1) together with additional hydrogen peroxide (about 0. to oxidise the glucose. membranous or fibrous. A major application of the glucose oxidase/catalase system is in the removal of glucose from egg-white before drying for use in the baking industry. the choice depends on the cost of a predetermined productivity within the product's specifications. particle size and regenerability. Attention must also be paid to the scale of operation. These factors will be . the supply and removal of gases and the stability of the enzyme. by catalase action.1 % (w/w)) to ensure that sufficient molecular oxygen is available. compressibility.

a. b. Note that a batch reactor is one in which all of the product is removed.e. and may be difficult to scale-up. This procedure is not only expensive in itself but means that there are considerable periods when such reactors are not productive. ________________________________________ Figure 16. STRs can be used for processes involving non-immobilised enzymes. where the enzyme is held within membrane tubes which allow the substrate to diffuse in and the product to diffuse out. The operating costs of batch reactors are higher than for continuous processes due to the necessity for the reactors to be emptied and refilled both regularly and often. fed -batch operation). after a fixed time.discussed in more detail with respect to the different types of reactor. Primary amongst these is their simplicity both in use and in process development. STR). fluidised bed reactor (FBR). (c). (d) . continuous flow stirred tank reactor (CSTR) which is a continuously operated version of (a). All reactors would additionally have heating/cooling coils (interior in reactors (a). due to the changing power requirements for efficient fixing. although in some circumstances there may be a need for further additions of enzyme and/or substrate (i. Generally this means that the enzyme and substrate molecules must have identical residence times within the reactor. containing a settled bed of immobilised enzyme particles. and exterior. and conditions (e. (e) and (f)) and the stirred reactors may contain baffles in order to increase (reactors (a). however. in reactors (b). continuous flow stirred tank reactor (CSTR. which contains all of the enzyme and substrates) until the conversion is complete.g. This reactor may often be used in a semicontinuous manner. 2. using the same enzyme solution for several batches. Batch reactors also suffer from pronounced batch-to-batch variations. Continuous reactor Stirred tank batch reactor (STR). Packed bed reactor (PBR). pH. generally. where the flow of gas and/or substrate keeps the immobilised enzyme particles in a fluidised state. They offer a closely controllable environment that is useful for slow reactions. where the composition may be accurately monitored. For this reason they are preferred for small-scale production of highly priced products. Advantage of using Batch Bioreactor They do. Disadvantage of using batch reactor 1. it also makes uneven demands on both labour and services. coenzyme concentrations) varied throughout the reaction. as rapidly as is practically possible. Batch membrane reactor (MR). continuous flow membrane reactor (CMR) fluidised bed reactor (FBR) 16. Batch Reactor. as the reaction conditions change with time. have a number of advantageous features. 3. and (d). The tank is normally fitted with fixed baffles that improve the stirring efficiency. There are two type of Reactor 1. packed bed reactor (PBR). 2. 1 Diagrams of various important enzyme reactor types. especially where the same equipment is to be used for a number of different conversions. also called plug -flow reactor (PFR). continuous flow membrane reactor (CMR) which is a continuously operated version of (b). (b).1 Batch Reactor Batch reactors generally consist of a tank containing a stirrer (stirred tank reactor. temperature. Stirred tank batch reactor (STR). if the consequences of these contaminating the product are not severe. batch membrane reactor (MR). They are also of use when continuous operation of a process proves to be difficult due to the viscous or intractable nature of the reaction mix.

The rate of reaction (v) may be expressed in terms of the volume of substrate solution within the reactor (VolS) and the time (t): (16. The normalised time (i.e. Figure 16. of the product stream is mixed with the incoming substrate stream. P may be considered as the relative position within a PBR or the reactor's absolute length. A cheap example of such a membrane is the dialysis membrane used for . [S]0/Km = 10.5) The change in fractional conversion and concentrations of substrate and product with time in a batch reactor is shown in Figure 16.3) Let the fractional conversion be X. The reaction S P is assumed with the initial condition. where: (16. the actual time for complete conversion being longer due to the reduction in the substrate concentration at the reaction progresses. with the initial condition [S]0/Km = 10. when I° = 1) that contains sufficient enzyme to convert all the substrate at the given flow rate if the enzyme acted at its maximum velocity throughout.2 Membrane reactors The main requirement for a membrane reactor (MR) is a semipermeable membrane which allows the free passage of the product molecules but contains the enzyme molecules.4b) Also (16. All reactors may use immobilised enzymes. where Vmax is the maximum velocity for unit reactor length and I is the reactor length) is relative to the length (i. I° = lVmax/F.4c) and (5. assuming the validity of the non -reversible Michaelis -Menten reaction scheme with no diffusional control. (a) The change in substrate and product concentrations with time. ________________________________________ 16. if semipermeable membranes are used on their outlets) may be used with the soluble enzyme.2(a).e.4b) in (5. The dashed line also indicates the variation of the fractional conversion (X) with t°. t° = t Vmax/[S]0) is relative to the time (t° = 1) that would be required to convert all the substrate if the enzyme acted at Vmax throughout. in a batch reactor. The reaction S P is assumed. (b) and (e) (plus reactors (d) and (f). The normalised reactor length (i. In addition. inhibition or denaturation. (b) The change in substrate and product concentrations with reactor length for a PBR. (16. 2 This figure shows two related behaviours.1) Therefore: (16. The concentrations of substrate (———) and product (-----------) are both normalised with respect to [S]". or most. reactors (a). ________________________________________ c. The concentrations of substrate (——— and product (-----------) are both normalised with respect to [S]0.3): (16.4a) and (16. The continuous reactors ((c) -(f)) may all be used in a recycle mode where some. The expected productivity of a batch reactor The expected productivity of a batch reactor may be calculated by. the actual reactor length necessary for complete conversion being longer due to the reduction in the substrate concentration as the reaction progresses.and (e) or decrease (reactor (f)) the stirring efficiency.e.4c) Therefore substituting using (5.4) Therefore.2) On integrating using the boundary condition that [S] = [S]0 at time (t) = 0: (16.

This pressure drop is. a configuration that imposes a severe restriction on the flow rate through the reactor. Deviations from these models occur primarily in configurations where the substrate stream is on the side of the membrane opposite to the enzyme and the reaction is severely limited by its diffusion through the membrane and the products' diffusion in the reverse direction.[5] They allow the easy replacement of the enzyme in processes involving particularly labile enzymes and can also be used for biphasic reactions. Achievement of high enough Re may. given the assumption. The usual choice for a membrane reactor is a hollow-fibre reactor consisting of a preformed module containing hundreds of thin tubular fibres each having a diameter of about 200 m and a membrane thickness of about 50  m. in batch mode. If the substrate is able to diffuse through the membrane. therefore. substrate activation and reaction reversibility. Advantage of using Membrane reactor [1] Membrane reactors may be used in either batch or continuous mode and [2] they allow the easy separation of the enzyme from the product. 16.6) In order to produce ideal plug -flow within PBRs. with respect to its length. in continuous mode (see later). Ideally. that there are no diffusion limitations: (16. proportional to the time spent within the reactor. All material present at any given reactor cross -section has had an identical residence time. they are also called plug flow reactors (PFR) because material flows through the reactor as a plug. the linear flow . they are often used for production on a small scale (g to kg).removing low molecular weight species from protein preparations. avoiding the costs and problems associated with other methods of immobilization and some of the diffusion limitations of immobilized enzymes. all of the substrate stream flows at the same velocity. if used in continuous mode. The longitudinal position within the PBR is. Any required degree of reaction may be achieved by use of an idea PBR of suitable length. Therefore equation (16. all other factors being equal. The major disadvantage of these reactors concerns the cost of the membranes and their need to be replaced at regular intervals. however. in turn.5) may be converted to represent an ideal PBR. parallel to the reactor axis with no back -mixing. The conversion efficiency of a PBR. it may be introduced to either side of the membrane with respect to the enzyme. especially where a multi-enzyme pathway or coenzyme regeneration is needed. At low Re the flow rate is proportional to the pressure drop across the PBR. for processes involving product inhibition. not often realised in practice. Consequent upon the plug -flow characteristic of the PBR is that the substrate concentration is maximised. generally found to be proportional to the bed height. 16. behaves in a manner similar to that of a well -stirred batch reactor with respect to its reaction time (Figure 5.[4] Due to the ease with which membrane reactor systems may be established. [3] They are normally used with soluble enzymes. as this causes improved mixing and heat transfer normal to the flow and reduced axial back-mixing.2(b)) Each volume element behaves as a batch reactor as it passes through the PBR. or the CSTR. all product emerging with the same residence time and all substrate molecule having an equal opportunity for reaction. relative to the final conversion at every point within the reactor.4 Packed bed reactors (PBR) The most important characteristic of a PBR is that. and the product concentration minimised. This means that PBRs are the preferred reactors. a turbulent flow regime is preferred to laminar flow. be difficult due to unacceptably high feed rates. The flow rate (F) is equivalent to VolS/t for a batch reactor. otherwise it must be within the same compartment as the enzyme.3 The kinetics of membrane reactors The kinetics of membrane reactors are similar to those of the batch STR. Under these circumstances the reaction may be even more severely affected by product inhibition or the limitations of reversibility than is indicated by these models. the effectiveness factor being high on entry to the reactor and low close to the exit.

which may contain colloidal or insoluble substrates. In general PBRs are used with fairly rigid immobilised-enzyme catalysts (1 -3 mm diameter). there is total back-mixing and the product stream is identical with the liquid phase within the reactor and invariant with respect to time. If the reactor is behaving in an ideal manner. The substrate stream is continuously pumped into the reactor at the same time as the product stream is removed. versatile and cheap reactor. and a maximisation of the product concentration.5 Continuous flow stirred tank reactors (CSTIR) This reactor consists of a well -stirred tank containing the enzyme. particle deformation and restricted flow may eventually result in no flow at all through the PBR. relative . which involves stagnant areas with negligible flow rate. a problem that may be particularly noticeable in wide reactors (> 15 cm diameter). i. so long as the insoluble particles are not able to sweep the immobilised enzyme from the reactor. The design of PBRs does not allow for control of pH. because excessive increases in this flow rate may distort compressible or physically weak particles.e. 16. However. The mechanical nature of the stirring limits the supports for the immobilised enzymes to materials which do not easily disintegrate to give 'fines' which may enter the product stream. back-mixing may result in the kinetic behaviour of the reactor approximating to that of the CSTR (see below). This. however. and the consequent difficulty in achieving a high degree of conversion. the fraction of the PBR volume taken up by the liquid phase). which allows simple catalyst charging and replacement. if they are sufficiently dense to stay within the reactor. causing increased pressure drop. In an extreme case. and hold-up. the resulting product streams having a distribution of residence times. An ideal CSTR has complete back -mixing resulting in a minimisation of the substrate concentration. Some molecules of substrate may be removed rapidly from the reactor. has the advantage that there is very little resistance to the flow of the substrate stream. The distribution of residence times for molecules in the substrate stream is shown in Figure 16. It is necessary to use a guard bed if plugging of the reactor by small particles is more rapid than the biocatalysts' deactivation. by addition of acids or bases. fairly small particle (down to about 10 m diameter) may be used. which is normally immobilised. whereas others may remain for substantial periods. The use of a uniformly sized catalyst in a reactor with an upwardly flowing substrate stream reduces the chance and severity of non-ideal behavior. PBRs behave as deep-bed filters with respect to the substrate stream.rate and dynamic viscosity of the substrate stream and (1 . but inversely proportional to the cross-sectional area of the immobilised enzyme pellets. This is the example of continous reactor. CSTRs tend to be rather large as the: need to be efficiently mixed. Particle deformation results in reduced catalytic surface area of particles contacting the substrate-containing solution. Its well -mixed nature permits straightforward control over the temperature and pH of the reaction and the supply or removal of gases.2 Advantages The CSTR is an easily constructed. These deviations are caused by channeling. or for easy temperature control where there is excessive heat output. poor external mass transfer characteristics and a restriction to the flow. Channels may form in the reactor bed due to excessive pressure drop. They are also easily fouled by colloidal or precipitating material. irregular packing or uneven application of the substrate stream. Deviations from ideal plug-flow are due to back-mixing within the reactors. A vicious circle of increased back-pressure. causing flow rate differences across the bed. where some substrate passes through the reactor more rapidly. Their volumes are usually about five to ten time the volume of the contained immobilised enzyme.ε)2/ε3 (where ε is the porosity of the reactor. This minimises problems due to diffusional resistance.

Hence: (16.which obeys the relationship .4 and 16. Under continuous operating conditions (operating time > 4V/F). It can be seen that there is little difference between the two reactors at faster flow rates and lower conversions.10) This equation should be compared with that for the PBR (equation (16. They are also useful where the substrate stream contains an enzyme inhibitor. higher [S]0/Km giving the higher curves).9) Therefore: (16. the mean residence time within the reactor is V/F. The relative number of molecules resident within the reactor for a particular time N. It should be noted that 100% of the molecules in an equivalent ideal PBR might be expected to have residence times equal to their mean residence time.5).5.4. it may be noted from the graph that only a few molecules have a residence time close to this value (only 7% between 0. Deviations from ideal CSTR behaviour occur when there is a less effective mixing regime and may generally be overcome by increasing the stirrer speed.7) Therefore: (16. represents a 90% conversion.1 V/F or greater than 2. the average residence time of the product being greater than that for the substrate. at every point within the reactor the effectiveness factor being uniform throughout. Together these equations can be used for comparing the productivities of the two reactors (Figures 16.6)). The residence time distribution of non -reacting media molecules ( ----------.3V/F.1 and 0. Thus.to the final conversion. decreasing the solution viscosity or biocatalyst concentration or by more effective reactor baffling. t F/V. 3Figure 5. for processes involving substrate inhibition or product activation. Comparison of the changes in fractional conversion with flow rate between the PBR (———) and CSTR (-----------) at different values of [S]0/Km (10. ________________________________________ .1. The flow rate is normalised with respect to the reactor's volumetric enzyme content ( = FKm/Vmax. 4Figure 5.1 V/F) whereas 20% of the molecules have residence times of less than 0. However. This effect is most noticeable if the inhibitor concentration is greater than the inhibition constant and [S]0/Km is low for competitive inhibition or high for uncompetitive inhibition. This composition may be calculated from the relative areas under the curves and. and substrate molecules that have long residence times are converted into product.9V/F and 1. as it is diluted within the reactor. is plotted against the normalised residence time (i. product (——— ) and substrate (•••••••••) are shown. where V is the reactor volume. and F is the flow rate. ________________________________________ Figure 16.4): (16.e. CSTRs are the preferred reactors. The residence time distribution of a CSTR. Kinetics of CSTR The rate of reaction within a CSTR can be derived from a simple mass balance to be the flow rate (F) times the difference in substrate concentration between the reactor inlet and outlet. giving a half-life for remaining in the reactor of . when the inhibitor dilution has more effect than the substrate dilution. in this case.8) from equation (5. especially at high values of [S]0/Km. where [M] is the concentration of media molecules. it is the time relative to that required for one reactor volume to pass through the reactor). The composition of the product stream is identical with that of the liquid phase within the reactor. The reaction S P is assumed. ________________________________________ Figure 16. everything else being equal.

substrate inhibited [S]0/KS = 10. product inhibited KP/Km = 0.19) where kd is the first -order inactivation constant (i. (curve e) [S]0/Km = 1. If the flow rate is unchanged then the 'normalised residence time' may be thought of as the reactor volume needed to produce the required degree of conversion. kd1.8): a. necessary to achieve various degrees of conversion for a range of process conditions.8) of these reactors with time can be compared using these equations.17) These equations may be used to compare the size of PBR and CSTR necessary to achieve the same conversion under various conditions (Figure 16. Combined with equation (5. product inhibited KP/Km = 0. the following relationships are obtained on integration: PBR (16.7.18) CSTR (16. Comparison of the ratio.15) and (5. 1 and 0.g.13) Product -inhibited CSTR (16. or (5.6) for PBR.01.7) and fractional conversion (Figure 16.Figure 16. in equation (1. ________________________________________ Figure 16. (curve b) [S]0/Km = 1. (curve c) [S]0/Km < 0.15) Reversible reaction in a CSTR (16. substrate inhibited [S]0/KS = 10.16) is the fractional conversion for a reversible reaction. (16. The productivity Another useful parameter for comparing these reactors is the productivity. (curve g) [S]0/Km = 100.11) Substrate -inhibited CSTR (16.85) and (1.12) b. using .16) X in equations (5. The residence time is the reciprocal of the normalised flow rate (see Figure 5.14) Reversible reaction in a PBR (16. These reactors may be operated for considerably longer periods than that determined by the inactivation of their contained immobilised enzyme. This can be derived for each reactor assuming a first-order inactivation of the enzyme (equation (1.68).e. Comparison of the changes in fractional conversion with residence time between the PBR (———) and CSTR (-----------) at different values of [S]0/Km (10. The actual size of the CSTR will be five to ten times greater than indicated due to the necessity of maintaining stirring within the vessel.1 .6. (curve a) [S]0/Km = 100. Substrate -inhibited PBR (16. (1. This is independent of any enzyme stabilisation and is simply due to such reactors initially containing large amounts of redundant enzyme.8). The size of a CSTR becomes prohibitively large at high conversions (e. Product -inhibited PBR (16.1. 5 Figure 5. of the enzyme content in a CSTR to that in a PBR.25)) and the fractional conversion subscripts refer to time = 0 or t. 6Figure 5. •••••••••••• product inhibited.96) rather than (1. The change in productivity (Figure 16. higher [S]0/Km values giving the lower curves).6). ________________________________________ Equations describing the behaviour of CSTRs and PBRs utilising reversible reactions or undergoing product or substrate inhibition can be derived in a similar manner. (curve f) [S]0/Km = 1. particularly if they are capable of high conversion at low substrate concentrations (Figure 16. --------substrate inhibited. ———Uninhibited reaction.5).26)). using equations (1.01.10) for CSTR. (curve d) [S]0/Km = 1.

•-•-•-•-•-•-•-• PBR [S]0/Km = 100.2 -I.. there is little or no back -pressure to increased flow rate through the CSTR.0 cm s-1) as most immobilisedenzyme particles have densities close to that of the bulk liquid.2).99 at a substrate feed concentration of [S]0/Km = 0. shape. [S]o/Km = 0. 8Figure 5. CSTRs are not generally used in processes involving high conversions but a chain of CSTRs may approach the PBR performance.6 times greater for a CSTR than a PBR. At high flow rates and low reactor diameters almost ideal plug -flow characteristics may be achieved. The time is given in terms of the half -life of the free enzyme ( ). ————CSTR. there is little further energetic input needed to increase the flow rate of the substrate stream through the reactor (Figure 16. This chain may be a number (greater than three) of reactors connected in series or a single vessel divided into compartments. but yields only 2. but this increases to 18 times for 99% conversion. assuming initial fractional conversion (X0) of 0. The initial stability in the fractional conversion over a considerable period of time. the kinetic performance of the FBR normally lies between that of the PBR and the CSTR. They consist of a bed of immobilised enzyme which is fluidised by the rapid upwards flow of the substrate stream alone or in combination with a gas or secondary liquid stream. either of which may be inert or contain material relevant to the reaction. when the process may be made continuous.99 and [S]0/Km = 100. 5. including concentration polarization or inactivation of the enzyme on the membrane but may be preferable in order to achieve a combined reaction and separation process or where a suitable immobilised enzyme is not readily available. There is a minimum fluidisation velocity needed to achieve bed expansion. The change in productivity of a PBR (---------) and CSTR (———) with time. This causes a number of process difficulties. ________________________________________ Figure 16.9. assuming an initial fractional conversion (X0) = 0. ________________________________________ Figure 16.80. ________________________________________ In general. A gas stream is usually preferred as it does not dilute the product stream. once fluidised.. Although the CSTR maintains its fractional conversion for a longer period than the PBR. it should be noted that the CSTR contains 1.9 times more enzyme.2 times more product. porosity and density of the particles and the density and viscosity of the liquid. This minimum fluidisation velocity is generally fairly low (about 0.8.. The change in fractional conversion of PBRs and CSTRs with time. The units of time are half -lives of the free enzyme ( ) and the productivity is given in terms of (FXt1/2). 7Figure 5.. In this case the relative bed expansion is proportional to the superficial gas velocity and inversely proportional to the square root of the reactor diameter. However. •••••••••••• PBR [S]0/Km = 0. due to the enzyme redundancy. as the small fluid linear velocities allowed by most biocatalytic particles causes a degree of back- . The difference between the two types of reactor is increased if the effectiveness factor (η) is less than one due to diffusional effects..CSTR. Although the overall productivity is 1. It should be noted that a CSTR capable of X0 = 0. Fluidised bed reactors These reactors generally behave in a manner intermediate between CSTRs and PBRs.curve b. particularly at high X0.01.01 contains 22 times more enzyme than an equivalent PBR.01. which depends upon the size. Such reactors may be started up as batch reactors until the required degree of conversion is reached. should not be confused with any effect due to stabilisation of the immobilised enzyme. [S]0/Km = 100.99 or 0. a CSTR contains three times the enzyme in a PBR to achieve a 90% conversion. . in order to minimise back-mixing CSTRs may be used with soluble rather than immobilised enzyme if an ultrafiltration membrane is used to separate the reactor output stream from the reactor contents. Fluidising the bed requires a large power input but.

PBRs allow scale-up factors of greater than 50000 but. Cloned gene is expressed in the expression vector to obtain the 3-D structure of the enzyme. Established industrial enzymes are being used in as wide a variety of ways as can be conceived. one can do random mutagenesis and/or directed evolution.9). The FBR is normally used with fairly small immobilised enzyme particles (20-40 m diameter) in order to achieve a high catalytic surface area. for example. relative to the substrate stream. novel enzymes are being designed and produce by genetic engineering. For efficient operation the particles should be of nearly uniform size otherwise a non-uniform biocatalytic concentration gradient will be formed up the reactor. FBRs are chosen when these intermediate characteristics are required. resolved by Xray crystallography. the cofactor requirement (NADPH to NADH) or imparting novel properties to a protein (eg. and pH). FBRs are usually tapered outwards at the exit to allow for a wide range of flow rates. temperature. In the absence of detailed 3-D structure. relatively few enzymes are available on a large scale (i. adding a Mn binding site into horse heart myoglobin at UBC). The actual design of the FBR will determine whether it behaves in a manner that is closer to that of a PBR or CSTR (see Figures 5. The major disadvantage of development of FBR process is the difficulty in scalingup these reactors. There are many directions in which enzyme technologists are currently applying their art and which are at the forefront of biotechnological research and development. > kg) and are suitable for industrial applications. if it is baffled in such a way that substantial backmixing is avoided. that they are not swept out of the reactor. These shortcomings are being addressed in a number of ways: New enzymes are being sought in the natural environment and by strain selection.g.5 -5.e. FBRs can only be scaled-up by a factor of 10 -100 each time. Chapter-17 Protein Engineering Introduction Protein engineering is the branch of science in which novel enzymes are being designed and produce by genetic engineering methods to improve the stability of an enzyme (stability with respect to chemical oxidation. Very high flow rates are avoided as they cause channelling and catalyst loss. although never total. e. be made to behave in a manner very similar to that of a PBR. Less-dense particles must be somewhat larger. It can. changes in the flow rate of the substrate stream causes complex changes in the flow pattern within these reactors that may have consequent unexpected effects upon the conversion rate.In the genetic engineering methods the availability of a cloned gene is important. where a high conversion is needed but the substrate stream is colloidal or the reaction produces a substantial pH change or heat output. They are particularly useful if the reaction involves the utilisation or release of gaseous material. In addition. At present.mixing that is often substantial. because of the markedly different fluidisation characteristics of different sized reactors. . Therefore protein engineering includes two parts one large scale production of genes by modification in the gene by gene cloning methods and other modification in the structure of protein to improve the substrate specificity. These particles must be sufficiently dense.

The development of genetically improved enzymes is generally undertaken by molecular biologists and the design and synthesis of novel enzyme-like catalysts is done by the organic chemists. R. This principle has been used to increase the activity of penicillin-G-amidase in Escherichia coli. The cellular DNA from a producing strain is selectively cleaved by the restriction endonuclease Hind III. coli.E. Such colonies are isolated and the penicillin-G-amidase gene transferred on to pBR322 plasmids and recloned back into E. The DNA having gene of interest for specific enzyme can be isolated by partial digestion of gene or complete digestion of gene (incomplete or complete fragmentation of gene) and selection of specific gene can be done with the help of probe.New organic catalysts are being designed and synthesized. Figure 17. More complex enzyme systems are being utilised. In the future. Space requirements in this volume do not allow the full treatment of these related areas but will be discussed briefly here. Note that single stranded DNA is rarely cut by the Restriction enzyme. This hydrolyses the DNA at relatively rare sites containing the 5'-AAGCTT-3' base sequence to give identical 'staggered' ends. The product can be tested by its specific function. These fragments are individual cloned into a cosmid vector and thereby returned to E. Intact DNA cleaved DNA Figure 17. only one of which contains the required genetic information. These colonies containing the active gene are identified by their inhibition of a 6-amino-penicillanic acidsensitive organism. for example. making glucose isomerase less susceptible to inhibition by the Ca2+ present in the starch saccharification processing stream. coli. There are a number of properties which may be improved or altered by genetic engineering for example. produce penicillin-G-amidase constitutively and in considerably higher quantities than does the fully induced parental strain. The engineered cells. If the vector is expression type then large amount of protein will be obtained after the translation of vector. 17. 2 showing structure of vector and their site containing antibiotic resistance gene as the marker of the gene for selection of recombinant gene. Both groups of workers will. 17. base their science on data provided by the enzyme technologist. Such increased yields are economically relevant not just for the increased volumetric productivity but also because of .1 Enzyme engineering A most exciting development over the last few years is the application of genetic engineering techniques to enzyme technology. enzymes may be redesigned to fit more appropriately into industrial processes. 1 figure showing staggered cut of DNA double strand by the restriction endonuclease. however. the ease of downstream processing and various safety aspects. Enzymes from dangerous or unapproved microorganisms and from slow growing or limited plant or animal tissue may be cloned into safe high-production microorganisms. the yield and kinetics of the enzyme. aided by the plasmid amplification at around 50 copies per cell. The total DNA is cleaved into about 10000 fragments.2 Enzyme production can be increased by cloning of gene The amount of enzyme produced by a microorganism may be increased by increasing the number of gene copies that code for it by inserting a portion of gene for specific enzyme into the suitable vector.

This information is analyzed together with the database of known and putative structural effects of amino acid substitutions to produce a possible improved structure. successful or unsuccessful. 4 showing nature of cutting of gene one sticky end Figure 17. Another extremely promising area of genetic engineering is protein engineering. This factitious enzyme is constructed by site-directed mutagenesis.reduced downstream processing costs. 6 showing complete procedure of gene cloning. An outline of the process of protein engineering is shown in Figure 17. the resulting crude enzyme being that much purer. Apparently quite small sequence changes may give rise to large conformational alterations and even affect the rate-determining step in the enzymic catalysis. New enzyme structures may be designed and produced in order to improve on existing enzymes or create new activities. (NCBI) and growing rapidly together with the necessary software. the relative probability of success will increase over the coming years and the products of protein engineering will make a major impact on enzyme technology. the principal enzyme in the detergent enzyme preparation.1.2). Unfortunately from a practical point of view. The process starts with the isolation and characterization of the required enzyme. However it is reasonable to suppose that.2 Application of Enzyme Engineering 17. . ________________________________________ Protein engineering. Figure 17.1 Subtilisin Much protein engineering has been directed at subtilisin (from Bacillus amyloliquefaciens). This emphasis is likely to change in the future. it is presently insufficient accurately to predict three-dimensional changes as a result of such substitutions. are added to the database. The main problem is assessing the long-range effects. and the process repeated until the required result is obtained. including solvent interactions. isolated and characterised. 3 showing structure and location of plasmid DNA inside the E. therefore. As the many reported results would attest. much of the research effort in protein engineering has gone into studies concerning the structure and activity of enzymes chosen for their theoretical importance or ease of preparation rather than industrial relevance. Although a large database of sequence-structure correlations is available. is presently rather a hit or miss process which may be used with only little realistic likelihood of immediate success. 5 showing selection of recombinant by use antibiotic As indicated by the method used for site-directed mutagenesis . the preferred pathway for creating new enzymes is by the stepwise substitution of only one or two amino acid residues out of the total protein structure. the science is at a stage where it can explain the structural consequences of amino acid substitutions after they have been determined but cannot accurately predict them. on the new structure. Such factitious enzymes are produced by site-directed mutagenesis (Figure 17. Figure 17. Figure 17. 7 The protein engineering cycle.coli vector.2. Figure 17. given a sufficiently detailed database plus suitable software. The results. 17.

however. These effects are achieved mainly by corresponding changes in the Km rather than the Vmax. 3. giving a slightly more hydrophobic core. by positively charged lysine gives the predictable result of abolishing the activity against its normal substrates but unpredictably also gives no activity against substrates where these basic residues are replaced by aspartic acid or glutamic acid. Substitution of this methionine by serine or alanine produces mutants that are relatively stable. an internal salt link or extra internal hydrophobic residues. which has an active site closely related to that of subtilisin. All of these changes are small enough to be achieved by protein engineering. the consequential increase in volume occluding the oxyanion hole. The mutant DNA may be identified by hybridisation with radioactively labelled oligonucleotides of complementary structure. possessing a large hydrophobic binding site which may be made more specific relatively easily (e. 8 Figure 17. the secondary structure of the enzyme must be conserved and this . using a hypothetical example. which has a general base role.the methionine residue (Met222) which causes subtilisin's lability to oxidation. 2. as native subtilisin is unusual in being fairly non-specific in its actions.g. Most of the attempted improvements have concerned alterations to: The P1 cleft. which stabilises the tetrahedral intermediate. ________________________________________ Trypsin An example of the unpredictable nature of protein engineering is given by trypsin. To ensure a more predictable outcome. complementary to a section of the gene apart from the base mismatch. A putative enzyme primary structure is proposed with an asparagine residue replacing the serine present in the native enzyme. particularly for the glycine residue at the bottom of the P1 cleft (Gly166). They should not be thought of as the usual result of engineering enzymes. which is used for binding the basic side-chains of lysine or arginine. The oxyanion hole (principally Asn155). by reducing its size). have been found to increase the specificity of the enzyme for particular peptide links whilst reducing it for others. (b) The oligonucleotide primer is annealed to a single-stranded copy of the gene and is extended with enzymes and nucleotide triphosphates to give a double-stranded gene. This has been aimed at the improvement of its activity in detergents by stabilising it at even higher temperatures. A short piece of DNA (the primer). ________________________________________ Figure 17. Thermophilic enzymes Considerable effort has been spent on engineering more thermophilic enzymes. On reproduction. the neighborhood of the catalytic histidyl residue (His64). although possessing somewhat reduced activity. is synthesised. Substitution of the negatively charged aspartic acid residue at the bottom of its P1 cleft (Asp189). pH and oxidant strength. It has been found that thermophilic enzymes are generally only 20-30 kJ more stable than their mesophilic counterparts. Increases in relative specificity may be useful for some applications. the gene gives rise to both mutant and wild-type clones. and 4.Alcalase. 9 An outline of the process of site-directed mutagenesis. (a) The primary structure of the enzyme is derived from the DNA sequence. The inactivation of subtilisin in bleaching solutions coincides with the conversion of Met222 to its sulfoxide. This may be achieved by the addition of just a few extra hydrogen bonds. It has been found that the effect of a substitution in the P1 cleft on the relative specific activity between substrates may be fairly accurately predicted even though predictions of the absolute effects of such changes are less successful. which holds the amino acid on the carbonyl side of the targeted peptide bond. Many substitutions.

although covalently immobilised enzymes are not considered here.19) from Bacillus macerans). seven.2]) but also showed stereoselectivity for the L-amino acids. Cyclodextrins (Schardinger dextrins) are naturally occurring toroidal molecules consisting of six. shows synzymic properties. however. often called synzymes. The synzyme is almost as effective as natural ascorbate oxidases. being sensitive to denaturation. asparagine <aspartate and lysine < arginine.generally restricts changes in the exterior surface of the enzyme.5-1 nm) but all are about 0. oxidation and hydrolysis.4. It should be recognized that making an enzyme more thermostable reduces its overall flexibility and. a substratebinding site and a catalytically effective site. isoleucine < threonine. It was not as active as natural transaminases. Whenever there is small increases in the interior hydrophobicity. being water soluble. Synzymic cyclodextrins are usually derivatised in order to introduce catalytically relevant groups. Their overall characteristic is hydrophilic.3. All the C-6 hydroxyl groups project to one end and all the C-2 and C-3 hydroxyl groups to the other. glutamine. Such substitutions have a fair probability of success. Both sites may be designed separately but it appears that. This converts it from an oxygen carrier to an oxidase. a C-6 hydroxyl group of β-cyclodextrin was covalently derivatised by an activated pyridoxal coenzyme. in spite of the many charges and apolar side-chains. and Michaelis-Menten kinetics with a Km of 2 mm and Vmax of 10-4 to 10-5 s-1 for the hydrolysis of 4-nitrophenyl acetate. by attaching (Ru(NH3)5)3+ to three surface histidine residues. They must possess two structural entities. it is probable that the factitious enzyme produced will have reduced catalytic efficiency. hence. These are generally synthetic polymers or oligomers with enzyme-like activities. For example. but the presence of their hydrophobic pocket enables them to bind hydrophobic molecules of the appropriate size. Many such derivatives have been examined. The resulting synzyme not only acted a transaminase (see reaction scheme [1. with optimum activity at about pH 5. Suitable for exterior substitutions for increasing thermostability have been found to be aspartate < glutamate lysine. this often achieves both functions. Polyglutamic acid. Such proteins will also show the drawbacks of natural enzymes. These form hydrophobic pockets due to the glycosidic oxygen atoms and inwards-facing C-H groups. An example is the derivatisation of myoglobin. respectively: they may be synthesized from starch by the cyclomaltodextrin glucanotransferase (EC 2. It acts as an esterase in much the same fashion as the acid proteases.5] Some synzymes are simply derivatised proteins. Polyethylenimine is formed by polymerising ethyleneimine to give a highly branched .7 nm deep. valine < threonine. Synzymes generally obey the saturation Michaelis-Menten kinetics. as their conformations are not presently predictable from their primary structure. if the synzyme has a binding site for the reaction transition state. serine < asparagine. eight. They differ in the diameter of their cavities (about 0. nine or ten -1. showing a bell-shaped pH-activity relationship. serum albumin. oxidising ascorbic acid whilst reducing molecular oxygen. 4-linked D-glucose units joined head-to-tail in a ring and β-cyclodextrins. however. polylysine binds anionic dyes but only 10% as strongly as the natural binding protein. For example.1. For a one-substrate reaction the reaction sequence is given by synzyme + S (synzyme-S complex) synzyme + P [17. It has been found that producing the facility for substrate binding is relatively straightforward but catalytic sites are somewhat more difficult. Artificial enzymes Synzymes A number of possibilities now exist for the construction of artificial enzymes. Disadvantage of synzymes It is impossible to design protein synzymes from scratch with any probability of success. the oxygen carrier in muscle. for example by substituting interior glycine or serine residues by alanine may also increase the thermostability.

hydrophilic three-dimensional matrix. There are several advantages of using GMOs for the production of enzymes. although still 1000-fold higher than hydrolysis by background hydroxyl ions. 50% secondary and 25% tertiary: [17. proteases: subtilisin (detergent enzyme). phosphonate esters of general formula (R-PO2-OR'). rennet (cheese-processing). As might be expected from its apparently random structure. A similar strategy may be used to produce synzymes by molecular 'imprinting' of polymers. R and R' in (R-PO2-OR')-) of the antigens. The process takes place under well controlled conditions in closed fermentation tank. . Other synzymes may be created in a similar manner. The Km values may be quite low. pharmaceutically important enzymes: urokinase (plasminogen activator) is a protease for the treatment of thrombosis and embolism (Präve et al. which then produces the desired enzyme. Genetic engineering can be used to improve production and purification of recombinant enzymes. If 40% of them are alkylated with 1-iodododecane to give hydrophobic binding sites and the remainder alkylated with 4(5)-chloromethylimidazole to give general acid-base catalytic sites. 1987). After fermentation the enzyme is separated from the production strain. Due to higher production efficiency there is an additional environmental benefit through reducing energy consumption and waste from the production plants For enzymes used in the food industry particular benefits are for example a better use of raw materials (juice industry). The specificities of these catalytic antibodies (also called abzymes) depends on the structure of the side-chains (i.are stable analogues of the transition state occurring in carboxylic ester hydrolysis. carbohydrases: amylases (starch hydrolysis). About 25% of the resultant amines are primary. glucose isomerase (HFCS) C. occupational health or environmental reasons. the resultant synzyme has 27% of the activity of α-chymotrypsin against 4-nitrophenyl esters.6] Ethyleneimine polyethyleneimine The primary amines may be alkylated to form a number of derivatives. cellulases (bioconversions by Stake Technology). 17. It is possible to produce enzymes with a higher specificity and purity. purified and mixed with inert diluents for stabilisation. whereas the Vmax values are low (below 1 s-1).3 Advantages of genetic engineering Many enzymes are now produced by fermentation of genetically modified microrganisms (GMOs). including.e. The enzymes are produced by fermentation of the genetically modified microrganisms (the production strain). For example. ligninases and Mn peroxidases: pulp processing. often in the micromolar region. xylanases (bioconversions and biopulping). using the presence of transition state analogues to shape polymerising resins or inactive non-enzymic protein during heat denaturation. it has very low esterase specificity. eg. Antibodies to transition state analogues of the required reaction may act as synzymes. The Lilly Research Labs hold the patent on the production of urokinase from a continuous line of porcine kidney cells D. better keeping quality of a final food and thereby less wastage of food (baking industry) and a reduced use of chemicals in the production process (starch industry). Monoclonal antibodies raised to immunizing protein conjugates covalently attached to these phosphonate esters act as esterases. It is possible to obtain enzymes which would otherwise not be available for economical. For enzymes used in the feed industry particular benefits include a significant reduction in the amount of phosphorus released to the environment from farming. B. targeting enzymes for extracellular secretion to allow for their easy isolation and purification. Examples of industrially useful enzymes include: A.

48) assay for galactose which involves the oxidation of galactose by the redox coenzyme. For the reaction to be linear with respect to the galactose concentration. When the biological component of biosensor is a component of the immune system then it is referred to as Immunosensors. restriction endonucleases: used extensively in research. these rates of reaction (v) are proportional to the substrate concentrations ([S]) at low substrate concentrations.1. the galactose is kept within a concentration range well below the Km of the enzyme for galactose. in a 1 cm path-length cuvette. 19. For routine or on-site operation. Such assays are commonly used in analytical laboratories and are. but it should be recognised that other biological systems may be utilised by biosensors. Chapter-18 Biosensors and Immunosensors Introduction A biosensor is an analytical device which converts a biological response into an electrical signal (Figure 19. these disadvantages must be overcome. ligand binding and the antibody-antigen reaction. In contrast. The emphasis of this Chapter concerns enzymes as the biologically responsive material. The term 'biosensor' is often used to cover sensor devices used in order to determine the concentration of substances. spanning biochemistry.1] A 0. equation from Michaelis velocity constant simplifies to give v = (Vmax/Km)[S] The rates of reaction are commonly determined from the difference in optical absorbance between the reactants and products. The drawbacks to this type of analysis become apparent when a large number of repetitive assays need to be performed. accompanied by a large increase in absorption of light at this wavelength. nicotine-adenine dinucleotide (NAD+). time consuming. whole cell metabolism.1.2 A successful biosensor must possess at least some of the following beneficial features . therefore. 19. for example. of 0. β-D-galactose + NAD+ D-galactono-1.1).1 Km.4-lactone + NADH + H+ [19. indeed. selectivity and efficiency. Then. Clark and Lyons first demonstrated the modern concept of biosensors. they are seen to be costly in terms of expensive enzyme and coenzyme usage.622. the NAD+ concentration is kept within a concentration range well above the Km of the enzyme for NAD+. physical chemistry.1 mM solution of NADH has an absorbance at 340nm. in order to avoid limiting the reaction rate. They are often used to determine the concentration of their substrates (as analytes) by means of the resultant initial reaction rates. Research and development in this field is wide and multidisciplinary. An example of this is the β-Dgalactose dehydrogenase (EC 1.E. electrochemistry. When [S] < 0. The conversion (NAD+ NADH) is. electronics and software engineering. in which an enzyme was immobilized into an electrode to form a biosensor. labour intensive and in need of skilled and reproducible operation within properly equipped analytical laboratories. This is being achieved by the production of biosensors which exploit biological systems in association with advances in micro-electronic technology.1 The use of enzymes in analysis Enzymes make excellent analytical reagents due to their specificity. excellent where a wide variety of analyses need to be undertaken on a relatively small number of samples. If the reaction conditions and enzyme concentrations are kept constant. bioreactor science. whereas the NAD+ from which it is derived has effectively zero absorbance at this wavelength. Most of this current endeavour concerns potentiometric and amperometric biosensors and colorimetric paper enzyme strips.

The biocatalyst must be highly specific for the purpose of the analyses, be stable under normal storage conditions and, except in the case of colorimetric enzyme strips and dipsticks (see later), show good stability over a large number of assays (i.e. much greater than 100). The reaction should be as independent of such physical parameters as stirring, pH and temperature as is manageable. This would allow the analysis of samples with minimal pre-treatment. If the reaction involves cofactors or coenzymes these should, preferably, also be co-immobilised with the enzyme. The response should be accurate, precise, reproducible and linear over the useful analytical range, without dilution or concentration. It should also be free from electrical noise. If the biosensor is to be used for invasive monitoring in clinical situations, the probe must be tiny and biocompatible, having no toxic or antigenic effects. If it is to be used in fermentors it should be sterilisable. This is preferably performed by autoclaving but no biosensor enzymes can presently withstand such drastic wet-heat treatment. In either case, the biosensor should not be prone to fouling or proteolysis. The complete biosensor should be cheap, small, portable and capable of being used by semi-skilled operators. There should be a market for the biosensor. There is clearly little purpose developing a biosensor if other factors (e.g. government subsidies, the continued employment of skilled analysts, or poor customer perception) encourage the use of traditional methods and discourage the decentralisation of laboratory testing. 19.2.2 Some of the notable characteristics of biosensor Specificity: Biosensor has got the remarkable ability to distinguish between the analyte of interest and other similar substances. With biosensor, an analyte can be detected with great accuracy . Response time: Analytic tracers or catalytic product can be detected directly and instantaneously with the help of biosensors. Thus they are very much suitable for on-line monitoring. Simplicity: The sensing molecule and the transducer are integrated on to a single probe. Thus handling of biosensors is very easy. Continuous monitoring ability: Biosensors can be regenerated and reused. Thus they are suitable for continuous monitoringJ purposes which can never be done with conventional analysis methods. Reproducibility: Biosensors are very reliable; reproducibility of the values is also a great advantage. Portability: Portability of such sensors adds to the advantages which can never be the case with a spectrophotometer. Cost: The cost of the biosensor has too some extend limited its application. 19.3 Working of Biosensor Mechanism of Biosensor ________________________________________ Figure 19. 1 Schematic diagram showing the main components of a biosensor. The biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer (b) which converts it to an electrical signal. The output from the transducer is amplified (c), processed (d) and displayed (e). ________________________________________ A biosensor is a device that detects, transmits and records information regarding a physiological or biochemical change. Technically, it is a probe that integrates a biological component with an electronic transducer thereby converting a biochemical signal into a quantifiable electrical response. The sensor works by converting the signal produced by the biological sensing element on response to a specific analyte to a measurable electrical signal with the help of the transducer. The amplifier increases the intensity of the signal so that it can be

readily measured. The digital display then displays the reading in a suitable unit. All these components are generally integrated onto a single probe to make the handling easier. Biosensor is device that on matching with appropriate biological sample gives signal to electrical components. Biological component is bound or immobilized on the electronic component called as transducer. Figure 19. 2 The electrical signal from the transducer is often low and superimposed upon a relatively high and noisy (i.e. containing a high frequency signal component of an apparently random nature, due to electrical interference or generated within the electronic components of the transducer) baseline. The signal processing normally involves subtracting a 'reference' baseline signal, derived from a similar transducer without any biocatalytic membrane, from the sample signal, amplifying the resultant signal difference and electronically filtering (smoothing) out the unwanted signal noise. The relatively slow nature of the biosensor response considerably eases the problem of electrical noise filtration. The analogue signal produced at this stage may be output directly but is usually converted to a digital signal and passed to a microprocessor stage where the data is processed, converted to concentration units and output to a display device or data store. 19.4 Classification of Biosensor There are several type of Biosensor depends on the physical characteristic like heat , electrical signal . light, electron ,charge, and mass. Biosensors may be classified according to several criteria, such as transducers, bioactive components, or immobilization techniques used. The heat output (or absorbed) by the reaction (calorimetric biosensors), changes in the distribution of charges causing an electrical potential to be produced (potentiometric biosensors), movement of electrons produced in a redox reaction (amperometric biosensors), light output during the reaction or a light absorbance difference between the reactants and products (optical biosensors), or Effects due to the mass of the reactants or products (piezo-electric biosensors). There are three so-called 'generations' of biosensors First generation biosensors where the normal product of the reaction diffuses to the transducer and causes the electrical response. Second generation biosensors which involve specific 'mediators' between the reaction and the transducer in order to generate improved response. Third generation biosensors where the reaction itself causes the response and no product or mediator diffusion is directly involved. A typical biosensor has got two main parts Biological component which can be enzyme, antibody, nucleic acid, microorganism, tissue, cell, etc. Electronic device, i.e., transducer which can be electrochemical, optical, piezoelectric, thermal, etc. The biological component is generally immobilized near or onto the transducer surface in order to facilitate reuse, to minimize interference and to maximize response. In each case it is the ability of the biological component to react or respond specifically to an analyte (or a group of analytes) that makes the biomolecule suitable for use as the sensing element of a biosensor. For example, an antibody will only bind to the specific antigen under suitable conditions. The biological signal that is produced is converted by means of a suitable transducer into a quantifiable electrical signal.Biosensors are suitable for detecting a wide variety of analytes including pollutants, explosives, viruses, biochemical & pharmaceutical products, vitamins, amino acids, heavy metals, ions, gases etc . In fact, every single analyte, be it simple or complex can be detected provided a biomolecule that response specifically to it is identified. 1. Transducers

This is the component of biosensor which converts the biological signal to a quantifiable electrical signal. Electrochemical: In this configuration, sensing molecules are either coated onto or covalently bonded to a probe surface. A membrane holds the sensing molecule in place, excluding interfering species from the analyte solution. The sensing molecule reacts specifically with compounds to be detected, sparking an electrical signal proportional to the concentration of the analyte. The most common detection method for electrochemical biosensors involves measurement of current, voltage, capacitance, conductance and impedance. Among such sensors, amperometric (e.g. Oxygen or hydrogen peroxide) and potentiometric (e.g. pH and carbon dioxide) transducers have found the widest applications. Optical: In optical biosensors, the optical fibers allow detection of analytes on the basis of absorption, fluorescence or light scattering. Since they are nonelectrical, optical biosensors have the advantages of lending themselves to in vivo applications and allowing multiple analytes to be detected by using different monitoring wave-lengths. The versatility of fiber optics probes is due to their capacity to transmit signals that reports on changes in wavelength, wave propagation, time, intensity, distribution of the spectrum or polarity of light. Piezoelectric: In this mode, sensing molecules are attached to a piezoelectric surface-a mass to frequency transducer-in which interactions between the analyte and the sensing molecules set up mechanical vibrations that can be translated into an electrical signal proportional to the analyte. Example of such sensor is quartz crystals. Thermal: In this mode, the biocomponent is immobilized in proximity to the heat sensing transducer, generally a thermister. Most of the enzymatic or microbial reactions are accompanied by considerable heat evolution making this sensor applicable to a very wide range of detection. However the use of sophisticated and expensive instrumentation is the major drawback of this technique. 2. Bioactive components This is the biological part of the biosensor which specifically reacts with the analyte of interest sparking a signal that is detectable by the attached transducer. Enzymes: Purified enzymes have been commonly used in the construction of biosensors due to their high specific activities as well as high analytical specificity. They are mostly used in catalytic type biosensors. Purified enzymes, however, are expensive and unstable, thus limiting their application sin the field of biosensors. As most of the enzymes being employed are intracellular, isolation and purification becomes tough. Antibodies: The binding between an antigen and its corresponding antibody is very specific. This property of antibody is exploited while designing biosensors based on antibodies. The binding reaction between the antibody and antigen can be monitored as a time dependent change of fluorescence signal which is proportional to the reaction ratio of antibody to analyte. Cells: Either whole microorganisms or tissues can be used as the biocomponent. Whole cells can be used either in a viable or non-viable form. Viable microbes metabolize various organic compounds either anaerobically or aerobically resulting in various end products like ammonia, carbon dioxide, acids etc that can be monitored using a variety of transducers. Viable cells are mainly used when the overall substrate assimilation capacity of microorganism is taken as an index of respiratory metabolic activity, as in the case of estimation of BOD, vitamins, sugars, organic acids, etc. Another mechanism used for the viable microbial biosensor involves the inhibition of microbial respiration by the analyte of interest, like environmental pollutants. The major limitation to the use of whole cells is the diffusion of substrate and products through the cell wall resulting in a slow response as compared to enzyme-based sensors. Nucleic acids: The ability of a single stranded nucleic acid to hybridize with another fragment of DNA by complementary base pairing is the principle behind the

nucleic acid sensors. Technological innovation is introduced in the manner in which the nucleic acid oligomer is attached to the surface of the detector and the manner in which the hybridized nucleic acid is detected and transduced into a measurable signal. Ammonia derivetised oligonucleotides can be detected by attaching to glass surfaces such as fiber-optics cables, glass beads or microscopic slides through covalent bonding with a chemical linker. Lipids: An active biological receptor can be immobilized and stabilized in a polymeric film for determining an analyte of interest in a sample. 3. Immobilization techniques for the bio-component: The biological material should bring the physico-chemical changes in close proximity of a transducer. Immobilization not only helps in forming the required close proximity between the biomaterial and the transducer, but also helps in stabilizing it for reuse.The selection of a technique and/or support material would depend on the nature of the biomaterial and the substrate and configuration of the transducer used. Covalent binding, a commonly used technique for the immobilization of enzymes and antibodies, has not been useful for the immobilization of cells. On the other hand, cross-linking using bifunctional reagents like glutaraldehyde has been successfully used for the immobilization of cells in various supports. Thus crosslinking technique will be useful in obtaining immobilized non-viable cell preparations containing active intracellular enzymes. Entrapment and adsorption techniques are more useful when viable cells are used. The synthetic polymers used for microbial biosensor applications include polyacrylamide, polyurethane-based hydrogels, photo cross-linkable resins and polyvinyl alcohol. Natural polymers used for the entrapment of the cells include alginate, carrageenam, low-melting agarose, chitosan, etc. But entrapment technique adds another diffusional barrier.Passive trapping of cells into the pores or adhesion onto the surfaces of cellulose or other synthetic membranes has the major advantage of direct contact between the liquid phase and the cell, thus reducing or eliminating the problem of mass transfer Table 19.1 table gives detail account of different type of Biosensor. Analyte Microorganism Transducer/immobilization Detection limit BOD Trichosporum cutaneum Miniature O2 electrode 0.2-28 mg/L BOD Activated sludge (mixed microbial consortium) O2 electrode/flow injection system >3.5 mg/L Phenolic compounds Ps. Putida O2 electrode (reactor with cells adsorbed on PEI glass) 100 μM Nitrite Nitrobactor vulgaris O2 electrode (adsorption on Whatman paper) >10 μM Cyanide S. cerevisiae O2 electrode (PVA) 0.15-15 nM Organophosphate nerve agent GEM E.coli Potentiometric 0.055-1.8 mM Mercuric chloride Synechococcus sp. PCC 7942 Photoelectrochemical 0.2 and 0.06 μM Alcohol Candida vini O2 electrode (Porous acetyl cellulose filter) 0.20.02 mM Glucose A. niger O2 electrode (entrapment in dialysis membrane) >1.75 mM Glucose, sucrose, lactose G. oxydans, S. cerevisiae, K. marxianus O2 electrode (gelatine) Up to 0-0.8 mM Sugars (glucose) Psychophilic D.radiodurans O2 electrode (agarose) 0.03-0.55 mM Short chain fatty acids in milk A. nicotianae O2 electrode (Polyvinyl alcohol) 0.11-1.7 mM CO2 CO2 utilizing autotrophic bacteria O2 electrode (bound on cellulose nitrate membrane) 0.2-5 mM

Vitamin B-6 S. uvarum O2 electrode (adsorption on cellulose nitrate membrane) 0.5-2.5 ng/ml Vitamin B-12 E. coli O2 electrode (trapped in porous acetyl cellulose membrane) 5-25x10-9 mM Peptides B. subtilis O2 electrode (filter paper strip & dialysis membrane)0.070.6 mM Phenylalanine P. vulgaris Amperometric O2 electrode 2.5x10-2 -2.5mM Pyruvate Streptococcus faecium CO2 gas sensing electrode 0.22-32 mM Tyrosine A.phenologenes NH3 gas sensing electrode 8.2x10-2 -1.0 mM Monitoring toxicity of compounds to eukaryotes S. cerevisiae was genetically modified to express firefly luciferase On-line monitoring of microbial growth E. coli engineered for constitutive bioluminescence Toxicity of Zn, Cu and Cd, alone or in combination E. coli HB101 and Ps. fluorescens 10586 genetically modified with luxCDABE Polycyclic aromatic hydrocarbons Ps. fluorescens HK44 genetically modified with luxCDABE Ecotoxicity assessment of organotins and their initial breakdown products Microtox and luxCDABE modified Ps. fluorescens Ethanol as a model toxicant E. coli TV1061, harboring the plasmid pGrpELux5 Monitoring of biocides Bioluminescent strain of E. coli produced by rDNA technology Metals, solvents, crop protection chemicals E. coli heat shock promoters, dnaK & grpE were fused with lux gene of V. fischeri Assessment of the toxicity of metals in soils amended with sewage sludge luxCDABE modified Ps. fluorescens Ps. fluorescens 19.5Type of Biosensor 19.5.1 Optical biosensors There are two main areas of development in optical biosensors. These involve determining changes in light absorption between the reactants and products of a reaction, or measuring the light output by a luminescent process. The former usually involve the widely established, if rather low technology, use of colorimetric test strips. These are disposable single-use cellulose pads impregnated with enzyme and reagents. The most common use of this technology is for whole-blood monitoring in diabetes control. In this case, the strips include glucose oxidase, horseradish peroxidase (EC 1.11.1.7) and a chromogen (e.g. otoluidine or 3,3',5,5'-tetramethylbenzidine). The hydrogen peroxide, produced by the aerobic oxidation of glucose, oxidising the weakly coloured chromogen to a highly coloured dye. peroxidase chromogen(2H) + H2O2 dye + 2H2O [19.2] The evaluation of the dyed strips is best achieved by the use of portable reflectance meters, although direct visual comparison with a coloured chart is often used. A wide variety of test strips involving other enzymes are commercially available at the present time.A most promising biosensor involving luminescence uses firefly luciferase (Photinus-luciferin 4-monooxygenase (ATP-hydrolysing), EC 1.13.12.7) to detect the presence of bacteria in food or clinical samples. Bacteria are specifically lysed and the ATP released (roughly proportional to the number of bacteria present) reacted with D-luciferin and oxygen in a reaction which produces yellow light in high quantum yield. luciferase ATP + D-luciferin + O2 oxyluciferin + AMP + pyrophosphate + CO2 + light (562 nm) [19.3] The light produced may be detected photometrically by use of high-voltage, and expensive, photomultiplier tubes or low-voltage cheap photodiode systems. The sensitivity of the photomultiplier-containing systems is, at present, somewhat

greater (< 104 cells ml-1, < 10-12 M ATP) than the simpler photon detectors which use photodiodes. Firefly luciferase is a very expensive enzyme, only obtainable from the tails of wild fireflies. Use of immobilised luciferase greatly reduces the cost of these analyses. 19.5.2 Amperometric biosensors Amperometric biosensors function by the production of a current when a potential is applied between two electrodes. They generally have response times, dynamic ranges and sensitivities similar to the potentiometric biosensors. The simplest amperometric biosensors in common usage involve the Clark oxygen electrode (Figure 19.3). This consists of a platinum cathode at which oxygen is reduced and a silver/silver chloride reference electrode. When a potential of -0.6 V, relative to the Ag/AgCl electrode is applied to the platinum cathode, a current proportional to the oxygen concentration is produced. Normally both electrodes are bathed in a solution of saturated potassium chloride and separated from the bulk solution by an oxygen-permeable plastic membrane (e.g. Teflon, polytetrafluoroethylene). The following reactions occur: Ag anode 4Ag0 + 4Cl- 4AgCl + 4e[19.4] Pt cathode O2 + 4H+ + 4e- 2H2O [19.5] The efficient reduction of oxygen at the surface of the cathode causes the oxygen concentration there to be effectively zero. The rate of this electrochemical reduction therefore depends on the rate of diffusion of the oxygen from the bulk solution, which is dependent on the concentration gradient and hence the bulk oxygen concentration. It is clear that a small, but significant, proportion of the oxygen present in the bulk is consumed by this process; the oxygen electrode measuring the rate of a process which is far from equilibrium, whereas ionselective electrodes are used close to equilibrium conditions. This causes the oxygen electrode to be much more sensitive to changes in the temperature than potentiometric sensors. A typical application for this simple type of biosensor is the determination of glucose concentrations by the use of an immobilised glucose oxidase membrane. The reaction results in a reduction of the oxygen concentration as it diffuses through the biocatalytic membrane to the cathode, this being detected by a reduction in the current between the electrodes (Figure 19.3 ). Other oxidases may be used in a similar manner for the analysis of their substrates (e.g. alcohol oxidase, D- and L-amino acid oxidases, cholesterol oxidase, galactose oxidase, and urate oxidase) ________________________________________ Figure 19. 3. Schematic diagram of a simple amperometric biosensor. A potential is applied between the central platinum cathode and the annular silver anode. This generates a current (I) which is carried between the electrodes by means of a saturated solution of KCl. This electrode compartment is separated from the biocatalyst (here shown glucose oxidase, GOD) by a thin plastic membrane, permeable only to oxygen. The analyte solution is separated from the biocatalyst by another membrane, permeable to the substrate(s) and product(s). This biosensor is normally about 1 cm in diameter but has been scaled down to 0.25 mm diameter using a Pt wire cathode within a silver plated steel needle anode and utilising dip-coated membranes. ________________________________________ Figure 19. 4 An alternative method for determining the rate of this reaction is to measure the production of hydrogen peroxide directly by applying a potential of +0.68 V to the platinum electrode, relative to the Ag/AgCl electrode, and causing the reactions: Pt anode H2O2 O2 + 2H+ + 2e[19.6] Ag cathode 2AgCl + 2e- 2Ag0 + 2Cl-[19.7] The major problem with these biosensors is their dependence on the dissolved oxygen concentration. This may be overcome by the use of 'mediators' which

transfer the electrons directly to the electrode bypassing the reduction of the oxygen co-substrate. In order to be generally applicable these mediators must possess a number of useful properties. They must react rapidly with the reduced form of the enzyme. They must be sufficiently soluble, in both the oxidised and reduced forms, to be able to rapidly diffuse between the active site of the enzyme and the electrode surface. This solubility should, however, not be so great as to cause significant loss of the mediator from the biosensor's microenvironment to the bulk of the solution. However soluble, the mediator should generally be non-toxic. The overpotential for the regeneration of the oxidised mediator, at the electrode, should be low and independent of pH. The reduced form of the mediator should not readily react with oxygen. The ferrocenes represent a commonly used family of mediators (Figure 19.5 a). Their reactions may be represented as follows, Figure 19. 5 (a) Ferrocene (Δ5-bis-cyclopentadienyl iron), the parent compound of a number of mediators. (b) TMP+, the cationic part of conducting organic crystals. (c) TCNQ.-, the anionic part of conducting organic crystals. It is a resonancestabilised radical formed by the one-electron oxidation of TCNQH2. Electrodes have now been developed which can remove the electrons directly from the reduced enzymes, without the necessity for such mediators. They utilise a coating of electrically conducting organic salts, such as N-methylphenazinium cation (NMP+, Figure6.7b) with tetracyanoquinodimethane radical anion (TCNQ.Figure 6.7c). Many flavo-enzymes are strongly adsorbed by such organic conductors due to the formation of salt links, utilising the alternate positive and negative charges, within their hydrophobic environment. Such enzyme electrodes can be prepared by simply dipping the electrode into a solution of the enzyme and they may remain stable for several months. Figure 19. 6 The response of an amperometric biosensor utilising glucose oxidase to the presence of glucose solutions. Between analyses the biosensor is placed in oxygenated buffer devoid of glucose. The steady rates of oxygen depletion may be used to generate standard response curves and determine unknown samples. The time required for an assay can be considerably reduced if only the initial transient (curved) part of the response need be used, via a suitable model and software. The wash-out time, which roughly equals the time the electrode spends in the sample solution, is also reduced significantly by this process. ________________________________________ These electrodes can also be used for reactions involving NAD(P)+-dependent dehydrogenases as they also allow the electrochemical oxidation of the reduced forms of these coenzymes. The three types of amperometric biosensor utilising product, mediator or organic conductors represent the three generations in biosensor development (Figure 19.6). The reduction in oxidation potential, found when mediators are used, greatly reduces the problem of interference by extraneous material. The current (i) produced by such amperometric biosensors is related to the rate of reaction (vA) by the expression: i = nFAvA (19.8) where n represents the number of electrons transferred, A is the electrode area, and F is the Faraday. Usually the rate of reaction is made diffusionally controlled by use of external membranes. Under these circumstances the electric current produced is proportional to the analyte concentration and independent both of the enzyme and electrochemical kinetics. Substrate(2H) + FAD-oxidase Product + FADH2-oxidasefi [19.9] This is followed by the processes: (a) biocatalyst FADH2-oxidase + O2 FAD-oxidase + H2O2 [19.10] electrode

H2O2 O2 + 2H+ + 2e[19.11] (b) biocatalyst FADH2-oxidase + 2 Ferricinium+ FAD-oxidase + 2 Ferrocene + 2H+ electrode 2 Ferrocene 2 Ferricinium+ + 2e[19.13] (c) biocatalyst/electrode FADH2-oxidase FAD-oxidase + 2H+ + 2e[19.14] ________________________________________

[19.12]

Figure 19. 7 Amperometric biosensors for flavo-oxidase enzymes illustrating the three generations in the development of a biosensor. The biocatalyst is shown schematically by the cross-hatching. (a) First generation electrode utilising the H2O2 produced by the reaction. (E0 = +0.68 V). (b) Second generation electrode utilising a mediator (ferrocene) to transfer the electrons, produced by the reaction, to the electrode. (E0 = +0.19 V). (c) Third generation electrode directly utilising the electrons produced by the reaction. (E0 = +0.10 V). All electrode potentials (E0) are relative to the Cl-/AgCl,Ag0 electrode. The following reaction occurs at the enzyme in all three biosensors: 19.5.3 Potentiometer biosensors Potentiometric biosensors make use of ion-selective electrodes in order to transduce the biological reaction into an electrical signal. In the simplest terms this consists of an immobilised enzyme membrane surrounding the probe from a pHmeter (Figure 19.8), where the catalysed reaction generates or absorbs hydrogen ions (Table 19.2). The reaction occurring next to the thin sensing glass membrane causes a change in pH which may be read directly from the pH-meter's display. Typical of the use of such electrodes is that the electrical potential is determined at very high impedance allowing effectively zero current flow and causing no interference with the reaction. ________________________________________ Figure 19. 8 A simple potentiometric biosensor. A semi-permeable membrane (a) surrounds the biocatalyst (b) entrapped next to the active glass membrane (c) of a pH probe (d). The electrical potential (e) is generated between the internal Ag/AgCl electrode (f) bathed in dilute HCl (g) and an external reference electrode (h). ________________________________________ A. There are three types of ion-selective electrodes which are of use in biosensors Glass electrodes for cations (e.g. normal pH electrodes) in which the sensing element is a very thin hydrated glass membrane which generates a transverse electrical potential due to the concentration-dependent competition between the cations for specific binding sites. The selectivity of this membrane is determined by the composition of the glass. The sensitivity to H+ is greater than that achievable for NH4+, Glass pH electrodes coated with a gas-permeable membrane selective for CO2, NH3 or H2S. The diffusion of the gas through this membrane causes a change in pH of a sensing solution between the membrane and the electrode which is then determined. Solid-state electrodes where the glass membrane is replaced by a thin membrane of a specific ion conductor made from a mixture of silver sulphide and a silver halide. The iodide electrode is useful for the determination of I- in the peroxidase reaction (Table 19.2c) and also responds to cyanide ions. ________________________________________ Table 19.2. Reactions involving the release or absorption of ions that may be utilised by potentiometric biosensors. (a) H+ cation,

z is the signed ionic charge.5] D-glucose + O2 D-glucono-1. Typical response time are between one and five minutes allowing up to 30 analyses every hour.5-lactone + H2O2 D-gluconate + H+ penicillinase penicillin penicilloic acid + H+ [19.6] urease (pH 6. Figure 19. b Can also be used in an NH3 (gas) potentiometric biosensor. T is the absolute temperature (K).8] lipase neutral lipids + H2O glycerol + fatty acids + H+ [19. Their normal range of detection is 10-4 .5) H2NCONH2 + 2H2O + H+ 2NH4++ HCO3[19. Thus. Enzyme membranes are coated on the ion-selective gates of these electronic devices. A recent development from ion-selective electrodes is the production of ionselective field effect transistors (ISFETs) and their biosensor use as enzymelinked field effect transistors (ENFETs. However.5)b H2NCONH2 + 2H2O 2NH3 + HCO3.25) where E is the measured potential (in volts). for example. a urea-sensitive FET (ENFET containing bound urease .10-2 M.9] (b) NH4+ cation. R is the gas constant. L-amino acid oxidase L-amino acid + O2 + H2O keto acid + NH4+ + H2O2 [19. The logarithmic dependence of the potential on the ionic concentration is responsible both for the wide analytical range and the low accuracy and precision of these sensors. [i] should be the activity of the ion but at the concentrations normally encountered in biosensors.anion peroxidase H2O2 + 2H+ + 2I.0)a H2NCONH2 + H2O + 2H+ 2NH4+ + CO2 [19.24] a Can also be used in NH4+ and CO2 (gas) potentiometric biosensors. conditions can often be found where there is a linear relationship between the apparent change in pH and the substrate concentration.I2 + 2H2O [19. As an example.20] asparaginase ` L-asparagine + H2O L-aspartate + NH4+ [19.glucose oxidase H2O [19. these are potentiometric devices although they directly produce changes in the electric current. F is the Faraday. < 5 mM) if a significant change in potential is to be determined.21] urease (pH 7. ________________________________________ The response of an ion-selective electrode is given by (19. The relationship between pH change and substrate concentration is complex.23] (d) CN-anion -glucosidase amygdalin + 2H2O 2glucose + benzaldehyde + H+ + CN[19.+ H+ [19. this is effectively equal to the concentration). that there is an increase in the electrical potential of 59 mv for every decade increase in the concentration of H+ at 25°C. E0 is a characteristic constant for the ion-selective/external electrode system. This means.9).e.7] urease (pH 9. including other such non-linear effects as pH-activity variation and protein buffering. although a minority are ten-fold more sensitive. the biosensor responding to the electrical potential change via the current output. Biosensors which involve H+ release or utilisation necessitate the use of very weakly buffered solutions (i.22] (c) I. and [i] is the concentration of the free uncomplexed ionic species (strictly. The main advantage of such devices is their extremely small size (<< 0.1 mm2) which allows cheap mass-produced fabrication using integrated circuit technology.

it flows past the reference thermistor (e) and . but non-catalytic ISFET immersed in the same solution. in the opposite direction to the flow of electrons).0 mg ml-1) during its active lifetime of a month. This may be simply calculated from the enthalpy change and the amount reacted. Heat output (molar enthalpies) of enzyme catalysed reactions.1 m thick) of silica (SiO2) which forms the gate of the FET.0001°C for the biosensor to be generally useful. At 80% efficiency. Under such closely controlled conditions. The temperature changes are usually determined by means of thermistors at the entrance and exit of small packed bed columns containing immobilised enzymes within a constant temperature environment (Figure 19. If a 1 mM reactant is completely converted to product in a reaction generating 100 kJ mole-1 then each ml of solution generates 0.3) which may be used as a basis for measuring the rate of reaction and.1 J of heat.g. From there. tantalum oxide). This is about the temperature change commonly encountered and necessitates a temperature resolution of 0. The chip is insulated by a thin layer (0. a current flows through the FET dependent upon the positive potential detected at the ion-selective gate and its consequent attraction of electrons into the depletion layer. The main body of the biosensor is a p-type silicon chip with two n-type silicon areas. the negative source and the positive drain. however.5. Several analytes may be determined by miniaturised biosensors containing arrays of ISFETs and ENFETs. the biocatalyst and the analyte solution. hence. 19.16 Glucose Glucose oxidase 80 Hydrogen peroxide Catalase 100 Penicillin G Penicillinase 67 Peptides Trypsin 10 .with a reference electrode containing bound glycine) has been shown to show only a 15% variation in response to urea (0. This current (I) is compared with that from a similar. up to 80% of the heat generated in the reaction may be registered as a temperature change in the sample stream.3. Reactant Enzyme Heat output -ΔH (kJ mole-1) Cholesterol Cholesterol oxidase 53 Esters Chymotrypsin 4 . the analyte concentration.10. this will cause a change in temperature of the solution amounting to approximately 0.30 Starch Amylase 8 Sucrose Invertase 20 Urea Urease 61 Uric acid Uricase 49 ________________________________________ Figure 19. ________________________________________ Figure 19. When a potential is applied between the electrodes. The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d). which is separated from sensitive parts of the FET by an inert encapsulating polyimide photopolymer. The actual dimensions of the active area is about 500 m long by 50 m wide by 300 m thick.10). may be affected by the composition. 9 Schematic diagram of the section across the width of an ENFET. by convention. (Note that the electric current is.4 Calorimetric biosensors Many enzyme catalysed reactions are exothermic. a protective ion selective membrane. ionic strength and concentrations of the solutions analysed. Schematic diagram of a calorimetric biosensor. This represents the most generally applicable type of biosensor. The sensitivity of FETs. ________________________________________ Table 19.05 . generating heat (Table 19. Above this gate is an equally thin layer of H+-sensitive material (e. 10.02°C.

When the temperature change is very small. For example. Such reaction systems do. used to detect the temperature change. Four enzymes (hexokinase.26) therefore: (19. the overall process results in a 1000 fold increase in sensitivity. The sensitivity (10-4 M) and range (10-4 . Reactions involving the generation of hydrogen ions can be made more sensitive by the inclusion of a base having a high heat of protonation. before amplification. Figure 19. as in the present case. containing the biocatalyst. lactate dehydrogenase and lactate oxidase) are co-immobilised within the packed bed reactor. have the serious disadvantage in that they increase the probability of the occurrence of interference in the determination of the analyte of interest. It is clearly of great importance that such environmental temperature changes are avoided. ________________________________________ The thermistors. In spite of the positive enthalpy of the pyruvate kinase reaction.10-2 M) of thermistor biosensors are both quite low for the majority of applications although greater sensitivity is possible using the more exothermic reactions (e. An equal movement of only 1°C in the background temperature of both thermistors commonly causes an apparent change in the relative resistances of the thermistors equivalent to 0. Thus the sensitivity of the glucose analysis using glucose oxidase can be more than doubled by the co-immobilisation of catalase within the column reactor in order to disproportionate the hydrogen peroxide produced. between the thermistors. Reaction limitation due to low oxygen solubility may be overcome by replacing it with benzoquinone. phosphoenol pyruvate.27) Where R1 and R2 are the resistances of the thermistors at absolute temperatures T1 and T2 respectively and B is a characteristic temperature constant for the thermistor. (19. 11 ADP is the added analyte and excess glucose. A major problem with this biosensor is the difficulty encountered in closely matching the characteristic temperature constants of the measurement and reference thermistors. function by changing their electrical resistance with the temperature.g. they both may be replaced in the denominator by T1. The resistance change is converted to a proportional voltage change. the heat output by the penicillinase reaction may be almost doubled by the use of Tris .29) The relative decrease in the electrical resistance (ΔR/R) of the thermistor is proportional to the increase in temperature (ΔT).01°C and equal to the full-scale change due to the reaction. all of which contribute to the heat output. primarily due to the recycling between pyruvate and lactate.(1/T2). which accounts for inclusion of the well-insulated aluminium block in the biosensor design . The low sensitivity of the system can be increased substantially by increasing the heat output by the reaction. (19. The expectation that there will be a linear correlation between the response and the enzyme activity has been found to be borne out in practice. In the simplest case this can be achieved by linking together several reactions in a reaction pathway. pyruvate kinase. 1ml volume). and hence temperature.into the packed bed bioreactor (f. obeying the relationship (19. A typical proportionality constant (-B/T12) is -4%°C-1.(1/T2) is very much smaller than one and this relationship may be substantially simplified using the approximation when x<<1 that exÅ1 + x (x here being B(1/T1) . An extreme case of this amplification is shown in the following recycle scheme for the detection of ADP. where the reaction occurs. which is reduced to hydroquinone by flavo-enzymes. The change in temperature is determined by the thermistor (g) and the solution passed to waste (h). NADH and oxygen are present to ensure maximum reaction. B(1/T1) . using a balanced Wheatstone bridge incorporating precision wirewound resistors.28) As T1 Δ T2. however. External electronics (l) determines the difference in the resistance. catalase).

the main advantages of the thermistor biosensor are its general applicability and the possibility for its use on turbid or strongly coloured solutions. Enzymes with high turnover numbers are used in order to achieve rapid response. (ii) After a suitable period the antigen and antigen-enzyme conjugate will be distributed between the bound and free states dependent upon their relative concentrations.5. (a)(iii) The analyte antigen solution is passed into the tube. 19. The most important disadvantage is the difficulty in ensuring that the temperature of the sample stream remains constant (± 0.9. covalent immobilization through an alkanethiol self-assembled monolayer (SAM) Lowest detection limit is 50 ng/L Carbamate Surface plasmon resonance. fluorescence dye is used As low as 136 attomole.5 Immunosensors Biosensors may be used in conjunction with enzyme-linked immunosorbent assays (ELISA). specific for the antigen of interest is immobilised on the surface of a tube. those giving rise to highly coloured. A simple immunosensor configuration is shown in Figure 19. ________________________________________ Analyte Transducer/immobilization characteristics Low molecular weight analytes Surface plasmon resonance A monoclonal antibody against HBP is used. fluorescent or bioluminescent products.9 μg/L CRP Surface plasmon resonance. the amount of enzymelinked antigen bound to the immobilised antibody being determined by the relative concentration of the free and conjugated antigen and quantified by the rate of enzymic reaction. to form immunosensors. covalent immobilization through an alkanethiol self-assembled monolayer (SAM) Lowest detection limit is 0. ________________________________________ Principles of a direct competitive ELISA. Linear detection level is 2-5 μg/ml using two different antiCPR antibodies Insulin with femtomole detection Liposomal immunosensors. Lowest detection limit is 0. Recently ELISA techniques have been combined with biosensors. binding and releasing some of the antibody-enzyme conjugate dependent upon . response time is 15 min DDT Surface plasmon resonance.(tris-(hydroxymethyl)aminomethane) as the buffer.1 ng/ml. However more advanced immunosensors are being developed which rely on the direct detection of antigen bound to the antibody-coated surface of the biosensor. speed and sensitivity. 12 Principles of immunosensors. in order to increase their range. A mixture of a known amount of antigen-enzyme conjugate plus unknown concentration of sample antigen is placed in the tube and allowed to equilibrate. An excess of specific antibody-enzyme conjugate is placed in the tube and allowed to bind. (iii) Unbound material is washed off and discarded.12 where the biosensor merely replaces the traditional colorimetric detection system. Total assay time is less than 30 min Antibiotics Poly and monoclonal antibodies are used. (a)(i) A tube is coated with (immobilised) antigen. Assay kits using this technique are now available for a vast range of analyses. The principles behind the ELISA technique is shown in Figure 16.In conclusion. covalent immobilization through an alkanethiol self-assembled monolayer (SAM) Lowest detection limit is 20 ng/L Organophosphorus Surface plasmon resonance. Piezoelectric and FET-based biosensors are particularly suited to such applications. The sensitivity of such assays may be further enhanced by utilising enzyme-catalysed reactions which give intrinsically greater response. for instance. ________________________________________ Figure 19. (i) Antibody.01°C). (a)(ii) After a suitable period any unbound material is washed off. ELISA is used to detect and amplify an antigen-antibody reaction. The amount of antigen-enzyme conjugate that is bound may be determined by the rate of the subsequent enzymic reaction.

The transducer is immersed in a solution containing a mixture of a known amount of antigen-enzyme conjugate plus unknown concentration of sample antigen. Chapter-19 Enzyme and protein STABILITY Abstract:This report contains an overview of protein stability. the change in frequency is proportional to the mass of absorbed material.30) where Δf is the change in resonant frequency (Hz). . however. The amount of antibody-enzyme conjugate released is determined by the response from the biosensor. Thus the stability of a protein is determined by large number of small positive and negative interaction energies. Δ m is the change in mass of adsorbed material (g). obeying the relationshipes (19.the antigen's concentration. and A is the adsorbing surface area (cm2). The frequency of this oscillation (f) depends on their thickness and cut. specific for the antigen of interest. The amount of antigen-enzyme conjugate bound is determined directly from the transduced signal. They are. K is a constant for the particular crystal dependent on such factors as its density and cut. (b)(iii) Unbound material is washed off and discarded. quartz) vibrate under the influence of an electric field. (b)(ii) After a suitable period the antigen and antigen-enzyme conjugate will be distributed between the bound and free states dependent upon their relative concentrations. (b)(i) A transducer is coated with (immobilised) antibody.g. In the sections that follow I discuss some of the factors that give rise to these positive and negative interaction energies. This frequency change is easily detected by relatively unsophisticated electronic circuits. small and robust. This resonant frequency changes as molecules adsorb or desorb from the surface of the crystal. The major drawback of these devices is the interference from atmospheric humidity and the difficulty in using them for the determination of material in solution. Piezo-electric biosensors Piezo-electric crystals (e. I will also briefly discuss rates of unfolding as they relate to perceived stability (Kinetic Stability). the stability of a protein is small. up to about a 2% change. or takes away from. inexpensive. A simple use of such a transducer is a formaldehyde biosensor. For any piezo-electric crystal. utilising a formaldehyde dehydrogenase coating immobilised to a quartz crystal and sensitive to gaseous formaldehyde. and capable of giving a rapid response. The contribution each residue makes to. each crystal having a characteristic resonant frequency.

a large free energy barrier to unfolding is required and the factors affecting stability are the relative free energies of the folded (Gf) and the transition state (Gts) for the first committed step on the unfolding pathway. The physical biochemist. it is kinetic stability or the rate of unfolding that is important. In a kinetically stable protein. For example. In the case of irreversible or slowly unfolding proteins. the energy barrier to inactivation is lower than that to refolding. The Gibbs free energy. . and with a simple. it will refold and fully recover activity. The biotechnologist. From a functional standpoint this may be all that is required for it to be classified as thermostable. The folding free energy difference. from a thermodynamic standpoint (and in terms of this dissertation) it is classified as non-thermostable. G. but once it cools to room temperature. However. reversibly.15 kcal/mol for a globular protein (compared to e. of the order of 5. Both experimental design and also theoretical treatment of data are simplified by reversible systems. Note that once the Unfolded form is reached.I Definition of Stability Before entering into a discussion of stability in proteins. G.100 kcal/mol for a covalent bond). A protein that is kinetically stable will unfold more slowly than a kinetically unstable protein. Irreversible loss of protein folded structure is represented by: Where ki is the rate constant for some irreversible inactivation process. Gu. is made up the two terms enthalpy (H) and entropy (S). on the one hand. is more concerned with the practical utility of the definition: Is the protein stable enough to function under harsh conditions of temperature or solvent? While the answer to this question may lie in thermodynamic stability (discussed above). The word is used in different ways by different people. it may also lie either simply in reversibility or. between the folded and the unfolded states. related by the equation: Where T is the temperature in Kelvin. the more stable is the protein to denaturation. Thus. is typically small. for irreversibly or slowly unfolding proteins. The bulk of this dissertation will also focus on thermodynamic stability. see Kinetic Stability. is the equilibrium constant for unfolding. a physical biochemist and a biotechnologist may each mean something different when they speak of stability. it is no surprise that most of the literature reports about stability discuss this type of reversible system. would probably discuss protein stability primarily in terms of the thermodynamic stability of a protein that unfolds and refolds rapidly. The easiest proteins in which to study folding and stability are those that exhibit this sort of rapid reversibility. in kinetic stability.g. two-state mechanism: Where Ku. we must define exactly what we mean by stability. Kinetic stability is discussed in more detail in its own section. The free energy profile for a rapidly inactivating protein is shown below. the stability of the protein is simply the difference in Gibbs free energy. on the other hand. The larger and more positive Gu. If a protein unfolds reversibly it may be fully unfolded and inactive at high temperatures. ~30 . cooperatively. The only factors affecting stability are the relative free energies of the folded (Gf) and the unfolded (Gu) states. In these cases.

In a . However. small angle Xray scattering studies have shown that the radius of gyration of the unfolded state of ribonuclease A is smaller than if it is in a random coil (Sosnick & Trewhella. the enthalpy of unfolding. for a small subset of mutants. was reduced to 50% that of wild-type. where introduction of a few interactions in the unfolded mutant could significantly decrease both enthalpy and entropy vis-à-vis the wild-type. 1986). For example. There is further evidence of this type from unfolding studies with chaotropic denaturants which suggest that. 1995). Further. The same is true for those interactions which stabilize the unfolded state. they are important because there are so many of them. This random coil ensemble can be treated as a single state for a thermodynamic description. To simplify assume that all of the intramolecular Van der Waals and hydrogen bonds that stabilize the native state are fully disrupted in the unfolded protein. The most important of which is conformational entropy. to explain this result in terms of changes in the folded state alone is difficult: requiring (i) that over half the stabilizing interactions present in the wild-type be absent in the mutant and (ii) that there be a huge increase in the entropy of the (folded) mutant over the native protein. in which the protein chain is extensively hydrated and the individual residues do not interact with each other. This is a major reason for the difficulty of quantitative computational calculation of protein stability.II The Unfolded State Until recently. in the unfolded state and that these interactions can be non-native (e.conformational entropy ________________________________________ The literature is in general agreement that the two types of interaction that are most prevalent in proteins are (i) hydrophobic interactions and (ii) hydrogen bonds.g. there is strong evidence that mutations can affect the degree of interaction in the unfolded state (discussed in detail in Sturtevant. particularly between aromatic residues. an ensemble of extended conformations. Diagram showing the burial of hydrophobic moities and formation of intramolecular H-bonds upon protein folding. A more convincing explanation of the result is that the enthalpy and entropy changes between mutant and wild-type are due to changes in the unfolded state. the overall free energy of a folded protein is given by the small difference between two large numbers. Note the release of water molecules upon folding. 1996). Although possible. Thus. in one mutant of Staphylococcal nuclease. (Remember that ). the amount of change in accessible surface area upon unfolding is different to that in wild-type protein (Shortle & Meeker. The reaction of these bonds upon going from the unfolded to the folded state is summarized in the cartoon below. NMR studies have shown that interactions do occur between residues. Pan et al. most studies and theoretical treatments of protein stability have assumed that the unfolded state of a protein is a random coil.hydrophobic interactions 2. For example. One consequence of this view of the unfolded state is that any mutation that effects stability must have its effect almost exclusively via the native (folded) structure. there is now mounting evidence that this simplistic view of the unfolded state is not always true (reviewed by Shortle. In addition. but with no concomitant change in G for the unfolding process (Tanaka et al. H. 1994). III Major Factors Affecting Protein Stability 1. 1993). 1992). Although both these interactions have small free energies per residue.hydrogen bonds 3.

The Hydrophobic Effect The hydrophobic effect is considered to be the major driving force for the folding of globular proteins. Destabilising Free Energy (kcal/mol) Conformational Entropy -177 Peptide Groups Buried -81 Polar Groups Buried -28 Total Destabilising -286 Stabilising Histidine Ionisation +4 Disulphide Bonds +7 Hydrophobic Groups Buried +94 Hydrogen Bonding +166 Total Stabilising +271 ----------------------------------------------------------G (estimate) -15 G (measured) +9 Note that the while the difference between the experimental and estimated values is small. Hartley in 1936 . term. cyclic peptides. while the bulk of the destabilizing factors were attributed to loss of conformational entropy on folding. and unfavourable burial of peptide and polar groups. frequently misunderstood. Gtr. however. See table. The entropy however is negative.. blocked amino acids. nor is there any very strong attraction of paraffin chains for one another. a very strong attraction of water molecules for one another in comparison with which the paraffin-paraffin or paraffin-water attractions are slight. There is no question of actual repulsion between individual water molecules and paraffin chains. At room temperature. The enthalpy of transfer. however. The contribution of the hydrophobic effect to globular protein stability has been estimated empirically both by measuring the thermodynamics of transfer of model compounds (e. "The antipathy of the paraffin chain for water is. is now positive (unfavourable). At high temperatures (~ 110°C) these cages are no longer any stronger than bulk water. There is. into water. and the effect decreases at temperatures above and below this temperature. 1996). S. however. Hydrogen bonding and hydrophobic interactions were estimated to contribute 260 kcal/mol to the stabilizing interactions. there is some temperature at which the hydrophobic effect is strongest. the stabilizing and destabilizing interactions were estimated at 271 and 286 kcal/mol. The number arrived at . the interaction enthalpies are the same in both cases. the estimated value would yield an unfolded protein. such as an organic solution. It is exemplified by the fact that oil and water do not mix and was described well by G. The decrease in the strength of the hydrophobic effect with decreasing temperatures is probably the major cause of cold-denaturation in proteins.. H. It results in the burial of the hydrophobic residues in the core of the protein. -T S. and by site directed mutagenesis studies on proteins. Water tends to form ordered cages around the nonpolar molecule and this leads to a decrease in entropy.g. the enthalpy of transfer from organic solution into aqueous solution is negligible." The thermodynamic factors which give rise to the hydrophobic effect are complex and still incompletely understood. and entropy. respectively (Pace et al. is made up of an enthalpy. and the entropy contribution tends to zero. The free energy of transfer of a non-polar compound from some reference state. Because the temperature dependence of entropy and enthalpy are not the same.) from organic solvents to water..recent analysis of the factors contributing to the stability of RNase T1.

is usually given as a function of the change in the solvent accessible non-polar surface area upon going from the unfolded to the folded state.8 kcal/mol for methylene) and suggests that the mutation introduced some smaller stabilizing influence. The finding was a strong correlation between the degree of destabilization (which ranges from 0. 1995). and one might think .60 to 4.8 kcal/mol (in Pace. I51V. in a radius of 6 Å of the group deleted from wild-type. such as nitrogen and oxygen. I55V. and the changes in the free energy of unfolding for mutations of hydrophobic residues in barnase. the strongest hydrogen bonds are collinear (Creighton. so two deletions such as this would be enough to destabilize a protein completely). V45A. 1993 and references therein).5±0.6 kcal/mol (corrected to 100% burial). 1992): the loss of this methyl group gave rise to a decrease in stability of 0. albeit before the pdb database was as large as it is today. However. They found that 90% of N-H---O bonds in proteins lie between 140 and 180°. such that Ile or Leu to Ala can destabilize a protein by up to 5 kcal/mol.91) (Serrano et al. the range is more broadly distributed between 90° and 160° and centred around 129°. the "hydrophobic effect" measured by SDM includes both an entropic component due to solvent ordering and a (primarily) enthalpic component due to loss of VDWs contacts within the protein. This is additive.3 kcal/mol. Thus. such as N-H---O. cavities created by residue substitution have an additional destabilizing effect: the loss of favourable VDWs interactions (as compared to the wild-type). the acceptor.. The model compound studies predict that the hydrophobic effect of exposing one buried methylene group to bulk water is 0. and that they are centred around 158°C. The occurrence of hydrogen bonds in protein structure has been extensively reviewed by Baker & Hubbard (1984). bifurcated hydrogen bonds with non-linear angles are preferred. See figure below. I109A.8 kcal/mol . The hydrogen is normally covalently attached to one atom. (Remember that many mesophilic proteins are stable by <10 kcal/mol. I25A. the result at zero cavity size is 0. 1996 and references therein).in agreement with the value found for the transfer of model compounds from octanol to water (Pace et al. I76A. There is a geometric component involved in hydrogen bonds. Such an SDM study of T4 lysozyme replaced the 80% buried Ile3 residue by Val (Eriksson et al. I76V & I109V). In barnase. interact with the same hydrogen. V36A. 0. I4A. The site directed mutagenesis studies yielded a larger number with greater statistical variation: the average hydrophobic effect estimated by SDM for a buried methylene group is about 1. 1960). 1992. and for single donor acceptor systems. In double acceptor systems. Hydrogen Bonds A hydrogen bond occurs when two electronegative atoms.6 kcal/mol. I4V.71 kcal/mol) and the number of methyl or methylene side chain groups surrounding the methyl or methylene group that was deleted (r = 0. Correlation between the number of side chain methylene and methyl groups. when the SDM results for methylene were plotted against the size of the cavity created by the residue substitution. The strength of a hydrogen bond is between 2 and 10 kcal/mol. and extrapolated to zero. with permission) The average free energy decrease for removal of a completely buried methylene group was found to be 1. perhaps such as the alleviation of strain within the protein. I51A. the donor. This is smaller than expected (c. This interaction is due to the dipole between the electronegative atoms and the proton. I55A. Electrostatic calculations suggest that deviation of 20° from linearity leads to a decrease in binding energy of approximately 10% (Pimentel & McClellan. but interacts electrostatically with the other. For C=O---H. In the SDM studies. 1992). I25V. (Taken from Serrano et al. 15 mutants were constructed in which a hydrophobic interaction was deleted (V10A.f.

80% of main chain carbonyls fail to form a second hydrogen bond. In globular proteins. one must also consider entropy. An estimation has been made of a positive contribution of 1. and only some are replaced by (often sub-optimal) intra-protein Hbonds. Dissection of the latter contributions from those due only to hydrogen bonds is not trivial. 1994). mutating Asn to Ala creates a cavity of 37.. However.8% of carbonyl groups in proteins fail to H-bond (without any obviously compensating interactions). Despite the small contribution made to protein stability by hydrogen bonds. and those hydrogen bonds that the protein made to bulk water are broken.8 and 0. But. one must take into account the contribution of side chain entropy and the hydrophobic effect to the ( G) values. Thus. that protein will be destabilized. 1996). This cavity may then be filled by water. There is evidence that hydrogen bonds contribute to stability in hyperthermostable proteins. It has been proposed that decreasing the conformational flexibility of the unfolded chain (by substitution with proline. much of the H-bonding potential of the backbone amide and carbonyl groups is satisfied by the formation of regular structure such as alpha helix and beta sheet (links to PPS). if one considers enthalpy terms alone.that this is the amount of energy one hydrogen bond contributes towards stabilization of a folded protein. Fersht. 1996). in order to form. However. replacing the hydrogen bonding of the asparagine NH group. 1987) from the formation of a buried intramolecular uncharged hydrogen bond. all potential hydrogen bonding partners in the extended polypeptide chain are satisfied by hydrogen bonds to water. charged-to-charged > charged-toneutral > neutral-to-neutral.6 kcal/mol. McDonald & Thornton (1994) showed that while only 1. Thus. For example. and in the recent past it was considered that H-bonds made no contribution overall to protein stability. A comparison of glyceraldehyde-3-phosphate dehydrogenase (GADH) from four organisms with a range of thermostabilities and more than 50% sequence identity found that the strongest correlation to thermostability was with the number of buried charged residues H-bonded to buried neutral residues (Tanner et al. The balance between the entropy and enthalpy terms are close.4 Å3 (Harpaz et al . a cavity is created. The difficulty with the SDM studies is that when a smaller residue replaces a larger one. these protein-to-water H-bonds are broken.5±1. However. it would appear that hydrogen bonding is destabilizing to folded protein structure. Estimation of the contribution of hydrogen bonding to protein stability has been made by a combination of experiments on model compounds and site-directed mutational (SDM) studies. The rationalization given for this preference of charged-to-neutral over neutral-to-neutral or charged-to-charged residue H-bonding was as follows: The enthalpy of H-bond formation is in the order. in the unfolded state. 1987) . but the entropic cost of desolvation is in the inverse order.Two such substitutions (A82P and G77A) in T4 lysozyme gave rise to only a small amount of stabilization (0. the entropy of the solvent increases. Even in more conservative mutations such as Thr to Val (which is isosteric) or Ser to Ala. When a protein folds. or by replacement of glycine) should lead to an increase in the stability of the folded relative to the unfolded protein (Matthews et al. the net energy gain for formation of a buried H-bond is approximately 0. we must remember that if we break or delete an intramolecular hydrogen bond in a protein without the possibility of forming a compensating H-bond to solvent. The greatest overall free energy benefit is proposed to be for the charged-to-neutral H-bonds. it is now generally accepted that H-bonds make a positive contribution to protein stabilisation (reviewed in Pace et al. possibly due to the counterbalancing removal . Conformational Entropy of Unfolding The factor that makes the greatest contribution to stabilization of the unfolded state is its conformational entropy.. 1996.90% of globular protein structure.0 kcal/mol (Pace et al. regular structure comprises 80 .3% of backbone amino groups and 1. When the protein folds.4 kcal/mol). the unfavourable interaction energy from burial of a polar group must be overcome.

the mutants were either equal in stability to wildtype or destabilized (A. Asp12 and Arg110 on the surface of barnase by construction of a thermodynamic cycle of all possible combinations of 1. The free energy contribution to the stability of the protein is only 1. When they substituted prolines at these positions in the homologous mesophilic enzymes they observed an increase in thermostability. When I tried this approach in the thermostable alpha amylase from Bacillus lichinoformis. 1996.f. the T4 lysozyme case described above by Matthews et al. suggesting that these factors outweighed the decrease in conformational stability (Dixon et al. or 3 alanine mutants. 1987) A similar mutation that also eliminated a hydrogen bond and some hydrophobic interactions was destabilizing. Shaw unpublished) revealed the creation of a hydrophobic surface cavity upon mutation. Dekker et al. On the other hand are mutational studies showing that the contribution of salt bridges to stability is small. Structural analysis of at least one destabilized mutant (A. but are still have some importance for protein stability. but to a slowing of the rate of unfolding. 1987). This suggests that. which make contributions to the stability of proteins. Watanabe et al ascribe their increase in stability to a decrease in conformational entropy of the unfolded state (c. IV Other Factors Affecting Protein Stability There are a number of other interactions. They include: • Salt Bridges • Aromatic-Aromatic Interactions • Metal Binding and • Disulphide Bonds Salt Bridges Salt bridges or ion-pairs are a special form of particularly strong hydrogen bonds made up of the interaction between two charged residues. Removal of Arg 110 has no effect on stability! Other studies have had similar results (Akke & Forsen. it is equally likely that any observed increase in stability is due. Horovitz et al . 2. not to an overall stabilization of the protein. proline substitution for stabilization should be used with some care. Structural analysis suggests that the optimal placement is at the N-cap of alpha helices and at the second position beta type I and beta type II turns. Watanabe et al (1994) have found a correlation between the number of proline residues in oligo-1. based on structural parameters alone.of favourable interactions upon mutation.. 1975. (1990) measured the stability of a surface salt bridge triad between Asp8. However.6-glucosidases from a number of bacterial species and their thermostability. Day & A. Perhaps at higher temperatures salt bridges make more of a contribution to stability. which presumably counterbalanced any stabilizing effect of the conformational rigidity. One reason that these contributions are not as large as might be expected from the strength of such an ion pair is that. Hydrogen Bonds. strong hydrogen bonds with . unpublished). in order form a salt bridge. As their enzyme (and ours) is irreversibly inactivated upon heating. The contribution of salt bridges to protein stability is a somewhat contentious issue in the literature. 1978. Perutz. Perutz & Raidt. Day. These interactions are few relative to the major factors. in the absence of any hints from nature in terms of homology.25 kcal/mol for the Asp12/Arg110 pair and 0. in addition to those already discussed (The Hydrophobic Effect. 1991).98 kcal/mol for the Asp8/Arg110 pair. On the one hand is the observation that thermophilic and hyper-thermophilic analogues of mesophilic proteins tend to have increased numbers of salt-bridges (Tanner et al.(Matthews et al. they made their substitutions based on recruitment from the more stable enzyme.. Conformational Entropy of Unfolding). 1992). 1990).

6 kcal/mol.. this is a little misleading as the comparisons made are between the apo. Aromatic-Aromatic Interactions About 60% of the aromatic side chains (Phe.41 kcal/mol. it was also shown that the wild-type arc repressor folds between 10 and 1250 more slowly than the mutant (Waldburger et al. The mutant is R31M. Mutation of the Tyr13 or Tyr17 to Phe leads to a decrease in stability of 0. Hydrophobic interactions have less of a steric requirement. They observed that the sequence and tertiary structure of alpha-lactalbumin (Ca2+ binding) and c-type . found in proteins are involved in aromatic pairings.. This is illustrated below and is presumably due to the cost of desolvating the charged groups on going from the unfolded to the folded state. in the apo-enzyme there is often a destabilising cluster of negative charge from coordinating acidic side chains.30 or 0.61 kcal/mol in the double mutant.and the holo. it is possible that most of the energy of stabilization comes from the increase in solvent entropy upon formation of the ion pair. However. Interestingly. From these observations.5 kcal/mol in the arc repressor (Waldburger et al. In contrast to surface salt bridges. As both hydroxyl groups are exposed to solvent (as they would be in the unfolded state).enzyme. it has been found that replacing a buried salt-bridge triad by well packed hydrophobic residues (found by random mutagenesis at the three charged residue positions) leads to an increase in stability of 4. it might be expected that such interactions make a contribution to protein stability. Metal Binding Another method by which the folded state of proteins can be stabilized is metal binding. and 0. 1995).9 kcal/mol (Braxton. it is unclear as to the source of this small destabilization. removal of one partner from a buried salt bridge leads to destabilization of 3-4 kcal/mol. 1984. usually by lone pair donation from oxygen or nitrogen atoms. The transition state would have a particularly high energy if one charged residue had to be buried before the other. in which metal ions are coordinated. In fact.. the geometry of the salt bridge might well be sub-optimal until that part of the protein attained its native conformation. Experiments have shown that metal binding can contribute 6 . analysis of the data using thermodynamic cycles (Carter. and Trp).3 kcal/mol to the protein stability. Perhaps a fairer estimation of the contribution of metal binding to protein stability comes from an experiment in which a metal chelating site was introduced into an alpha helix of iso-cytochrome c and gave rise to a 1 kcal/mol increase in stability in the presence of saturating Cu(II) (Kellis et al.. 1996). Horovitz & Fersht. 1996 and references therein) to stability. therefore. 1991). R40L.water have to be broken. hydrophobic interactions contribute more to stability than a salt bridge triad. However. Thus. E36Y. Tyr. 1991). Studies with model compounds suggest that the optimal geometry is perpendicular. This has been tested on the solvent exposed Tyr13 / Tyr17 pair on the surface of barnase (Serrano et al. This is proposed to be due to the high energy barrier to burying charged residues. This is only very slightly higher than the stabilization expected from the hydrophobic contribution from burying surface area between them. Most of this destabilization can be explained in terms of the loss of interactions between the tyrosine residues and the rest of the protein. However. Another interesting study was that of Kuroki et al. mutation to the double alanine mutant leads to a decrease in stability of 4. In this case. Even if the salt bridge had formed prior to the transition state. such that the partially positively charged hydrogens on the edge of one ring can interact favourably with the pi electrons and partially negatively charged carbons of the other. 1990) indicates that the interaction energy between the two aromatic groups contributes only 1. however. Tyr13 and Tyr17 were mutated to both Ala and Phe. (1989). there is little apparent extra stabilization due to the aromatic pair.

1996). Two are destabilizing. The mutant protein binds one mole of calcium ions and has optimal activity at about 10°C higher than the wild-type.5 . or otherwise have more consequences than just removal of the disulphide bridge. when the geometry is optimal. If the stabilization is purely due to conformational entropy. as described in Conformational Entropy of Unfolding.2 and 4. and three are stabilizing relative to their reduced form. and Xaa-Pro peptide bonds undergo cis-trans isomerisation (where Xaa is any amino acid). Interestingly.. Thus. there have been successful stabilizations by introduction of disulphides: The stability of RNase Hn is increased by 2. but all five are destabilized relative to wild-type (Betz. the former disulphide encompasses a loop of 34 residues. It is worth noting that small proteins are often naturally rich in disulphide bonds. 1993)! However. Engineering disulphides into the ribonuclease barnase gave rise to increased stability of 1.3. the apo-enzyme is about 5 °C less stable than wild-type. Experiments in which naturally occurring disulphides are either mutated to alanine. because these experiments also leave cavities. Disulphide linkages are rare in hyper-thermostable proteins. However.lysozymes (non-Ca2+ binding) are homologous. Interestingly. it is clear that we are still far from understanding the role of disulphides in protein structure.(exemplified by insulin at this PPS link) molecular bridges.1 kcal/mol) is higher than would be predicted by the theory (Betz.5 kcal/mol of stabilization. this bond is present in both the folded and the unfolded state. the upper limit for protein chemical thermostability may be higher than one would calculate from studies involving model mesophilic enzymes. One might imagine that as the enthalpy of a covalent disulphide bond is very high. it contributes a great deal to stability. while the latter encompasses 17. They recruited the binding site from alpha-lactalbumin into human c-type lysozyme (Q86D/A92D). it is hard to estimate the stabilization due to crosslinks alone.120 °C) Asn and Gln are susceptible to deamidation.8 kcal/mol upon introduction of a disulphide. perhaps. thus its enthalpic contribution to the free energy difference is negligible. or chemically reduced and blocked. Apart . 1995). the ultimate limit of protein stability must come from covalent degradation. disulphides bonds rupture. the magnitude of stabilization would be the opposite way round. All of the stabilizing effect of a disulphide bond is proposed to come from the decrease in conformational entropy of the unfolded state. Introduction of novel disulphides into proteins has also had mixed results.(examples are shown in Jane Richardson's Protein Tourist kinamage) or inter. Five disulphides have been introduced into T4 lysozyme (which has no disulphides in the wild-type). Unfortunately. Asp-Xaa peptide bonds are susceptible to hydrolysis. 1993 ). Interestingly. lead to decreased stability ranging from 2 . At high temperatures (80 . Calculations suggest that a disulphide bond should give rise to 2. Disulphide Bonds Disulphide bonds are formed by the oxidation of two cysteine residues to form a covalent sulphur-sulphur bond which can be intra. depending on the primary sequence separation between the crosslinks (Braxton. above. further. 1993 and references therein). V Chemical Degradation It should be mentioned that however stable a protein can be made by stabilizing the folded state. or buried polar groups. Disulphides can also contribute a great deal to Kinetic Stability . they compensate for the small number of non-covalent interactions.8 kcal/mol (Betz. the degree of stabilization observed for the 17 residue loop (4.1 kcal/mol (Clarke et al. presumably because they are chemically labile at high temperatures .

is and continues to be. in fully folded hyper-thermophilic structures (Vielle & Zeikus. presumably by steric constraint. an exciting one. Furthermore. VI Conclusions This dissertation has discussed some of the many forces that make small and conflicting contributions to protein stability. mostly DNA is targeted in an . A number of molecules have been artificially created that mimic the interactions between the DNA and these regulatory proteins.from the trivial response of just avoiding these residues (disulphides are absent and Asn/Gln content is reduced in hyper-thermophiles). DNA replication and transcription are the basic steps in the processes of cell division and gene expression. Chapter -20 Study of drug stratgies INTRODUCTION DNA is the carrier of all genetic information in most organisms and thus a biological molecule of paramount importance. It is the sum of these various stabilising and destabilising interactions that gives rise to the final stability of a protein. and the search for the solution to The Folding Problem. DNA replication and transcription are regulated by several small DNA binding proteins such as transcription factors and polymerases. 1996 and references therein). activate or modulate the processes of DNA replication and transcription by binding to the DNA instead of the regulatory proteins. as often as not mutations have the opposite effect to that which had been predicted. Though the actions of activation and modulation are possible. The total destabilising and the total stabilising energies are both large. it also clear that great strides have been made. our understanding of these many forces is incomplete. These molecules have served as useful drugs that can be used to inibit. one of the great remaining questions in biology. However. This is one of the reasons that current computational methods struggle to predict protein stability from structure. In addition. it is observed that the deamidation rate of Gln and Asn residues is reduced. the activation energy for folding is an important determinant of both kinetic stability and whether a protein will fold to a global minimum. and their difference is small.

On intercalation of DNA by Daunomycin the following structural changes occur: CG and GC base pairs bend by ca. 9o and 15o respectively to prevent excessive van der Waal’s contacts.inhibitory mode. These polyamides are linked systems that recognize DNA base pairs through specific orientation of imidazole (Im) and pyrrole (Py) rings. to destroy cells for antitumor and antibiotic action. These drugs can also form hydrogen bonds with N-3 of adenine and O-2 of thymine. The complex. The chromophores spaced at 3. For example.5A. a few synthetic polyamides like lexitropsins and imidazole-pyrrole polyamides specific forG-C and C-G regions in the grooves have been designed. Recently. Intercalators induce strong structural distortions in DNA. Example: Berenil. Non covalent interactions are generally reversible. Daunomycin is a DNA intercalator that is site specific for GC base pairs. Triostin A has two quinoxaline units connected by a cyclic peptide structure with a cystein pair (disulfide bond) at its center. sandwich two base pairs between the two quinoxalines. Chemical structure of triostatin A Triostin A is an antibiotic possesing cross-linked octapeptide rings attached to two quinoxaline chromophores. The binding mainly is promoted by van der Waals interactions. DNA binding drugs interact with NON COVALENT INTERACTIONS Crick model of DNA double helix in which strands running spirally around each other them base pairs stacked one above the DNA either non-covalently or covalently. Example: The eight ring hairpin polyamide ImPyPyPy-g-ImPyPyPy-b-Dp (Dp dimethylamino propylamide) has been found to inhibit the expression of 5S RNA in fibroblast cells (skin cancer cells) by blocking the transcription factor IIIAbinding site. Generally minor groove binding has negligible influence on the conformation of DNA. INTERCALATORS Intercalating agents contain planar heterocyclic groups which plug in between adjacent DNA base pairs. . Most minor groove binding drugs bind to A/T rich sequences. Drugs that undergo non covalent interactions with the DNA can be classified into two main classes: • Minor Groove binders • Intercalators MINOR GROOVE BINDERS Minor groove binding drugs are usually shaped such that they easily fit into the groove. is mainly stabilized by π-π stacking interactions between the drug and DNA bases. TYPES OF DNA BINDING DRUGS We are already aware of the Watson and there are two sugar phosphate backbone in an antiparallel fashion and between other. thus formed.

The predisposition of minor groove binders like Berenil towards A/T rich regions could be explained by the following facts: A/T regions are narrower than G/C regions. COVALENT INTERACTIONS Covalent binders bind the DNA irreversibly. Cisplatin (cis-diamine-dichloro-platinum) . The counter ions can effectively screen the negative backbone surface thereby allowing non electrolytes as well as positively charged drug ligands to interact more strongly with the target base pairs. In the case of intercalator triostin A it has been proposed that the linker peptide structure could be responsible for specific interaction with the DNA surface. small positively charged counter ions such as Na+.8 Å. These distortions lead to a total DNA unwinding angle of ca. . Anthramycin . due to the C-2 amino group of the guanine base. invariably leading to complete inhibition of DNA processes and subsequent cell death Important drugs belonging to this category are: Mitomycin C .Also. FACTORS AFFECTING DRUG-DNA INTERACTIONS BASE PAIR SEQUENCE Base pair sequence plays a large role on the specific nature of most DNA binding drugs.anticancer drug. The major group specific readout sequence of H-bond donor and acceptor could be involved in triostatin A binding.antitumor antibiotic The activated antibiotic forms a cross links guanine bases on adjacent strands of DNA thereby inhibiting single strand formation. Therefore. or Ca++ and Mg++ ions may be present around the DNA. SOLVENT In the process of drug DNA binding. It platinates N-7 of guanine on the major groove site of DNA double helix. There also occurs a partial compensation of charges as the DNA and drug are oppositely charged which causes some partially solvated counterions to be released into the bulk solvent and become fully solvated. 8o. thus. This leads to the cross-linking of two adjacent guanines on the same DNA strand hindering the mobility of DNA polymerases. COUNTER IONS DNA carries a large negative charge because of the phosphate backbone. better van der Waals contacts. These changes have the effect of inhibiting the association of DNA with the DNA helicase and topoisomerase. a displacement of solvent from the binding site on both the DNA and drug takes place. The nature and strength of the interaction occurring between the drug and the DNA depends greatly upon the the specific bases involved. the base pairs are wedged out to a separation of 6.antitumor antibiotic It binds covalently to N-2 of guanine located in the minor groove of DNA. High steric hindrance in the G/C groove regions.

it cannot distinguish cancer cells from the stem cells. A few moleculare features of cancer cells that may serve as molecular targets in chemotherapy are listed belowCancer cells have been found to be much less adhesive to other cells and non cellular substrates. They lose the property of contact inhibition and do not undergo appoptosis. some structural deformation/adaptation occurs in both. The properties of cancer cells that are unique to them should be exploited for drug targetting. Chemotherapy makes use of DNA binding drugs to kill cancerous cells. which leads to a number serious side effects. Some of the important DNA binding drugs used in chemotherapy are cisplatin. Unfortunately. fast dividing healthy cells such as stem cells also become severely affected by the drugs. hydration/dehydration energies. chemotherapy cannot differentiate cancer cells from healthy cells. SIDE EFFECTS OF CHEMOTHERAPY Current mechanisms target chemotherapeutic drugs to cells that are rapidly divivding. It is this lack of adhesiveness that permits cancer cells to . Thus.Hydrophobic effect is the major driving force of hydrophobic ligand receptor interaction. Side effects caused by chemotherapt may be from mild to severe such as: Hair loss Nausea Low RBC count Decreased immunity Faliure of certain organs Even death. COUNTERACTING SIDE EFFECTS DESIGNING MORE ACCURATE DRUG DELIVERY STRATEGIES* For designing better drug delivery strategies. etc. it can be concluded that some of the important forces contributing to drug DNA interactions are. van der waals interactions and hydrogen bonds. When the cancer cells fail to divide they die. ultimately causing the entire tumor to shrink. Conventional methods target rapidly dividing cells. aromatic sidechains interact favorably with the aromatic environment of the base pair stacking. The disadvantge of this approach is that. It is seen in the case of intercalating drugs as the hydrophobic. STRUCTURAL MODIFICATIONS So that the DNA and the drug molecule can accommodate each other. CHEMOTHERAPY. The chemotherepeutic drugs bind to the DNA of the cancer cells and halt the cell division. we should focus upon the differences between cancer cells and normal cells at olecular level.hydrophobic force. ion effects. As a result.DNA BINDING DRUGS AGAINST CANCER Cancer cells exhibit rapid cell division. amifosatine and mitomycin C.

The whole concept rounds upto the fact that the product (drug DNA complex) must be more stable than the starting elemnt . These differences could be employed for transpoting the DNA binding drugs to the cancer cells without affecting the healthy cells of the body. This happens because of the expression of new cell surface proteins reffered to as tumor associated antigens in cancer cells that can induce modiications at the cell surface.metastasize. DESTABLISING DRUG DNA INTERACTIONS IN HEALTHY STEM CELLS* A DNA binding drug binds with the DNA based on simple rules of chemistry. For this reason they have become targets of clinical research across the globe. there several hurdles . thereby leading to the loss of adhesiveness. CONCLUSION Thus. This synthetic agent can now be targetted to the stem cells where it will act so as to pull anthracycline from the DNA and itself form a stabler complex with it. As a tumor grows. As a consequence. without undergoing ageing this seemingly immortal nature of cancer cells can be attributed to the presence of the telomerase enzyme which is absent in normal cells. leaving portions of the tumor with regions where the oxygen concentration is significantly lower than in healthy tissues. hydrophobic effects and electrostatic interations. However. tumor cells in these hypoxic zones rely on glycolysis for energy production and therefore further increase the levels of proteins responsible for glucose transport and metabolism. Cancer cells require large amounts of glucose for energy production and growth. Cancer cells continue to divide indefinitely. The interactions that stablise most drug-DNA complexes are simple hydogen bonds. thus allowing cells to continue to divide. DNA binding drugs such as cis-Platin have revolutionised chemotherapy so that now it is possible to completely root out cancers. Let us assume that a patient of cancer was administerred with anthracycline(an inercalator). If we could design synthetic molecules that offered a particular drug a set of more stablising interactions inside the cell then it could be thought of using this molecule to destablise the drug-DNA interactions in the healthy cells so that they can resume their normal cell cycle. we see that DNA binding drugs of all categories have displayed a promising potential in the treatment various deadly viral diseases and cancers. side effects begin to manifest. Let us consider a situation for understanding this conept better. When cells become cancerous.unbound drug. Now the stem cells are free of the drug and can undergo their normal cellular functions. Now. it rapidly outgrows its blood supply. they require more energy and the level of proteins needed for glucose transport and metabolism increases. which due to inefficient drug delivery found its way into stem cells and got bound to their DNA. In order to nullify the side effects a synthetic molecule is designed that provides a greater affinity to the drug bound to stem cell DNA than the DNA itself. The telomerase enzyme matains teloeres at the ends of the chromosomes.

such as cis-Platin.1 The DNA molecule . etc • Sveral DNA binding drugs have played an invaluable role in cancer chemotherapy. Stretches of DNA called ‘genes’ have the extremely important function of coding for proteins. Fig.hydrophobic force. The ‘Genome’ is unique to an organism. daunomycin. • Important forces contributing to drug DNA interactions are. DNA being the form in which this information is stored. van der waals interactions and hydrogen bonds. ion effects. • These molecules are used as drugs that can be bound physically to the DNA for interrupting key cellular activities such as DNA replication and gene expression. is not very clearly known. The total DNA content of a cell is termed the ‘Genome’. DNA. SUMMARY • Certain molecules are able to mimic the naturally occuring DNA binding proteins such as transcription factors and DNA polymerases. • DNA binding drugs can be grouped into 3 categories – minor groove binders.still to be cleared before these drugs can be considered as a safe cure for cancer. The scientific community understands this and therefore full fledged research work is being conducted in several big research institutes and laborataries to design better and safer drugs. • Side effects of chemotherapy can be minimised by designing strategies for efficient drug targetting and destabilising the drug-DNA complexes in healthy cells. hydration/dehydration energies. The function of the rest of the genome. and so on. intercalators and covalent binders. and is the information bank governing all life processes of the organism. • DNA drug interactions can be covalent or non covalent. is a molecule of great biological significance. Assignment Structural Biology DNA And Drug Interaction Introduction to DNA Deoxyribonucleic acid. loosely termed as ‘non-gene’ regions.

2 Watson Crick Base pairing. carriers. thymine (T). lymphomas and germ cell tumors. Thus. enzymes.e. which now also includes carboplatin and oxaliplatin. and an acceptor. DNA is present in the body in the form of a double helix. or replication. some carcinomas (e. Upon administration.. 1. adenine (A). and could induce cell death. A-T and G-C base pairing Specific recognition of DNA sequences by proteins/ small molecules is achieved via the combination of hydrogen bond acceptor/donor sites available on the major groove or minor groove. Replication: DNA is responsible for its own regeneration. Transcription and replication are vital to cell survival and proliferation as well as for smooth functioning of all body processes. regulators etc. if the binding specificity and strength of this regulatory protein can be mimicked by a small molecule. fig.DNA has two main functions. 2. RNA. DNA and Drug Binding DNA activation would produce more quantities of the required protein. small cell lung cancer. i. or could induce DNA replication. which is often in the form of a regulatory protein binding to a particular region of the DNA. and makes an intra/interstrand cross-link via the chloro groups with the nitrogens on the DNA bases. It was the first member of its class. guanine (G) and cytosine (C). e. Transcription: Information is retrieved from the DNA by ribonucleic acid. DNA inhibition would restrict protein synthesis. Thus. mostly DNA is targeted in an inhibitory mode. and ovarian cancer). a donor N6.g. cisplatinum or cis-diamminedichloroplatinum(II) (CDDP) is a platinumbased chemotherapy drug used to treat various types of cancers. DNA self replicates. then DNA function can be artificially modulated. Cisplatin. O4 on the major groove side. Covalent Binding Drugs Covalent binding in DNA is irreversible and invariably leads to complete inhibition of DNA processes and subsequent cell death. Drugs bind to DNA both covalently as well as non-covalently. where each strand is composed of a combination of four nucleotides. inhibited or activated by binding this molecule instead of the protein. as hormones. Within a strand these nucleotides are connected via phosphodiester linkages. N7. DNA starts transcribing or replicating only when it receives a signal. The two strands are held together primarily via Watson Crick hydrogen bonds where A forms two hydrogen bonds with T and C forms three hydrogen bonds with G (Figure2). receptors. Though both these actions are possible.g.g. a chloride ligand undergoes slow displacement with water (an . depending on which site the drug is targeted. to destroy cells for antitumor and antibiotic action. structural proteins. Cis-platin (cisdiamminedichloroplatinum) is a famous covalent binder used as an anticancer drug. e. and utilized to synthesize proteins in the body. Proteins are involved in all body processes and play many roles. this synthetic/natural small molecule can act as a drug when activation or inhibition of DNA function is required to cure or control a disease (Table 1). including sarcomas. the A-T base pair offers a hydrogen bond acceptor.

SNo 1 2 3 4 5 6 7 8 9 10 Drug Action Mode of Binding PDB Hoechst 33258 Antitumor Minor groove binding 264D Netropsin Antitumor. the high binding strength of covalent binders is a major advantage. allowing cisplatin to coordinate a basic site in DNA. carinii Minor groove binding 227D Netropsin Antitumor. Minor groove binders. presented by the C2 amino group of the guanine base.[1] Cisplatin crosslinks DNA in several different ways.Minor groove binding drugs are usually crescent shaped. Non-covalently bound drugs : 1. carinii Minor groove binding 1D64 Berenil Antitrypanosomal Minor groove binding 1D63 Guanyl bisfuramidine Active against P.aqua ligand) molecules. Drug. among other factors. which in turn activate apoptosis when repair proves impossible. is thought to be stabilized by π-π stacking interactions between the drug and DNA bases. The aqua ligand in the resulting [PtCl(H2O)(NH3)2]+ is easily displaced. However. Intercalators. However. with binding constants in the nanomolar range. action and mode of binding for some DNA binding drugs. The damaged DNA elicits DNA repair mechanisms. falciparum Minor groove binding 144D Nogalamycin Antitumor Intercalation 182D . Non-covalent binding is reversible and is typically preferred over covalent adduct formation keeping the drug metabolism and toxic side effects in mind. and remains a major challenge to the design of drugs for DNA. Antiviral Minor groove binding 2DND SN7167 Antitumor. Most minor groove binding drugs bind to A/T rich sequences. 2. the platinum cross-links two bases via displacement of the other chloride ligand. since A/T regions are narrower than G/C groove regions and also because of the steric hindrance in the latter. which complements the shape of the groove and facilitates binding by promoting van der Waals interactions. typically to N3 of adenine and O2 of thymine. Additionally. Proteins are large molecules and bind quite strongly to the DNA.These contain planar heterocyclic groups which stack between adjacent DNA base pairs. Subsequently. Antiviral Minor groove binding 121D Pentamidine Active against P. The complex. It has been difficult to achieve similar specificity and affinity using small non-covalent binders. Antiviral Minor groove binding 121D Distamycin Antitumor. a few synthetic polyamides like lexitropsins and imidazole-pyrrole polyamides have been designed which have specificity for G-C and C-G regions in the grooves. Some DNA binders are listed in the following table Table 1. these drugs can form hydrogen bonds to bases. Antiviral Minor groove binding 328D SN6999 Active against P. This preference in addition to the designed propensity for the electronegative pockets of AT sequences is probably due to better van der Waals contacts between the ligand and groove walls in this region. interfering with cell division by mitosis. Intercalators introduce strong structural perturbations in DNA. in a process termed aquation.

solvation van der Waals. When binding occurs. Simulations of DNA with solvent and the attendant counterion atmosphere require careful consideration to ensure system stability. We are attempting to understand the energetics of DNA-drug interaction by theoretically estimating the above contributions employing classical and statistical mechanical methods. the design of sequence specific drugs having requisite affinity for DNA requires a knowledge how the structure of the drug is related to the specificity/affinity of binding and what structural modifications could result in a drug with desired qualities. the comprehensive estimation of which is a major challenge. All these events are associated with some energetic gains/losses. Developing a theoretical protocol for detailed quantitative analysis of DNA-ligand binding in solution is a daunting task due to some major challenges. identifying the forces/energetics involved in such processes is fundamental to unraveling the mystery of molecular recognition in general and DNA binding in particular.11 12 13 14 15 Menogaril Antitumor. it results in a displacement of solvent from the binding site on both the DNA and drug. which can be theoretically estimated from ensembles of structures generated via simulations.Topoisomerase II poison Intercalation Mithramycin Anticancer antibiotic Minor groove binding Plicamycin Anticancer antibiotic Minor groove binding 1BP8 Chromomycin A3 Anticancer antibiotic Minor groove binding cis -Platin Anticancer antibiotic Covalent cross-linking 202D 146D 1EKH 1AU5 Forces involved in DNA-drug recognition: Understanding the forces involved in the binding of proteins or small molecules to DNA is of prime importance due to two major reasons. Some of the forces that are known to contribute to biomolecular recognition and also to DNA-drug binding are direct electrostatic interactions. Consider DNA-drug binding in an aqueous environment. Also. ion effects and entropy terms. Structural and conformational changes in the DNA and drug on binding in solution are associated with enthalpic and entropic contributions to the binding free energy. evolving a computationally efficient technique using statistical mechanical principles for quantitative estimates of binding free energies in large biomolecular systems is an equally challenging task. Secondly. the binding process would be associated with some structural deformation/adaptation of the DNA as well as the drug molecule in order to accommodate each other. The associated counterions lie near the charged groups and are also partially solvated. since there would be partial compensation of charges as the DNA and drug are oppositely charged. DNA is polyanionic in nature and the drug molecule is also often charged. complex hydration/dehydration contributions composed of hydrophobic component. Also. Our study is aimed at providing such a theoretical protocol for complementing experimental techniques and facilitating a minute study of the structure-energy relationships in DNA-drug complexes. Firstly. DNA-drug binding may be described in the following manner. solvation electrostatics. some counterions would be released into the bulk solvent and are solvated fully. direct van der Waals/packing interactions. Also. The only drawback of this approach is the long time .

(1998) Drug--DNA interactions. Ther. Westhof. Thurston. D. Struct... J. 13.L. Biol. Biophys. Wemmer. van der Waals. 509-516. 7. 18. S.taken for the simulations. Biophys. R277-279. B. PreDDICTA.. Chaires. 227–236. 44. 355-61. B. H.. J. (1996) Modeling DNA in aqueous solutions: theoretical and computer simulation studies on the ion atmosphere of DNA. 5. Med. S. 7. Opin. Neidle. S. Dervan. Biopolymers. Prod. 8.. electrostatics. D. 291-309. P. M. 25. B. Struct. J.. Structure Fold Des. Berman. E. Hurley. 9. Annu.. Geierstanger.E. B. Curr. Sondhi. hydrophobic component. thus providing a swift method for evaluation of potential lead candidates for researchers pursuing structure based drug design for DNA.. (2000) The genome as a drug target: sequence specific minor groove binding ligands Curr.. Struct. 287. Curr. 1-14. and correlates it with experimental binding free energy and ΔTm. Goodsell. P. Mol. D. Denny. L. 314320. 15. J. P. 3. 285-96. Rev. 7. 8. References and Further Reading 1. H.. Curr. Reddy. Jen-Jacobson. W. Nat. Annu. (2003) Recognition of the DNA minor groove by pyrrole-imidazole polyamides. (1998) Simulations of the molecular dynamics of nucleic acids. Beveridge. Struc. Thornton. (2002) DNA and its associated processes as targets for cancer therapy. B. S. J.E. 367-394. 877-896. Edelson. (1997) Targeting the minor groove of DNA. 11. 10. (2001) Sequence recognition of DNA by lexitropsins.. 13. 463-493. STRUCTURAL BIOLOGY ASSIGNMENT DNA-DRUG INTERACTION . Pharmacol. Curr. Biol. Janin. Biol.H.. P. S. Chem. Curr. 2. 8. (2001) DNA minor-groove recognition by small molecules. Turner. 284-299. P. Struct. 24. Nature Reviews Cancer. Opin. 188-200. rotational and translational entropy can be estimated from single structures. van Heyningen. Jones... 5. (1999) Wet and dry interfaces: the role of solvent in protein-protein and protein-DNA recognition. Opin. 14. 12. 6. (1999) Protein-DNA interactions: A structural analysis. The other terms. (1997) Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state.. 2. Opin. 8. Biol. M. S. 1. A.. (1995) Complexes of the minor groove of DNA. 84..153-180. R. Wemmer. Jayaram. M. B. (1999) Synthetic DNA minor groovebinding drugs. The web tool.. Biomol. Biomol. D. Struc. estimates the components of DNA-drug binding free energy which can be calculated from a single structure. 1-111. (2005) Chemical approaches to the discovery and development of cancer therapies Nat Rev Cancer. Dervan. Rep.. Biol.. S.. Rev. E.. L. D. namely. Drug Targ. 4. W. Auffinger. B. Lown. Neidle.

The latter is central for tumorigenesis and pathogenesis. Third.. The latter cause little distortion of the DNA backbone. There are three principally different ways of drug binding: First. enzymes. 2. Replication: DNA is responsible for its own regeneration. The synthesis and screening of synthetic compounds that do not exist in nature. e. and utilized to synthesize proteins in the body. Here. Second. is a molecule of great biological significance. Fig. the drugs interact with the proteins that bind to DNA. is not very clearly known. DNA self replicates. DNA as carrier of genetic information is a major target for drug interaction because of the ability to interfere with transcription (gene expression and protein synthesis) and DNA replication. Proteins are involved in all body processes and play many roles.MADE BY: Name: VASU R SAH No. and is the information bank governing all life processes of the organism. small aromatic ligand molecules that bind to DNA double helical structures by (i) intercalating between stacked base pairs thereby distorting the DNA backbone conformation and interfering with DNA-protein interaction or (ii) the minor groove binders. Both work through non-covalent interaction.e. i. work much like Roll Section: R .: 38038 Semester: Vth (AIB) DNA Drug Interaction Deoxyribonucleic acid. DNA. The function of the rest of the genome. loosely termed as ‘non-gene’ regions. as hormones. The ‘Genome’ is unique to an organism. through RNA binding to DNA double helices to form nucleic acid triple helical structures or RNA hybridization (sequence specific binding) to exposed DNA single strand regions forming DNA-RNA hybrids that may interfere with transcriptional activity.g. Transcription: Information is retrieved from the DNA by ribonucleic acid. Stretches of DNA called ‘genes’ have the extremely important function of coding for proteins. The small ligand drug approach offers a simple solution. through control of transcription factors and polymerases. carriers. DNA being the form in which this information is stored. a major step in cell growth and division. RNA. The total DNA content of a cell is termed the ‘Genome’. structural proteins.1 the DNA molecule DNA has two main functions: 1. regulators etc. receptors.

Minor groove bindersAdditionally. Thus. Thus. or replication. thymine (T). depending on which site the drug is targeted. DNA activation would produce more quantities of the required protein. Most minor groove binding drugs bind to A/T rich sequences. which is often in the form of a regulatory protein binding to a particular region of the DNA. where each strand is composed of a combination of four nucleotides. if the binding specificity and strength of this regulatory protein can be mimicked by a small molecule. however. this synthetic/natural small molecule can act as a drug when activation or inhibition of DNA function is required to cure or control a disease (Table 1). the A-T base pair offers a hydrogen bond acceptor. Within a strand these nucleotides are connected via phosphodiester linkages. mostly DNA is targeted in an inhibitory mode. O4 on the major groove side. guanine (G) and cytosine (C). A-T and G-C base pairing Specific recognition of DNA sequences by proteins/ small molecules is achieved via the combination of hydrogen bond acceptor/donor sites available on the major groove or minor groove. a donor N6. Drugs bind to DNA both covalently as well as non-covalently. typically to N3 of adenine and O2 of thymine. then DNA function can be artificially modulated. Cis-platin (cisdiamminedichloroplatinum) is a famous covalent binder used as an anticancer drug. DNA is present in the body in the form of a double helix. N7. but rather certain cellular states or physiological and pathological conditions. and makes an intra/interstrand cross-link via the chloro groups with the nitrogens on the DNA bases. Though both these actions are possible. these drugs can form hydrogen bonds to bases.pharmacological ligand for cell surface receptors in excitable tissue. DNA starts transcribing or replicating only when it receives a signal. DNA inhibition would restrict protein synthesis. e. Non-covalently bound drugs mostly fall under the following two classes: 1. The two strands are held together primarily via Watson Crick hydrogen bonds where A forms two hydrogen bonds with T and C forms three hydrogen bonds with G (Figure2). Covalent binding in DNA is irreversible and invariably leads to complete inhibition of DNA processes and subsequent cell death. to destroy cells for antitumor and antibiotic action. and could induce cell death. Fig. . and an acceptor. like rapid cell growth and division that can be selectively suppressed as compared to non growing or slowly growing healthy tissue. DNA-Drug Interaction : Transcription and replication are vital to cell survival and proliferation as well as for smooth functioning of all body processes. adenine (A). does not allow to target specific genes. or could induce DNA replication. The lack of sequence specificity for intercalating molecules.2 Watson Crick Base pairing. and appear to be more readily delivered to cellular targets than large RNA or protein ligands. inhibited or activated by binding this molecule instead of the protein.g.

This preference in addition to the designed propensity for the electronegative pockets of AT sequences is probably due to better van der Waals contacts between the ligand and groove walls in this region. This structure fits between to adjacent base pair planes and can have some. double aromatic rings) units linked through a cyclic peptide structure (center left) which is stabilized at its center by a cystein pair (disulfhydril covalent bond). the high binding strength of covalent binders is a major advantage. Fig. a few synthetic polyamides like lexitropsins and imidazole-pyrrole polyamides have been designed which have specificity for G-C and C-G regions in the grooves. the stronger the expected binding affinity. As a rule. However. is thought to be stabilized by π-π stacking interactions between the drug and DNA bases. and remains a major challenge to the design of drugs for DNA. 2. rotational freedom within the plane of the ring. IntercalatorsThese contain planar heterocyclic groups which stack between adjacent DNA base pairs. Chemical structure of triostatin A The space filled side view indicates how the two quinoxaline rings are positioned by the linker peptide in co-planar fashion suitable for intercalating with DNA base pairs. However. The ligand itself may have flexibility of structural parts outside the DNA binding site and may contain more than one intercalating sidechain: The structure of the antibiotic triostin A shows the presence of two quinoxaline (groups to the right. Non-covalent binding is reversible and is typically preferred over covalent adduct formation keeping the drug metabolism and toxic side effects in mind. the more intercalating sidechains are linked within a single ligand structure. presented by the C2 amino group of the guanine base. It has been difficult to achieve similar specificity and affinity using small non-covalent binders. Since the spacing . since A/T regions are narrower than G/C groove regions and also because of the steric hindrance in the latter. with binding constants in the nanomolar range. Intercalators introduce strong structural perturbations in DNA. The major requirement for intercalating agents is the planar aromatic ring structure. Proteins are large molecules and bind quite strongly to the DNA. although much restricted. among other factors. Modeling DNA-ligand interaction of intercalating ligands: The following properties have been identified as important for the successful modeling of ligand-DNA interaction: Degrees of freedom Role of base pair sequence Counter ion effects Role of solvent ligand-receptor binding Degrees of freedom this problem is analogous to that of protein ligand interaction. Triostatin A belongs to a family of antibiotics which are characterized by crosslinked octapeptide rings bearing two quinoxaline chromophores. The complex.

5A. aromatic sidechains interactive favorably with the aromatic environment of the base pair stacking. the intercalation process sandwiches two base pairs between the two quinoxalines. hydration shell). since the counter ions can screen and shield the negative backbone surface allowing non electrolytes as well as positively charged ligand to interact more strongly with the DNA target. there is no directionality at the minor groove side. The molecular basis of specific recognition between echinomycin and DNA is due to the hydrogen bonding between the ligand alanine carbonyl groups and the 2-amino group of guanine. This phenomenon is called bis-intercalation and has first been described for echinomycin by showing that bis-intercalating drugs cause twice the DNA helix extension and unwinding seen as compared to single intercalating molecule like ethidium. AT vs TA). or Ca++ and Mg++ ions as well as basic residues of proteins. however. As the structure of triostatin A suggests. Counter ion effect DNA is a negatively charged polyanion attracting counter ions. The latter is a chromophore which is activated by UV light and is used by molecule biologists to label nucleic acids in gel electrophoresis or ion gradient centrifugation. The three classes basically describe the sequence of events of free ligand interacting with its receptor and the change in overall solvent interaction before and after binding. Rational for drug design . This type of interaction is found in intercalating substrates because the hydrophobic. and (c) ligand-DNA complex with solvent interaction. This is consistent with the observation that the preferred binding site is the sequence CG. (b) receptor solvent interaction. Role of base pair sequence Experimental evidence suggests that base pair sequence does not play a large role on the specific mature of most intercalating complexes.between the chromophores is 3. Role of solvent ligand-receptor binding There are three general classes of interactions that must be considered in solvated ligand-receptor binding : (a) ligand solvent interaction (e. The figure graphically shows the direct readout of the DNA base sequence on a double helical structure. The presence of small counter ion affect drug binding.g. however. The major group specific readout sequence of H-bond donor and acceptor could be involved in triostatin A binding. positively charged Na+.g. reduces non covalent interaction mediated by hydrogen bonds and electrostatic interactions. Fig. We have seen that the hydrophobic effect is completely described by this system and the contribution of the entropy of free bulk water is the major driving force of hydrophobic ligand receptor interaction. strong hydrophobic While the interaction on the major groove side is distinct for the direction of the base pair (e. the linker peptide structure may well promote specific interaction with the DNA surface. Overlap of AT and GC base pairs The following characteristics of non covalent bond formation are associated with the binding sites indicated above: binding site GC base pair AT base pair W1 H-bond acceptor H-bond acceptor W2 blank blank W3 H-bond acceptor H-bond donor W3' blank blank W2' H-bond donor H-bond acceptor W1' C-H weak hydrophobic CH3. High ionic strength. The total amount of surface bound water is reduced in the after complex formation.

The optimal goal of polyamide ligand design has been reached with finding structures able to recognize DNA sequences of specific genes.When a compound intercalates into nucleic acids.g. The binding is of course an equilibrium process because no covalent bond formation is involved. and Anthramycin. Correlating these biophysical parameters with cytotoxicity is used to support the antitumor activity of these drugs as based on their ability to intercalate in DNA double helical structures. . Solid phase synthesis of polyamides of variable length has produced efficient ligands. the eight ring hairpin polyamide ImPyPyPy-g-ImPyPyPy-b-Dp (Dp dimethylamino propylamide) shown in the figure above. The binding constant can be determined by measuring the free and DNA bound form of the ligand. Modeling DNA-ligand interaction of minor groove binders: Hairpin minor grove binding molecules have been identified and synthesized that bind to GC reach nucleotide sequences. but also on tissue distribution and toxic side effects on the heart (cardiac toxicity) due to redox reduction of the aromatic rings and subsequent free radical formation. The activated antibiotic forms a cross-linking structure between guanine bases on adjacent strands of DNA thereby inhibiting single strand formation (this is essential for mRNA transcription and DNA replication). DNA double helix structures are found to be more stable with intercalating agents present and show a reduced heat denaturation. The hairpin polyamides originated from the discovery of the three-ring Im-Py-Py molecule that bound to minor groove DNA as an antiparallel side by side dimer. WP631 is a dimeric analog of the clinically proven anthracycline antibiotic daunorobuicin. there are changes which occur in both the DNA and the compound during complex formation that can be used to study the ligand DNA interaction. Free radical species are thought to induce destructive cellular events such as enzyme inactivation. Structure of WP631 This new synthetic compound shows an affinity of 10pM and also showed to be resistant against multidrug resistance mechanisms often encountered in antitumor therapy. Structure of hairpin ligand (right) on DNA minor groove (left) The compound was found to recognize GC base pairs. Hairpin polyamides are linked systems that exploit a set of simple recognition rules for DNA base pairs through specific orientation of imidazole (Im) and pyrrole (Py) rings. Improvement of anticancer drugs based on intercalating activity is not only focussed on DNA-ligand interaction. This small synthetic molecule has an binding constant in the order of 0. Also. Multidrug resistance is a phenomenon where small aromatic compounds are efficiently expelled from the cell by cell membrane transport proteins commonly referred to as ABC transporters (or ATP Binding Cassette proteins). Mitomycin C is a well characterized antitumor antibiotic which forms a covalent interaction with DNA after reductive activation. The structure shown above inhibits the expression of 5S RNA in fibroblast cells (skin cancer cells) by interfering with the transcription factor IIIA-binding site. Cisplatin.03nM. A new strategy of rational drug design exploits the combination of polyamides with bis-intercalating structures. this can be done spectroscopically. e. Fig. Drugs that form covalent bonds with DNA targets Drugs that interfere with DNA function by chemically modifying specific nucleotides are Mitomycin C. DNA strand cleavage and membrane lipid peroxidation. Since many of the intercalating substrates are aromatic chromophores. Fig.

Anthramycin has a preference of purine-Gpurine sequences (purines are adenine and guanine) with bonding to the middle G. some counterions would be released into the bulk solvent and are solvated fully. Our study is aimed at providing such a theoretical protocol for complementing experimental techniques and facilitating a minute study of the structure-energy relationships in DNA-drug complexes. solvation electrostatics. Developing a theoretical protocol for detailed quantitative analysis of DNA-ligand binding in solution is a daunting task due to some major challenges. which can be theoretically estimated from ensembles of structures generated via simulations. solvation van der Waals. Some of the forces that are known to contribute to biomolecular recognition and also to DNA-drug binding are direct electrostatic interactions. Structural and conformational changes in the DNA and drug on binding in solution are associated with enthalpic and entropic contributions to the binding free energy. All these events are associated with some energetic gains/losses. the binding process would be associated with some structural deformation/adaptation of the DNA as well as the drug molecule in order to accommodate each other. When binding occurs. evolving a computationally efficient technique using statistical mechanical principles for quantitative estimates of binding free energies in large biomolecular systems is an equally challenging task. Secondly. Also. Cisplatin is a transition metal complex cis-diamine-dichloro-platinum and clinically used as anticancer drug. DNA is polyanionic in nature and the drug molecule is also often charged. DNA-drug binding may be described in the following manner: Consider DNA-drug binding in an aqueous environment. it results in a displacement of solvent from the binding site on both the DNA and drug. The associated counterions lie near the charged groups and are also partially solvated. Also. Forces involved in DNA-drug recognition: Understanding the forces involved in the binding of proteins or small molecules to DNA is of prime importance due to two major reasons. direct van der Waals/packing interactions.Anthramycin is an antitumor antibiotic which bind covalently to N-2 of guanine located in the minor groove of DNA. since there would be partial compensation of charges as the DNA and drug are oppositely charged. the comprehensive estimation of which is a major challenge. ion effects and entropy terms. Also. Firstly. complex hydration/dehydration contributions composed of hydrophobic component. the design of sequence specific drugs having requisite affinity for DNA requires a knowledge how the structure of the drug is related to the specificity/affinity of binding and what structural modifications could result in a drug with desired qualities. The only drawback of this approach is the long time . This chemical modification of platinum atom cross-links two adjacent guanines on the same DNA strand interfering with the mobility of DNA polymerases. identifying the forces/energetics involved in such processes is fundamental to unraveling the mystery of molecular recognition in general and DNA binding in particular. We are attempting to understand the energetics of DNA-drug interaction by theoretically estimating the above contributions employing classical and statistical mechanical methods. Simulations of DNA with solvent and the attendant counterion atmosphere require careful consideration to ensure system stability. The effect of the drug is due to the ability to platinate the N-7 of guanine on the major groove site of DNA double helix.

The other terms. Therefore. Each unit cell contains exactly one unique set of the crystal's components. The set of diffracted. many molecules are in the same orientation with respect to the incoming X-rays. The X-ray beam enters the crystal and a number of smaller beams emerge: each one in a different direction. is placed on the opposite side of the crystal from the X-ray source. Many diseases. The basic building block of a crystal is called a unit cell. Illuminating a protein's structure also paves the way for the development of new agents and devices to treat a disease. But sometimes a protein twists into the wrong shape or has a missing part. an important component of any X-ray diffraction instrument is a device for accurately setting and changing the orientation of the crystal. enabling them to carry out an extraordinarily diverse range of biological functions. therefore. . each one with a different intensity. all of the unit cells present the same face to the beam. namely. When the crystal is placed in an X-ray beam. Chapter-21 Technique to study protein Introduction: Proteins are fundamental components of all living cells. electrostatics. produce a complex pattern of X-ray diffraction. It often takes scientists working in the laboratory months. The web tool. sometimes years. If an X-ray detector. such as a piece of film. Scientists know that the critical feature of a protein is its ability to adopt the right shape for carrying out a particular function. like a protein. Identifying a protein's shape. Determination of Protein Structure Traditionally. or structure. emerging beams contains information about the underlying crystal structure.Nuclear Magnetic Resonance (NMR) Spectroscopy. will produce a spot on the film. Crystals of a complex molecule. or scattering of X-rays. such as Alzheimer's and "mad cow". because only a few reflections can be detected with any one orientation of the crystal. They exhibit an enormous amount of chemical and structural diversity.taken for the simulations. 2. a protein's structure was determined using one of two techniques: 1. thus providing a swift method for evaluation of potential lead candidates for researchers pursuing structure based drug design for DNA. is a key to understanding its biological function and its role in health and disease. The challenge lies in developing methods for accurately and reliably understanding this intricate relationship. to experimentally determine a single structure. estimates the components of DNA-drug binding free energy which can be calculated from a single structure. PreDDICTA. hydrophobic component. rotational and translational entropy can be estimated from single structures. Yet solving the structure of a protein is no easy feat. and correlates it with experimental binding free energy and ∆Tm. called a reflection. each diffracted ray. are now known to result from proteins that have adopted an incorrect structure.X-ray Crystallography. van der Waals. preventing it from doing its job. scientists have begun to turn toward computers to help predict the structure of a protein based on its sequence. However. the smallest possible set that is fully representative of the crystal. X-ray Crystallography .

rather than the electron density in a molecule. When the radio waves hit the spinning nuclei. These resonating nuclei emit a unique signal that is then picked up on a special radio receiver and translated using a decoder. In this technique. A complete structure can thus be calculated by sequentially assigning cross peak correlations in 2-D spectra. Currently. With NMR. NMR has the advantage over crystallographic techniques in that experiments are performed in solution as opposed to a crystal lattice. they tilt even more. These radio waves encourage the nuclei of the molecule to resonate. The thermal motion of the molecule—the movement of the molecule associated with the temperature of the material—further creates a torque. or twisting force. pairs of H that can be close in space. or Nuclear Overhauser Effect). C-13. This decoder is called the Fourier Transform algorithm. even if they are from residues that are not close in sequence (NOE spectra. NMR measures the distances between atomic nuclei. nucleic acids. or spin. that makes the magnetic moment "wobble" like a child's top.H nuclei is the most sensitive to detect 1-D spectra contain the information about all the chemical shifts of all the H in the protein.and medium-sized molecules. a complex equation that translates the language of the nuclei into something a scientist can understand. the moving charge creates what is called a magnetic moment. By measuring the frequencies at which different nuclei flip. The frequency resolution is often not enough to distinguish individual chemical shifts. 2-D NMR solves these problems by containing information about the relative position of H in molecular structures. This dependence on next neighbors known as chemical shift (or spin-spin coupling constant) and reflects the local electronic environment and the information contained in 1-D NMR spectra. NMR has proven to be a powerful alternative to X-ray crystallography for the determination of molecular structure. In the past 10 years. Strategy behind NMR Nuclear magnet resonance obtains the same high resolution using a very different strategy. As the positively charged nucleus spins. scientists can determine molecular structure. a strong. For proteins. the size limit for proteins amenable to NMR solution structure analysis is about 200 amino acids. sometimes flipping over. The following reasons make the H-1 NMR spectroscopy the method of choice for biological macromolecules: . 2-D NMR spectra contain information about interaction between H that is covalently linked through one or two other atoms (COSY or correlation spectroscopy). the principles that make NMR possible tend to make this technique very time consuming and limit the application to small. The distance and type of neighboring nuclei determines the resonance frequency of the stimulated atomic nuclei. However. NMR usually measures the spin of protons. An important feature of the identification of cross peaks is that regular patterns can be recognized that stem from secondary structure elements such as alpha helices and parallel or anti- . a sample is immersed in a magnetic field and bombarded with radio waves. D-2. as well as many other interesting properties of the molecule. or N-15 (they have a magnetic spin) and measures the frequency of the magnetic field of the atomic nuclei during its oscillation period back to the initial state.H has a high abundance for each site . high frequency magnetic field stimulates atomic nuclei of the isotopes H-1. and polysaccharides .Nuclear Magnetic Resonance(NMR)Spectroscopy The basic phenomenon of NMR spectroscopy was discovered in 1945.H is present at many sites in proteins. The important step is to determine which resonance comes from which spin. Alternatively.

is driven by magic angle spinning at a speed omega r that matches the rotational resonance condition delta = n omega r. The distances measured in this way for both the 6-s-cis. primarily as residual dipolar couplings.2 angstroms (A)] and inconsistent with that for a 6-s-cis configuration (3.parallel beta sheets because they contain typical hydrogen bonding networks. the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. this represents a convergence of solid-state and solution NMR approaches to structure determination X-ray crystallography and NMR are complementary techniques. Fig. . NMR also requires the knowledge of the amino acid sequence. In bacteriorhodopsin the exchange trajectory between C-8 and C-18 was in good agreement with the internuclear distance for a 6-s-trans configuration [4. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. which are experimentally accessible through experiments performed on weakly and completely aligned samples. was used to determine the distance between the C-8 and C-18 carbons of retinal in two model compounds and in the membrane protein bacteriorhodopsin. The results illustrate that rotational resonance can be used for structural studies in membrane proteins and in other situations where diffraction and solution NMR techniques yield limited information. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. a new nuclear magnetic resonance (NMR) technique for measuring internuclear distances between like spins in solids. Rotationally resonant magnetization exchange. Since weakly aligned samples of proteins display these same effects. where n is a small integer.and 6-s-trans-retinoic acid model compounds agreed well with crystallographically known distances. but the protein does not have to be in an ordered crystal. yet high concentrations of solubilized protein must be available (NMR structures are therefore also called solution structures). Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. This is true not only for proteins. In biopolymers.1 A). the primary structure (sequence) logically breaks up the molecule into groups of coupled spins normally one or two groups per residue. Observed NOEs in antiparallel and parallel sheets Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin. delta. Structure of bacteriorhodopsin: General: Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions. Importantly. in solution NMR spectra. Magnetization transfer between inequivalent spins with an isotropic shift separation. but also for nucleic acids and polysaccharides.

This information is read using the genetic code. complex functional active site active or inactive domains domains atomic nuclei.116. The code is read by copying stretches of DNA into the related . domain any size. with a backbone made of sugars and phosphate groups joined by ester bonds. purity < 20kD. Biochemistry by Lehninger. which specifies the sequence of the amino acids within proteins. It is the sequence of these four bases along the backbone that encodes information. 783 . DNA is a long polymer of simple units called nucleotides.1126/science. 4995. or are involved in regulating the use of this genetic information. purity single crystal.5Å resolution limit 2-3. and RG Griffin. R Gebhard. K van der Hoef. A McDermott. protein folding long time scale. Chapter-22 Enzymology of DNA folding and unfolding WHAT IS DNA??? Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms. The main role of DNA molecules is the long-term storage of information and DNA is often compared to a set of blueprints. MB Spijker-Assink.5Å primary structure must be known primary structure must be know (except if resolution is 2Å or better for every single residue) References: 1.4: “The three dimensional structure of Proteins”. 251. no. The DNA segments that carry this genetic information are called genes. since it contains the instructions needed to construct other components of cells. such as proteins and RNA molecules. Attached to each sugar is one of four types of molecules called bases.NMR spectroscopy X-ray crystallography short time scale. Chapter no. but other DNA sequences have structural purposes. Science 15 February 1991: Vol. page no. MH Levitt. J Herzfeld.1990439 ARTICLE Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin F Creuzet. domain. chemical bonds electron density resolution limit 2-3. 2. J Lugtenburg. Wikipedia. Chemically. pp.786 DOI: 10. 3. static structure solution.

[1][2] The DNA chain is 22 to 26 Ångströms wide (2.[6] The DNA double helix is stabilized by hydrogen bonds between the bases attached to the two strands. These bases are classified into two types. The four bases found in DNA are adenine (abbreviated A). Eukaryotic organisms such as animals.[3] Although each individual repeating unit is very small.3 Ångstroms (0. These four bases are shown below and are attached to the sugar/phosphate to form the complete nucleotide. the largest human chromosome. These asymmetric bonds mean a strand of DNA has a direction. For instance. If multiple nucleotides are linked together. DNA does not usually exist as a single molecule. chromatin proteins such as histones compact and organize DNA. this polymer is referred to as a polynucleotide. Within cells. One of the major differences between DNA and RNA is the sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. in a process called DNA replication. but others are used directly in structures such as ribosomes and spliceosomes. but instead as a tightly-associated pair of molecules. In general. adenine and guanine are fused five. DNA is organized into structures called chromosomes and the set of chromosomes within a cell make up a genome. which helps control its interactions with other proteins and thereby control which genes are transcribed.[4] In living organisms.6 nanometres). called uracil (U). which interacts with the other DNA strand in the helix. while cytosine and thymine are six-membered rings called pyrimidines. The nucleotide repeats contain both the segment of the backbone of the molecule.2 to 2. is 220 million base pairs long. and one nucleotide unit is 3. in a process called transcription. which holds the chain together.[6] A fifth pyrimidine base. The asymmetric ends of DNA strands are referred to as the 5′ (five prime) and 3′ (three prime) ends.33 nanometres) long.nucleic acid RNA. and a base. in the shape of a double helix. while in prokaryotes such as bacteria it is found in the cell's cytoplasm. guanine (G) and thymine (T).[7] The backbone of the DNA strand is made from alternating phosphate and sugar residues. These chromosomes are duplicated before cells divide. and fungi store their DNA inside the cell nucleus. with 2-deoxyribose being replaced by the alternative pentose sugar ribose in RNA.and six-membered heterocyclic compounds called purines.[8] The sugar in DNA is 2-deoxyribose. a base linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. plants. chromosome number 1. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand.[5][6] These two long strands entwine like vines. as in DNA. DNA polymers can be enormous molecules containing millions of nucleotides. Most of these RNA molecules are used to synthesize proteins. cytosine (C). DNA STRUCTURE DNA is a long polymer made from repeating units called nucleotides. usually takes the place of thymine in RNA and differs from thymine by lacking . This arrangement of DNA strands is called antiparallel. which is a pentose (five carbon) sugar. Within the chromosomes. as shown for adenosine monophosphate.

proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.[13] Replication Cell division is essential for an organism to grow. they leave gaps between each set of phosphate backbones. The double-stranded structure of DNA provides a simple mechanism for DNA replication.[71] In this way. There are two of these grooves twisting around the surface of the double helix: one groove. but a very rare exception to this rule is a bacterial virus called PBS1 that contains uracil in its DNA. occurring only as a breakdown product of cytosine. As the DNA strands wind around each other. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction. This enzyme makes the complementary strand by finding the correct base through complementary base pairing. and the cell ends up with a perfect copy of its DNA MECHANISM OF DENATURATION AND RENATURATION OF DNA DNA DENATURATION : . revealing the sides of the bases inside (see animation). the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. the base on the old strand dictates which base appears on the new strand. As a result.[9] In contrast.[ 0] Major and minor grooves Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. a significant number of the uracils are converted to thymines by the enzymatic addition of the missing methyl group. the minor groove. Large version[11] The double helix is a right-handed spiral. is 12 Å wide. following synthesis of certain RNA molecules.a methyl group on its ring. is 22 Å wide and the other. Here. the major groove. but when a cell divides it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. Uracil is not usually found in DNA. different mechanisms are used to copy the antiparallel strands of the double helix. and bonding it onto the original strand.[12] The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. This occurs mostly on structural and enzymatic RNAs like transfer RNAs and ribosomal RNA.

It is thought that this phenomenon facilitates the access of regulatory proteins to the inner part of the DNA molecule. the complementary in 3'-5'direction) and are connected by hydrogen bonds (see DNA structure). Inside the cell. The rupture of these hydrogen bonds causes the DNA to separate or to "denature". A=T rich zones melt at lower temperatures than G=C rich zones.3 is able to denature DNA. which form the double helix. In the moment that the denaturating agent disappears. replication and transcription begins in the A=T rich regions. because it builds covalent bonds with the NH2 groups of the bases. observable as a change in light absorption. In both cases. DNA will renature again. It shows . a partial separation of the DNA strands is necessary for replication and transcription and is caused by several proteins that use ATP. alcaline conditions or chemical compounds The denaturation by heat is complete at 90ºC. since the heterocyclic rings of the bases absorb more light if they are not piled up or connected by hydrogen bonds insight the double helix. Distilled water can also cause some degree of denaturation. Concerning alcalic conditions. 40% higher absorption than double stranded DNA. This will prevent it from forming again the hydrogen bonds in an ordered manner. It inhibits the neutralisation of the phosphate groups by salts like magnesium or sodium. whereas GC rich zones tends to have a higher Tm. is a very valuable mechanism to evaluate the genetic relation between organisms.The two DNA strands. The negative charge of the phopsphate group causes a repulsion and therefore a physical separation of the DNA strands. It can react with the DNA at room temperatures. The denaturation of the DNA can be observed by a change of light absorption at a wavelength of 260 nm. DNA RENATURATION : The process of renaturation. it needs to be transferred immediately from 100ºC to ice for fast cooling. because even in native DNA the bases are in a dynamic equilibrium of pairing and unpairing in discrete regions. Chemical compounds like urea or formamide denature the DNA by directly reacting with the bases. Urea is used in denaturing gels to sequence DNA. But in vitro the rupture of the hydrogen bonds can be caused by heat. The content of G+C can therefore be calculated from the Tm value of the DNA. Single stranded DNA has an appr. The Tm is reduced proportionally to the concentration of denaturating chemical agents. If we decrease the concentration of either urea or formamide. This phenomenon is called "hipercromicity". the DNA renaturalizes ( reassociates. The temperature at which half of the molecules are denatured is called Tm (melting temperature). a pH above 11. the process of denaturation is reversible. thus preventing base-pairing. Also in biological systems. The Tm varies among organisms depending on their porcentage of G+C. called the respiration or palpitation of the DNA. are in opposite orientations (one runs in 5'-3'direction. Formaldehyde causes an irreversible denaturation. Denaturation at an elavated pH > 11 is caused by a rupture of the hydrogen bonds between the complementary bases. reanneals) and it gets back to its native structures of a double helix. The alcalic conditions change the charges of the many side groups that are involved in the non-covalent binding between the bases. thereby preventing normal base-pairing. If DNA is to be kept in a denatured state.

although mismatching occurs quite often. The Base-pairing is extended over the whole strand in form of a "zipper". A DNA that contains a high quantity of repetitive sequences (satellite sequences) will renaturate much faster than DNA that is mainly single sequences.higher the GC content. since the genetic sequences are unlikely to be exactly the same. Double helical DNA is stabilised by cations. Unstacked bases (random orientation) absorb more light than neatly stacked (oriented) base-pairs. But it must be said that not all mismatching is bad. In long molecules. The process of renaturation is also called hybridization.the degree of similarity. 2. The renaturation occurs is two steps: 1.to bacterial DNA. This results in single stranded DNA. This is an important feature of DNA which is utilised in the laboratory when carrying out DNA hybridisation. by observing the change in absorbance at 260nm. This fact is valuable for example in compairing phage. correspondance and divergance between DNAs of different origin. and therefore Tm. Shorter DNA molecules reassociate faster than longer molecules. What is Denaturation of DNA ?? As DNA is heated. What is Reannealing of DNA ?? If melted DNA is cooled slowly. Ionic Strength . "Stringency" is the term given to the conditions of annealing which control the . Perfect annealing requires the perfect matching of base pairs. then they would need to allow some mismatch. Tm is a measure of the stability of DS-DNA under a given set of conditions. even so it is 30 time bigger. This process is called "annealing". If the Tm lowers by 1 o.formamide for instance lowers the Tm by weakening the hydrophobic interactions. then 1% mismatch has occurred. but also with another sequence that has high homology but not complete. a gene was found in a rat. If. for instance. salt concentration and temperature.. The temperature at which DNA is half unfolded is called the melting temperature. it reaches a temperature where the strands seperate (DNA melts). the higher the Tm. The base pairs are separated as the H-bonds between them are broken and the strand unwind. so does Tm. It is possible to follow this process in a spectrophotometer.. Hybridization is the basis of many molecular techniques studied in This course. Stability. The conditions of annealing. can determine the amount of mismatch that occurs. This gives less stable DNA.. and it can happen with its original complementary strand. Organic Solvents .as the ionic strength increases. Divalent cations (eg Mg2+) are more effective than monovalent cations (<NA+ or K+). Base Composition . and can be monitored by watching the Tm. Mice DNA for example renatures faster that bacterial DNA.Two complementary sequences collide by incidence and they combine forming a helix structure. many small fragments appear as a result of shering forces and the possibility that the complementary segments "find each other" is lower. Only complementary strands can anneal. is affected by. complementary strands will pair up again. because it contains many repetitive sequences. This results in a melting curve. and researchers needed to know if a similiar gene was found in humans.

If the stringency of hybridisation is high. Renaturation of DNA by a Saccharomyces cerevisiae Protein That Catalyzes Homologous Pairing and Strand Exchange* A protein from mitotic Saccharomyces cerevisiae cells that catalyzes homologous pairing and stranedx change was analyzed for the ability to catalyze other related reactions. there is little or no mismatch with 95-100% of base-pairs matched correctly.h oeing a higher affinity for single-stranded DNA. by analogy. Incubation of the yeast protewini th complementary single-strandedD NA resulted in the rapid formation ofl arge aggregates which did noetn ter agarose gels. These aggregates contained many branched structures consisting of both single-stranded and doublestranded DNA. At low stringency. The protein formed stable complexesw ith both single-stranded and double-stranded DNAs. The binding to single-stranded DNA resulted in the formation of large protein:DNA aggregates. Trheessuel ts demonstrate that the S. gives further indication that it might be implicated in homologous recombination Mechanism of DNA denaturation in the presence of manganese ions . only 40-50% of base-pairs are correctly repaired. These reactions required stoichiometric amounts of protein but shonwoe Ad TP requirement. cerevisiae strand-exchange protein shares additional properties with the Escherichia coli recA protein which. The proteiwna s capable of renaturing complementary single-strandedD NA as evidenced by S1 nuclease assays and analysis of the reaction products by agarose gel electrophoresis and electron microscopy. These aggregates were also formed in strand-exchange reactions and contained both substrate and product DNAs.extent of mismatching allowed.

RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The usefulness of the PCR MP for molecular typing was shown for clinical strains of Escherichia coli. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0. 2. The use of nitrocellulose paper for the .The unwinding of DNA strands in the presence of small concentrations of Mn2+ ions (2 × 10-4-4 × 10-4M) has been studied. The RNA is stably bound to the nitrocellulose paper by this procedure. excellent reproducibility and may be applied for epidemiological studies. The Mn2+ ions are responsible for the binding of the unwound strands. We found that PCR MP technique is a rapid method that offers good discriminatory power. which is currently considered to be the gold standard for epidemiological studies. shifts the equilibrium towards the melted state. There is no true renaturation in the partially melted DNA. and causes slow unwinding at a constant temperature. the DNA strands are gradually unwound at a constant temperature corresponding to the beginning of the melting curve. The process of unwinding is nonequilibrium. Enterococcus faecium VRE and Stapylococcus aureus.HYBRIDIZATION OF DENATURED RNA AND SMALL DNA FRAGMENTS TRANSFERRED TO NITROCELLULOSE A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. It is shown in the paper that these effects are due to the aggregation of the unwound DNA regions. The binding of denaturated regions seems to occur through the guanines ADVANTAGES OF DENATURATION… 1. The aggregation precludes renaturation. allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram).PCR MELTING PROFILE(PCR MP) The performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR) of bacterial DNA is shown. Results from strain genotyping illustrate that PCR MP is useful for the study of intraspecific genetic relatedness of strains and is as effective in discriminating closely related strains as the PFGE method.

SUPERCOIL SEQUENCING:A FAST AND SIMPLE METHOD FOR SEQUENCING PLASMID DNA A method for obtaining sequence information directly from plasmid DNA is presented. In addition. as well as RNA. and nucleic acid-protein interactions at single nucleotide resolution 4. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt. its complete denaturation by alkali. The procedure involves the rapid preparation of clean supercoiled plasmid DNA from small bacterial cultures. and sensitive. The method is simple. using LiAc to yield competent cells. Each band in these sequences represents 3 fg of DNA complementary to the probe. reproducible. denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. or heat denaturated double stranded. inexpensive. 6. The advantages of the method include speed. HIGH EFFICIENCY TRANSFORMATION OF INTACT YEAST CELLS USING SINGLE STRANDED NUCLEIC ACIDS AS A CARRIER A method. avoidance of additional cloning steps into single-stranded phage M13 vectors.GENOMIC SEQUENCING Unique DNA sequences can be determined directly from mouse genomic DNA. simplicity.analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). was effective. indicating a difference in the mechanism of transformation with the two methods. and sequence determination using oligodeoxyribonucleotide-primed enzymatic DNA synthesis in the presence of dideoxynucleoside triphosphates. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA. 5. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. Numerous different sequences can be obtained from a single membrane by reprobing. and hence applicability to sequencing large numbers of samples. The method is simple and rapid: the recombinant plasmid DNA is . Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake. 3.5% transformants per viable cell. nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. DNA methylation at deoxycytidines. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms. The use of single stranded. is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1. under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.DIDEOXY SEQUENCING METHOD USING DENATURED PLASMID TEMPLATES The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved.

Addition of dextran sulfate beyond 10-12% in 2. The formation of common high molecular weight complexes during joint reannealing of two DNA's with complementary sequences was used as a method to detect sequence homology in . The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers. (ii) a heptadecamer rather than a pentadecamer is recommended as a primer. 3) Renaturation and Hybridization Studies of Mitochondrial DNA The products of the renaturation reaction of mitochondrial DNA from oocytes of Xenopus laevis have been studied by electron microscopy and CsCl equilibrium density gradient centrifugation.4M Na+. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.) Renaturation of DNA in the presence of ethidium bromide The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The acceleration in 2M NaCl occurs without loss of the normal concentration and temperature dependence of DNA renaturation and is also independent of dextran sulfate concentration if sufficient dextran sulfate is used. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.4M Et4NCl fails to increase the acceleration beyond approximately 10-fold. and 75°C and for native DNA at 65°C in 0. Accelerations of 100-fold may be achieved with 3540% dextran sulfate in 1M NaCl at 70°C. the rate drops off precipitously approaching zero at 0. although acceleration may be achieved in solvents containing formamide or other denaturants. Dextran sulfate may be selectively precipitated by use of 1M CsCl. No other mixed solvent system was found to be more effective.03 moles per mole of nucleotide. 65. ADVANTAGES OF RENATURATION OF DNA 1.03 moles. and (iii) the sequencing reaction should be done at 37 degrees C or higher rather than at room temperature. and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA. The formation of circular molecules of the same length as native circles of mitochondrial DNA was also observed. 2) One hundred-fold acceleration of DNA renaturation rates in solution Solvents which accelerate DNA renaturation rates have been investigated. We examined each step of the procedure.06 moles bound ethidium per nucleotide at 65°C respectively.extracted and purified by rapid alkaline lysis followed by ribonuclease treatment. The reaction leads to the formation of intermediates containing single-stranded and double-stranded regions. Volume exclusion by dextran sulfate is the most effective method of accelerating DNA renaturation with concentrated DNA. It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55.4M Et4NCl initially increases renaturation rates at 45°C and then leads to a loss of second-order behavior.08 and 0. Further reactions of these intermediates result in large complexes of interlinking doublestranded filaments. The greatest accelerations are seen with LiCl and dilute DNA. The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA. Addition of NaCl or LiCl to DNA in 2. Above 0.

These are allergenicity. allergenic responses by factory workers are a very significant problem particularly when fine-dusting powders are employed. being proteins. dry enzyme preparations have been replaced to a large extent by liquid preparations. Although this method does not produce quantitative data it offers several advantages in the present study.different DNA samples. a procedure that has been applied most successfully to the proteases and other enzymes used in detergents. Enzyme producers and users recognise that allergenicity will always be a potential problem and provide safety information concerning the handling of enzyme preparations. and chemical toxicity. The problem has been largely overcome by encapsulating and granulating dry enzyme preparations. laevis and yeast. They stress that dust . laevis and the chick. All enzymes. Once an individual has developed an immune response as a result of inhalation or skin contact with the enzyme. Workers in such environments are usually screened for allergies and respiratory problems. activity-related toxicity. laevis and the mouse and between the mitochondrial DNA's of X. are potential allergens and have especially potent effects if inhaled as a dust. Chapter-23 Safety and regulatory aspects of enzyme use Introduction Only very few enzymes present hazards. to those handling them in normal circumstances but there are several areas of potential hazard arising from their chemical nature and source. re-exposure produces increasingly severe responses becoming dangerous or even fatal. Where dry preparations must be used. but none between the mitochondrial DNA's of X. No homologies could be detected between the nuclear DNA and the mitochondrial DNA of X. residual microbiological activity. These results are interpreted as indicating the continuity of mitochondrial DNA during evolution. Because of this. as in the formulation of many enzyme detergents. sometimes deliberately made viscous to lower the likelihood of aerosol formation during handling. laevis or of Rana pipiens. In interspecies comparisons homologies were found between the nuclear DNA's of X. because of their catalytic activity.

rennet and trypsin. Aspergillus oryzae. Substances for which the available evidence suggests toxicity and which ought not to be permitted for use in food until adequate evidence of their safety has been provided to establish their acceptability. Aspergillus niger. Rhizopus oryzae. glucose oxidase. Bacillus licheniformis. and Klebsiella in8aerogenes. glucose isomerase. Bacillus coagulans. Activity-related toxicity is much rarer but it must be remembered that proteases are potentially dangerous. enzymes must be Generally Regarded As Safe (GRAS) by the FDA (Food and Drug Administration) in order to be used as a food ingredient. many of which have been used in food or food processing for many hundreds of years. Kluyveromyces lactis and Mucor javanicus. Class cin8 microorganisms that are not included in Classes b and c. invertase. Substances for which inadequate or no toxicological data are available and for which it is not possible to express an opinion as to their acceptability for use in food. Group E. Liquid preparations are inherently safer but it is important that any spilt enzyme is not allowed to dry as dust formation can then occur. particularly in concentrated forms and especially if inhaled. No enzyme has been found to be toxic. lactase. after first moistening it with water. However. Actinoplanes missouriensis. catalase. . rennet and various other proteases. including Mucor miehei. pepsin. In the USA. Saccharomyces cerevisiae. The enzymes that fall into group A are exclusively plant and animal enzymes such as papain. including Bacillus subtilis. Such enzymes include α-amylase. Any waste enzyme powder should be dissolved in water before disposal into the sewage system.in the air should be avoided so weighing and manipulation of dry powders should be carried out in closed systems. enzyme preparations cannot be regarded as completely safe as such dangerous materials may be present as contaminants. the Food Additives and Contaminants Committee (FACC) of the Ministry of Agriculture. Group B. derived from the enzyme source or produced during its processing or storage. Trichoderma reesei. mutagenic or carcinogenic by itself as might be expected from its proteinaceous structure. lipase. In the UK.β-amylase. Fisheries and Food classified enzymes into five classes on the basis of their safety for presence in the foods and use in their manufacture. Enzyme on the skin or inhaled should be washed with plenty of water. bromelain. Group D. glucoamylase. Substances that on the available evidence may be regarded as provisionally acceptable for use in food but about which further information must be made available within a specified time for review. by poor operating procedures in centrifugation) must be avoided as these are at least as harmful as powders. The Association of Microbial Food Enzyme Producers (AMFEP) has suggested subdivisions of the FACC's group B into: Class ain8 microorganisms that have traditionally been used in food or in food processing. The formation of aerosols (e. Streptomyces albus. lipase. galactosidase. catalase. cellulase. pectinase. Group B contains a very wide range of enzymes from microbial sources. Substances for which the available information indicates definite or probable toxicity and which ought not to be permitted for use in food. Substances that the available evidence suggests are acceptable for use in food. Class bin8 microorganisms that are accepted as harmless contaminants present in food. This classification takes into account the potential chemical toxicity from microbial secondary metabolites such as mycotoxins and aflotoxins. The growing body of knowledge on the long-term effects of exposure to these toxins is one of the major reasons for the tightening of legislative controls. including Bacillus stearothermophilus. ficin. papain. Group A. The organisms used in the production of enzymes may themselves be sources of hazardous materials and have been the chief focus of attention by the regulatory authorities.g. Any spilt enzyme powder should be removed immediately. Group C. Kluyveromyces fragilis.

Mechanism of Catalysis. This is an extremely safe technique. Module II Specificity of enzyme action. teratogenicity. Nomenclature. ENZYMOLOGY AND ENZYME TECHNOLOGY Course Code: BTBBT 30602 Course Objective: The course aims to provide an understanding of the principles and application of proteins. Course Contents: Module I Enzymes: Introduction and scope. Some of the safety problems associated with the use of free enzymes may be overcome by using immobilised enzymes . Linear plots. It may prove more satisfactory to clone such an enzyme into one of AMFEP's Class a organisms but this will first require new legislation to regulate the use of cloned microbes in foodstuffs. Module III Enzyme Kinetics: Single substrate steady state kinetics. ping-pong mechanism. The cost of the various tests needed to satisfy the legal requirements are very significant and must be considered during the determination of process costs. in vivo mutagenicity. Sigmoidal kinetics . Alberty equation. and in vitro mutagenicity. The theoretical understanding of biochemical systems would certainly help to interpret the results of laboratory experiments. Michaelis Menten equation. Multisubstrate systems. In addition Class c should be tested for microorganism pathogenicity and. secondary metabolites and enzyme biochemistry in therapeutic applications and clinical diagnosis. leak into the product stream. monomeric and oligomeric enzymes. nor the immobilised enzymes. King-Altman’s method. so long as the materials used are acceptable and neither they. It was proposed that Class a should not be subjected to testing and that Classes b and c should be subjected to the following tests: acute oral toxicity in mice and rats. subacute oral toxicity for 4 weeks in rats. Enzyme inhibition. Inhibitors and activators. Plainly the introduction of an enzyme from a totally new source will be a very expensive matter.and Penicillium emersonii. and carcinogenicity. oral toxicity for 3 months in rats. under exceptional circumstances.

B. Mieras.A. ENZYMOLOGY AND ENZYME TECHNOLOGY LAB Course Code: BTBPL 30622 Course Objective: The laboratory will help the students to isolate enzymes from different sources. smbhatt_bhu@rediffmail. References • Enzymes Biochemistry. Module V Immobilization of Enzymes. enzyme utilization in industry.D. John Wiley and Sons Inc. Trevor Palner Dr. Carriers. Copeland. Cambridge University Press. Module VI Biotechnological applications of enzymes: Large scale production and purification of enzymes. nitrate reductase. Uhlig. Butterworth Heinemann.H. R. covalent coupling. R. Course Contents: Module I Isolation of enzymes from plant and microbial sources. • Enzyme Technology. Mechanism and Data Analysis. Biotol Partners Staff. cellulase. M. activity and specific activity – determination of amylase.F. Clinical Chemistry. Chaplin and C. Bhatt smbhatt@amity.com phone 9313993840 Amity University Uttar Pradesh • • Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. Micro-environmental effects. Bucke. Advantages. V. Biotechnology. • Enzymes: A Practical Introduction to Structure.and Allosteric enzymes Module IV Extraction & purification of enzymes. protease Module III Purification of Enzyme by ammonium sulphate fractionation . S. I. enzyme assays and studying their kinetic parameters which have immense importance in industrial processes. Currell.C. Wiley-Interscience • Industrial Enzymes & their applications. John Wiley and Sons Inc. M. Segel. enzymes and recombinant DNA technology Examination Scheme: Component Codes Weight age (%) H/Q 10 S 10 CT2 20 EE 60 Text & References: Text • Biotechnological Innovations in Chemical Synthesis. Module II Enzyme assay. adsorption.edu . cross-linking and entrapment methods. H.

Lock and Key Model for Enzyme Activity B. Sawhney and Singh Chapter. determination of Michaelis-Menten constant (Km) and Maximum Velocity (Vmax. Simple enzymes B.1 Enzymes an introduction 9 Definition Introduction Historical aspects in enzyme discovery Classification of Enzymes. Catalysis by Proximity and Orientation c. Induced Fit (Hand and Glove) Model of Enzyme Activity Mechanisms of Enzyme Catalysis Chapter 4 Enzyme-Substrate Interactions 24 a. Complex enzymes Complex enzymes Role of Coenzymes A. Catalysts B. Catalysis by Bond Strain b. Text: Practical Biochemistry. Module V Effect of Temperature and pH on enzyme activity Module VI Enzyme immobilization Examination Scheme: Major Experiments: Minor Experiments: Spotting: Viva: Records: Total: 40 20 10 20 10 100 Note: Minor variation could be there depending on the examiner. Biocatalyst or Enzymes How enzymes speed up the reaction without taking part Unit of activity Chapter 3 Enzyme Specificity 20 ENZYME MECHANISM MODELS A. application of enzymes Chapter-2 Types of Enzymes 16 A.) using Lineweaver-Burk plot. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases) Concerted acid-base catalysis d. Covalent Catalysis: .Module IV Enzyme Kinetics: Effect of varying substrate concentration on enzyme activity.

Noncompetitive inhibition Determination of Vmax and Km Chapter-8 Multisubstrate enzymes 83 Enzyme Catalysis Mechanisms of Chemical Catalysis one-substrate enzymes: Linear plots for enzyme kinetic studies FYI . Cell breakage by osmotic shock B. Uncompetitive inhibition C. Catalysis in organic solvents Some advantages to using enzymes in non-aqueous solvents include (Dordick. Test of purity is done by following method Source of enzyme Media for enzyme production Cell can be break down byfollowing methods A. These include Abzymes Exercises Chapter-5 ENZYME KINETICS & INHIBITION 46 Chemical Reactions and Rates Chemical Reaction Order Michaelis-Menton constant a. ULTRACENTRIFUGATION A. Cell lysis by High pressure homogenisers Method of separation of enzyme from lysed cell.Covalent Intermediates e) Metal Ion Catalysis Catalytic Power a) Proximity b) Orientation f. Steady state:-Derivation: Catalytic efficiency and Turnover number. Method of separation. Chapter-6 Factor affecting the rate of catalysis 58 Substrate concentration Effect of temperature and pressure Effect of pH on Enzyme Chapter-7 Enzyme inhibition 68 A. Use of enzymic lytic methods c. Pre-steady state:-b.The Eadie-Hofstee Plot ENZYME KINETICS AND INHIBITION What's exciting about enzyme inhibition? Multi-substrate Enzymes Chapter-9 ISOLATION of ENZYME 102 INTRODUCTION strategies for enzyme purification. Competitive inhibition B. Ultrasonic cell disruption d. CentrifugationB. Differential centrifugation Filtration of enzyme after separation Separation of enzyme in Aqueous biphasic systems . 1989): Importance and relevance of whole cell enzymes in biotechnology There may be some drawbacks to using whole cells in catalysis.

Polyacrylamide Gel Electrophoresis c.Preparation of enzymes from clarified solution. Agarose Gel Electrophoresis b. Ultrafiltration Chapter-10 Concentration and purification of enzyme 123 Concentration by precipitation A. SDS-PAGE f. AGAROSE GEL ELECTROPHORESIS OF DNA Preparation of the gel Polyacrylamide gel Chapter -12 Enzyme Immobilization 160 127 . Chromatography Principles of chromatography Adsorption Chromatography COLUMN CHROMATOGRAPHY History Principle Mechanism of separation by size in gel permeation chromatography ADVANTAGE OF GEL –CHROMATOGRAPHY Application of GPC or gel permeation chromatography PAPER CHROMATOGRAPHY Ion –exchange chromatography There are two type of ion exchanger Choosing an Ion Exchanger Gas / Liquid Chromatography Reversed Phase Chromatography HIGH PRESSURE LIQUID CHROMATOGRAPHY [HPLC] Partition Chromatography Affinity chromatography Principle METHOD OF LIGAND IMMOBILIZATION 1.11 Maintaining Enzyme Activity Chapter-11 Techniques used in Enzyme characterization 132 HOW TO KNOW THE PROPERTY OF ENZYME Introduction A. Carbomyl diimidazole activated agarose. Heat treatment 1.6 DIAMINO HEXANE. 3. Iso-electric focusing: IEF gel: g. 6-AMINO-HEXENOIC ACID & 1. Chromatofocussing 2D-PAGE DETECTION. Applications Thin layer chromatography Principle ADVANTAGE OF TLC OVER OTHER CHROMATOGRAPHY TECHNIQUE HPTLC and HPPLC Selection of chromatographic system Electrophoretic technique a. Nucleic acid removal B. Enzyme purification by Chromatography 2 Affinity chromatography .3 Hydrophobic interaction chromatography (HIC) or affinity elution CHOICE OF MATRIX Electrophoresis 2. i. ESTIMATION AND RECOVERY OF PROTEIN GELS. CNBR activated agarose 2.

Use of immobilised lactase .In Heart disease α-amylase Creatine Kinase and fructose bisphosphate aldolase. In Treatment of cancer Enzyme deficiencies Enzyme inhibitors and drug design 183 Use of enzyme in Dignosis Blood glucose Disadvantages of Using Enzyme as Therapeutic Agents Thermozymes The large-scale use of enzymes in solution The use of enzymes in detergents Chapter-15Application of Enzyme in Industries . 2. 3-Use of immobilised raffinase 4-Use of immobilised Invertase 5-Production of amino acids 6.Introduction Aim of Enzyme Immobilization [a] Physical properties [b] Chemical properties [c] Stability [d] Resistance [e] Safety [f] Economic Limitations of Immobilized Enzyme Advantages of Immobilizations Methods of immobilizations Carrier matrices Adsorption of enzymes Some disadvantages of adsorption include Covalent coupling Functional groups that affects the covalent coupling A.7 Production of antibiotics Preparation of acrylamide Chapter-14 Application of Enzyme in clinics 233 Introduction Determination of enzyme activities 1. Alkaline phosphates Acid phosphatase.Clinical Enzymology of liver disease. cyanogen bromide (b) Ethyl chloroformate (c) Carbodiimide (d) Glutaraldehyde (e) 3-aminopropyltriethoxysilane Entrapment and Encapsulation Entrapment of enzymes Membrane confinement Encapsulation of Enzymes Crosslinking Chapter-13 Introduction to Industrial biotechnology 171 Application of Immobilised-Enzyme Processes Introduction 172 1-High-fructose corn syrups (HFCS) 2-GLUCOSE ISOMERASE a Treatment with activated carbon.

different sector of industry where enzymes are used Food Textile and other indsutries Chapter-16 Enzyme Reactors all type of reactors detailed dscription kinetics involved in reactors Chapter-17 Protein Engineering 215 Artificial enzymes Chapter-18 Biosensors and Immunosensors 225 Chapter-19 Enzyme and protein STABILITY 246 I Definition of Stability II The Unfolded State III Major Factors Affecting Protein Stability The Hydrophobic Effect Hydrogen Bonds Conformational Entropy of Unfolding IV Other Factors Affecting Protein Stability Salt Bridges Aromatic-Aromatic Interactions Metal Binding Disulphide Bonds V Chemical Degradation VI Conclusions Chapter -20 Study of drug and molecule binding stratgies 260 TYPES OF DNA BINDING DRUGS NON COVALENT INTERACTIONS Minor Groove binders INTERCALATORS COVALENT INTERACTIONS DNA-Drug Interaction : Chapter-21 Technique to study protein 284 Nuclear Magnetic Resonance(NMR)Spectroscopy Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin. Structure of bacteriorhodopsin: Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin Chapter-22 Enzymology of DNA folding and unfolding 290 WHAT IS DNA Major and minor grooves Replication Enzyme involved in DNA folding and unfolding Invitro-DNA folding and unfolding Folding unfolding kinetics Chapter-23 Safety and regulatory aspects of enzyme use 305 .

. Chapter.. depending on its electrical charge. vertically down a gel by net charge and horizontally by molecular mass).g. Each unique protein mixture produces a characteristic pattern or fingerprint of protein separation on a 2D gel... In onedimensional (1D) gel electrophoresis.Module I Enzymes: Introduction and scope. which can be used to detect the number of binding sites on an enzyme.. iron (Fe2+ or Fe3+). which monitors. Automated sorting devices. Enzyme: Protein that acts as a catalyst..g. freeenergy change. and the scattering pattern of the X rays is used to create 3D representations of the crystal with atomic resolution. zinc (Zn2+). entropy change) for molecular interactions. X-ray crystallography: Technique used to obtain structural information for a substance (e.g. Steady State: Growth state in which the concentration of bacterial cells is in equilibrium with the concentration of nutrients or substrates (i. Electrophoresis: Method of separating large molecules (such as DNA fragments or proteins) in a sample. Metalloprotein: Protein that incorporates one or more metals into its molecular structure by binding individual metal ions [e.g. how quickly reactants are converted into products) under various conditions. Metalloprotein are important components of electron transport chains. Nomenclature. and physical processes (e. each molecule travels through the medium at a different rate. primarily by size.g.e.g. . Two-dimensional (2D) gel electrophoresis is used in proteome analyses to separate complex protein mixtures using two separation planes (e. An electric current is passed through a medium containing the sample. Flow Cytometry: Analysis of biological material by detection of light-absorbing or fluorescing properties of cells or subcellular components (e. speeding the rate of a biochemical reaction but not altering its direction or nature. chemical. shape and size. chromosomes) passing in a narrow stream through a laser beam. protein. enthalpy change. Agarose and acrylamide gels are commonly used media for electrophoresis of proteins and nucleic acids. and differential scanning calorimetry (DSC).. Two kinds of calorimetry important to biomolecular studies are isothermal titration calorimetry (ITC). the concentrations remain constant over time). or magnesium (Mg2+)] or nonprotein organic compounds containing metals. An absorbance or fluorescence profile of the sample is produced. Mechanism of Catalysis. separate successive droplets of the analyzed stream into different fractions depending on the fluorescence emitted by each droplet. molecular complex) that has been crystallized. proteins and nucleic acids are separated in one direction on a gel. dissociation constant. A beam of X rays is focused on the crystals. Kinetics: Field of study that deals with determining the rates of biological.1 Enzymes an introduction Definition Calorimetry: Measurement of heat released or taken up in a chemical reaction or physical process to derive thermodynamic data (e. used to fractionate samples.

Ribonuclease was artificially synthesized from amino acid precursor. They were called as ribozymes. They are also regulated very precisely.Introduction Enzymes are the wonderful creations of the biological system. structural orientations and strain. Homones in several cases also play important role in regulations like in formation of lactose by progestron and prolactin before and after the birth of baby. Enzymes have specificity not only toward the substrate but also recovered in the end of the reactions. Various biological reactions go on smoothly without single mistakes. e. In several cases certain organic molecule or metals termed as co-enzyme or cofactor that either or electron or proton acceptor or by electrostatic mechanism are needed to convert a substrate to product. Ribonuclease amino acid was sequenced. 1920 Sumner crystallized Urease first time that hydrolyze the urea into CO2 and NH3. Modern techniques like MOLDI-TOF. three dimensional structure of Lysozyme was deduced. x-ray crystallography along with sequencer helped a lot to scientist in deciphering the mechanism of enzyme activity. or by allosteric mechanism. 1897 Buchner isolated yeast extract used for formation of ethanol. 1958 and 1960 1961 1965 1986 Koshland proposed induced fit model to account for enzyme catalytic power specificity. Any deficiency in the formation of these pockets like structure may render the whole enzyme to become inactive that leads to certain deficiency diseases. Total activity of enzyme depends on several factors like acid base. Historical aspects in enzyme discovery 1860 Bertholate disrupted yeast cell and isolated extract that catalyzed conversion of sucrose to glucose and fructose. RNase was found to work as catalyst by Cech. electrostatic interactions (by metal ions) and formation of covalent bond. 1894 Fisher proposed lock and key hypothesis to explain the mechanism of Enzyme operating between substrate and enzyme. Normal monomeric enzyme like trypsin and pepsin are regulated by removing or adding a small piece of peptide that make them inactive or active while the complex enzymes are regulated by end product . The system (complex enzymes are termed as system) specifically and precisely binds to the substrate and convert them into the required product that sometimes becomes the substrate for next reaction for next enzyme to act on. Knowledge of enzyme activity and its detailed mechanism made it possible to design artificial enzyme in the lab to cop up with many enzyme related metabolic problems or complex viral enzyme like AIDS.g. 1878 Word enzyme first used by kuhne. Actually enzymes activity depends on the precise function of a small part called as the active site that is a small pocket like structure that contains various helper amino acid residues that by electron or proton transport make bond of the substrate molecule weaker and rearrange them into a stable product at normal temperature and pressure. Ribonuclease –P .

and transmethylases. For Fe+3 3. amylase. most enzymes are still called by their common name. Its second digit has been denoted in the table. Examples such as Phosphatases.+ NAD+ ---------.7. Each enzyme has assigned 4 digit classification number and a systematic name.C.These are enzymes that catalyze oxidations or reductions. Examples are the digestive enzymes such as sucrase. 3 for ether bond.1.U.These enzymes catalyze the transfer of a group from one molecule to another. In 1955 International Union of Biochemistry classified the enzyme on the basis of kind of reaction they performed.+ NADH+ + H+ 2. For example alkaline phophatases is a hydrolase that hydrolyse variety of substrate at alkaline pH.e. while 4th digit is the actual substrate. They are classified according to the type of bond hydrolysed 1 for ester 2 for glycosidic bond. For example 1-for NAD+ or NADP+ 2. For e. third and four digits is the group transfer or accepted as shown in figure. system also specifies a textual name for each enzyme. and the second digit denotes the subclass. the product(s) and the enzyme's functional class. 4 for peptide bond etc. Thus 1 denotes transfer of CH3 group while 2 denotes the transfer of CH2OH and 3 denotes the carboxyl or carbamoyl transferases. 1 for single carbon unit) that catalyses the transfer of phosphate from ATP to D hexose resulting the D hexose-6-phosphate. EC 2.1 or D-hexose-6-phosphotransferase. Classes of enzyme: there are six classes of enzyme according to reaction type they performed 1. transaminases. 2. and peroxidases. 1.1 means first digit indicates the class transferases. Enzymes such as dehyrogenases. D-hexose-6-phosphotransferase (hexokinase) D hexose + ATP-----------------------------------phosphate + ADP D hexose-6- 3. maltase. 4 for phosphoric diester hydrolases. third digit denotes the type of bond hydrolysed like 1 for hydrolysis of ester bond. In EC number 1 digit denotes type of reaction i.27). There are major 6 classes and each with the subclass. Its second digit has been provided in the table. 2 for thiol ester bond. position of group or functional group and number of amino acid. oxidases. The I.1.1. Its third digit denotes the group transferred. 3rd digit denotes NAD as electron acceptor NAD+. Lactate dehydrogenase CH3CH(OH)COO. Oxidation means transfer of oxygen atom and reduction means transfer of H atom.Classification of Enzymes. In everyday usage.U.B. and lactase. such as alcohol dehydrogenase. the change to I.approved nomenclature has been slow. For clear understanding we can take example of S-latate NAD+ oxidoreductase (E. 7 for phosphate group. Here S lactate is the substrate and NAD+ is electron acceptor/ hydrogen acceptor and the reaction is of type oxidation reaction. . Because many enzymes.1.g. The enzyme's name is comprised of the names of the substrate(s). 3 for monoester bond.CH3C(O)COO. Its third digit refers to the hydrogen or electron acceptor.B. Therefore they divided the enzyme into six major classes. are widely known in the scientific community by their common names. Transferases. Phosphotransferases are given trivial name as kinase for example hexokinases (E. Oxidoreductases.7. Suffix added in the enzyme is –ase to the name of substrate or to word. Hydrolases-These enzymes catalyze hydrolysis reactions. substrate they act. Urea hydrolysis is done by the Urease. For oxygen. oxidoreductase 2nd digit is the functional group of lactate which is alcohol therefore it is denoted by subclass 1.C.

These are also called synthetases which are enzymes that catalyze condensation reactions where smaller molecules are connected with the resulting removal of a water molecule.8 Transferring sulfur-containing groups EC 2.6 Acting on NADH or NADPH EC 1. This is accompanied with the formation of a high energy Phosphate link that stores energy. Lyases.15 Acting on superoxide radicals as acceptor EC 1.1 Acting on the CH-OH group of donors EC 1. Ligases.18 Acting on iron-sulfur proteins as donors EC 1. Its second digit denotes the bond broken and third digit denotes type of group removed like 1 for carboxyl group and 2 for aldehyde group and 3 for ketoacid group.14 Acting on paired donors.4 Glycosyltransferases EC 2.1 Transferring one-carbon groups EC 2.These enzymes catalyze the removal of groups and create double bond in non-aqueous media.97 Other oxidoreductases EC 2 Transferases EC 2.2 Transferring aldehyde or ketonic groups EC 2.8 Acting on a sulfur group of donors EC 1. 2nd digit refers to the type of reaction like 1 for recemization or epimerization (if inversion occurs at assymitric carbon having four different attached group. other than methyl groups EC 2.7 Acting on other nitrogenous compounds as donors EC 1.2 Acting on the aldehyde or oxo group of donors EC 1.10 Acting on diphenols and related substances as donors EC 1.19 Acting on reduced flavodoxin as donor EC 1.3 Acyltransferases EC 2. Subclass Name EC 1 Oxidoreductases EC 1. L histidine carboxy lyase histidine -----------------------------------histamine + CO2 5.5 Transferring alkyl or aryl groups.6 Transferring nitrogenous groups EC 2.16 Oxidising metal ions EC 1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases) EC 1.7 Transferring phosphorus-containing groups EC 2.3 Acting on the CH-CH group of donors EC 1.11 Acting on a peroxide as acceptor EC 1. with incorporation or reduction of molecular oxygen EC 1.20 Acting on phosphorus or arsenic in donors EC 1.5 Acting on the CH-NH group of donors EC 1. An example would be the decarboxylases. 6.12 Acting on hydrogen as donor EC 1. Isomerases.4 Acting on the CH-NH2 group of donors EC 1.17 Acting on CH or CH2 groups EC 1.Enzymes that catalyze the isomerization of molecules means L isomer will be D isomer after catalysis and cis isomer will be trans isomer after the catalysis for Examples L alanine will be converted into the D alanine by alanine racemases.21 Acting on X-H and Y-H to form an X-Y bond EC 1.9 Transferring selenium-containing groups .4.9 Acting on a heme group of donors EC 1. An example would be the amino acid RNA Ligases.

Oxidoreductases Transferases .10 EC 3.12 EC 3.3 EC 5.2 EC 6.11 EC 3. other than peptide bonds Acting on acid anhydrides Acting on carbon-carbon bonds Acting on halide bonds Acting on phosphorus-nitrogen bonds Acting on sulfur-nitrogen bonds Acting on carbon-phosphorus bonds Acting on sulfur-sulfur bonds Acting on carbon-sulfur bonds Carbon-carbon lyases Carbon-oxygen lyases Carbon-nitrogen lyases Carbon-sulfur lyases Carbon-halide lyases Phosphorus-oxygen lyases Other lyases Racemases and epimerases cis-trans-Isomerases Intramolecular isomerases Intramolecular transferases (mutases) Intramolecular lyases Other isomerases Forming Forming Forming Forming Forming Forming carbon—oxygen bonds carbon—sulfur bonds carbon—nitrogen bonds carbon—carbon bonds phosphoric ester bonds nitrogen—metal bonds Class Reaction catalyzed.1 EC 3.4 EC 4.3 EC 6.6 SN 1 2 3 4 5 6 Acting on ester bonds Glycosylases Acting on ether bonds Acting on peptide bonds (peptidases) Acting on carbon-nitrogen bonds.5 EC 5.5 EC 4.4 EC 3.2 EC 4.8 EC 3.6 EC 3.3 EC 4.EC 3 Hydrolases EC 3.9 EC 3.99 EC 6 Ligases EC 6.13 EC 4 Lyases EC 4.2 EC 3.2 EC 5.6 EC 4.7 EC 3.5 EC 6.5 EC 3.4 EC 6.1 EC 5.1 EC 6.3 EC 3.1 EC 4.99 EC 5 Isomerases EC 5.4 EC 5.

C-N. Cytochrome oxidase Carbonic anhydrase Alcohol dehydrogenase Hexokinase Glucose -6-phosphate Pyruvate kinase.C-S. Urease Dinitrogenase Glutathione peroxidase Fe+2 or Fe+3 Cu+2 Zn+2 Mg+2 . Formation of C-C. Arginase Ribonucleotide reductase Pyruvate kinase.C-O. Some metalloenzymes and their cofactor Enzyme Cofactor Cytochrome oxidase Catalase Peroxidase.Hydrolases Lyases Isomerases Ligase Electron transfer Group transfer Hydrolysis Addition of group to double bonds Transfer of group to yield isomeric forms.

and RNAase. Pepsin. Lysozyme. Lactase. single or multiple subunits. Other enzyme consisting of two or more polypeptide are termed as oligomeric enzyme. Actually Ogstien considered three sites on enzyme one or two binding site along with one catalytic subunit. Monomeric enzymes are of few 200-300 amino acid and molecular weight ranges from 15. Non-protein part called “coenzyme” or “prosthetic Group”.000 kd to 35. and others. Therefore multiple substrates can interact with .Enzymes that catalyze the addition of Hydrogen atoms • Oxidases. • Hydrolases-Enzymes that catalyze hydrolysis reactions. Thus they contain four subunit and substrate can bind to each of four subunit and they are termed as isozyme. Complex enzymes Complex enzymes They involve protein plus small organic molecule(s) or metal. Enzymes such as. HM3. Other enzymes have retained common names attributed to them. Chapter-2 Types of Enzymes A. H2M2. If they consist of single polypeptide unit they are termed as monomeric enzyme ( sometime like in chymotrypsinogen they are of three polypeptide and are inctive zymogen form and after cleavage from pie to delta and finally to alpha trypsinogen they become active and consist of two polypepide and still they are termed as monomeic enzyme since they are read in continous sequences).Enzymes that catalyze oxidations In addition each enzyme has a specific name which usually consists of part of the name of the substrate molecule. Like alcohol dehydrogenase (contains two polypeptide and Zn+2 and act by electrostatic meechansim) and lactate dehydrogenase that requires cofactor NAD+ or NADP+ for its activity and occurs in multiple unit like H4. Lactate is formed from pyruvate in aneorbic condition and thus pyruvate accumulation are avoided. Maltase. Alpha Amylase. and M4.Mn+2 K+ Ni+2 Mo Se In addition to the classification name most enzymes have group names. Simple enzymes They are composed solely of protein. elastases.000 kd (kilodalton) like trypsin . Lactate arev then transported to other organs. Examples are sucrase. Protein part called “apoenzyme”. They form the integral part of the enzyme and remain adjacent to the catalytic center to provide coordination for interacting with substrate functional group. B. Thus a slight decrease in entropy make free energy negative that is essential for stabilization of intermediate transition state). Trypsin. that are termed as serine proteases because of an active serine residue. galactosidase. Entire complex called “holoenzyme”. Catalase. H3M. Chymotrypsin. Serine residue become active due to relay mechanism of electron transfer (adjacent amino acid can transfer electron in series of pathway and make the serine active sufficient to active at electron deficient or carbocation center of substrate making breaking of bond possible. • hydrogenases.

A. and Iron serve as industrial catalysts that catalyze a wide variety of reactions such as Hydrogenation. Enzymes are also classified on the basis of their composition. Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions that is with very high affinity. The detailed mechanism will be discussed in next topic under the head of enzyme specificities. which are composed of protein plus a relatively small organic molecule. Certain transition state metals like Palladium. The catalyst and the reactant molecules are not in the same phase. are known as metal-activated enzymes. The chemical groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes.enzyme and react with them. Enzymes composed completely of protein are known as simple enzymes in contrast to complex enzymes. Role of Coenzymes The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. which are used up during the course of a reaction) coenzymes are generally regenerated. Heterogeneous Catalysts 2. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst. Those have a lower affinity for metal ion. Since coenzymes are chemically changed as a consequence of enzyme action. or second substrates. All current research suggests that enzymes are extremely specific in what a given enzyme catalyzes. it is often useful to consider coenzymes to be a special class of substrates. since (unlike the usual substrates. This is sometimes referred to as surface catalysts. There are two types of catalysts: 1. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. which lack sufficient cofactors derived from vitamins to maintain homeostasis. The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. when the binding between the apoenzyme and non-protein components is non-covalent. but still require the metal ion for activity. Nickel. the coenzymes donate the carried chemical grouping to an acceptor molecule and are thus regenerated to their original form. In all cases. These rules give each enzyme a unique number. Platinum. or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate. the small organic molecule is called a coenzyme. Homogeneous Catalysts Heterogeneous catalysts are those that provide a surface for the reaction to proceed upon. Enzymes are very different. Complex enzymes are also known as holoenzymes. Homogeneous catalysts are catalysts that exist in the same phase as the reactant molecules usually in a solution. Catalysts Catalysts are substances that increase product formation by lowering the energy barrier (activation energy) for the product to form and increase the favorable orientation of colliding reactant molecules for product formation to be successful. while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds. Acids and Bases in solution serve as catalysts in a wide variety of Organic reactions. which are common to many different holoenzymes. Indeed. Most industrial catalysts are responsible for more than one catalysis among reactants and are considered relatively non-specific in what they catalyze. most enzyme molecules catalyze only one specific reaction but it does so in a . In this terminology the protein component is known as the apoenzyme.

All the chemical reactions inside the cell take place with the help of enzyme. This difference of energy is called as activation energy. The macromolecular components of almost all enzymes are composed of protein. In recent years Enzymes are in more demand due to their application in various biotech and pharmacy industry.phenomenally efficient manner. In old days renine (new name chymosine) were utilized for the formation of cheese from milk. the taq polymerase isolated from the thermus aquaticus. Actually an enzyme decreases the free energy between the ground state and the transition state. Higher activation energy reflects the slower reaction. Enzyme mediates rearrangement of the bond in substrate molecule by breaking of one covalent bond and formation of other covalent bond. Because of their role in maintaining life processes. The rate of reaction can be increases by increasing . B. One enzyme molecule might be responsible for converting thousands of reactant molecules called substrate molecules into product. found inside the cell. and aqueous environment. Biocatalyst or Enzymes Enzymes are the protenaceous catalyst of the biosphere. An enzyme provides specific environments found inside the cells. Most of the biological molecules are stable in the neutral pH. One such enzyme is utilized in the PCR polymerase chain reaction. Plasma membrane enzymes (membrane bound enzyme) regulate catalysis within cells in response to extracellular signals (signal may be any chemical or any substrate molecule). They have pocket like structure called as active sites ( is only small portion of total protein and contains substrate binding site and catalytic site. Enzyme may occur anywhere inside the cell. Molecule is bounded by the active sites and acted upon by the enzyme is called as substrate. and their supernatant is utilized for further purification and isolation by NH4 SO4. Enzymes increase the rate of reactions without involving themselves being altered in the process of substrate conversion to product They mediate the formation of several biomolecules like Polynucleotide (DNA. They are found in all the living system except the viruses. Catalytic site contains various amino acid residue which reacts with substrate and form a complex structure). The Biological Catalysts are efficient in lowering down the activation energy of the reaction and thus they occur at very low temperature and pressure. The bond of the substrate molecule comes under strain due to presence of several functional group of different type over amino acid residue present inside the active site of the enzyme. except for a class of RNA modifying catalysts known as ribozymes. Almost every significant life process is dependent on enzyme activity. Enzymes are found in all tissues and fluids of the body. Enzymes can be active at higher temperature and pH if isolated from the extremophiles bacteria living in the harsh condition. starch in plants. and enzymes of the circulatory system are responsible for regulating the clotting of blood. like cell cytoplasm. RNA) protein enzyme. Most of the study done regarding the ES complex since this complex decides overall rate of reactions means formation of products. Intracellular enzymes (enzyme located inside the cell and can be isolated after breaking the wall of the cell) catalyze the reactions of metabolic pathways. and cell organelle. Enzymes mostly mediate formation of specific isomer either L (+) or D (-). the assay and pharmacological regulation of enzymes have become key elements in clinical diagnosis and therapeutics. pH. mild temp. Enzymes are active at certain optimum temperature. They are specific towards the substrate and remains in inactive form inside the cell. vacuoles. Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Their isolation involves the breaking of cell membrane. ATP etc. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the Phosphodiester bond of other RNAs.

E . P Reaction coordinate R= reactant P = product ∆G = ∆H – T ∆S this is the thermodynamical term used to explain the stability of reactions.sometimes by as much as one millionfold. This complex is known as the activated state or transition state complex for the reaction. The result is that the reaction rate is increased. In unstabilized or uncatalysed system ∆S is positive and formation of transition complex renders them lowering of entropy leading to make overall free energy negative. A negative free energy stabilizes the system and makes the reaction possible. but more typically by about one thousand fold. although the magnitude of the rate constants of the forward and reverse reactions is are increased. it is apparent that enzymes and other catalysts have no effect on the equilibrium constant of the reactions they catalyze. which are regenerated during the course of a reaction. Catalysts speed up the forward and reverse reactions proportionately so that. The amount of energy required to achieve the transition state is lowered. Enzymes and other catalysts accelerate reactions by lowering the energy of the transition state. consequently. Enzymes increase reaction rates by decreasing the amount of energy required to form a complex of reactants that is competent to produce reaction products. SI unit is ‘katal’ that is defined as Micromole substrate consumed or product formed per second. at any instant a greater proportion of the molecules in the population can achieve the transition state. Catalyst or enzymes decreases this requirement by lowering down the activation energy. Unit of activity Micromole substrate consumed or product formed per minute is referred as IU international unit. See graph in the figure. Enzymes never disturb the equilibrium. ES Free energy G Enzyme lowers the activation energy.the temperature. ∆H denotes the enthalpy and ∆S denotes the entropy. A more ordered system also has lower entropy for example free amino acid has more entropy than the amino acid linked with the peptide bond. The free energy required to form an activated complex is much lower in the catalyzed reaction. the ratio of the rate constants remains the same in the presence or absence of enzyme. These biological catalysts are physiologically important because they speed up the rates of reactions many folds (up to 10 6 to 10 15 times than normal one) that would otherwise be too slow to support life. Since the equilibrium constant is equal to a ratio of rate constants. Enzymes increase reaction rates--. Transition state. How enzymes speed up the reaction without taking part? In cells and organisms most reactions are catalyzed by enzymes.

LDH is a tetrameric enzyme composed of all possible arrangements of two different protein subunits. often. . in fact. various isozymes of a group are expressed in different tissues of the body. They may be of product specific or substrate specific or stereospecific act on specific stereo molecules like alanine recemase acts on only L–alanine to convert them into Dalanine. the role of racemases is to convert D isomers to L isomers and vice versa. it follows that a given substrate may be acted on by a number of different enzymes. ethanol is the preferred substrate. and the all M isozymes is typically found in skeletal muscle and liver. Thus racemases attack both D and L forms of their substrate. They exhibit absolute specificity. Enzymes that attack D sugars will not attack the corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. These subunits combine in various combinations leading to 5 distinct isozymes. In the case of ADH. but they exhibit differing degrees of efficiency. each of which uses the same substrate(s) and produces the same product(s). The detection of specific LDH isozymes in the blood is highly diagnostic of tissue damage such as occurs during cardiac infarct. The best studied set of isozymes is the lactate dehydrogenase (LDH) system. Thus enzyme may be group specific if they act over on several closely related substrate for example alcohol dehydrogenase on which several alcohol are substrate. ranging from methanol to butanol. For example. the subunits are known as H (for heart) and M (for skeletal muscle). The enzymes known as racemases provide a striking exception to these generalities. The all H isozymes is characteristic of that from heart tissue.Chapter 3 Enzyme Specificity Although enzymes are highly specific for the kind of reaction they catalyze. These are the products of genes that vary only slightly. or act over by specific substrate like glucokinase that transfer phosphate from ATP to glucose. enzymes having broad substrate specificity are most active against one particular substrate. The individual members of a set of enzymes sharing such characteristics are known as isozymes. while succinic dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its substrate is invariably succinic acid. As enzymes have a more or less broad range of substrate specificity. Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. the same is not always true of substrates they attack. alcohol dehydrogenase (ADH) always catalyzes oxidation-reduction reactions but attacks a number of different alcohols. Generally. These isozymes all catalyze the same chemical reaction.

This is the reason why sometime in presence of non-polar solvent like DMSO dimethylsulphoxide some enzyme are more reactive. Therefore further cleavage occurs to make them active exposing the catalytic center. It will only use aspartate (not similar amino acids such as glutamate) and only the L-isomer. Aspartase catalyses the interconversion of aspartate and fumarate: L-Aspartate ↔ Fumarate + NH3 Fig .g. 1Lock and key hypothesis 2Induced fit hypothesis. trypsin and elastases having almost similar structure but differ in specificity due to variations in the side chain amino acids. Intermediate specificity is most common e. and character and must not block the substrate from binding. showing asymmetric binding site in the enzymes. shape. Nor will it add NH3 to maleate. A. Replacement of glycine by alanine make the enzyme more active. Most enzymes are very large relative to their substrates. The actual reaction occurs in the active site. or carboxypeptidase which snips the C-terminal amino-acid off many polypeptide chains. Three theory were proposed to explain the specificity of enzyme but all of them are correct and none of them are alone operative in the enzyme specificity. Specificity May Be Very Strict Or Relatively Broad Broad specificity is shown by many degradative enzymes e. alkaline phosphatase. The model of enzyme structure was proposed by fisher as three point one catalytic site and others are binding site. 3Transition state stabilization ENZYME MECHANISM MODELS There are two major considerations . the cis isomer of fumarate. and C4 alcohols. ie. For proper catalytic activity their side chains must be of suitable size. Such enzymes follows michaelis behaviour while others having complex and separate site behave in more complex way as allosteric site. Therefore both the subunit of enzyme like binding and catalytic site is the active center of the enzymes.Specificity is actually decided by amino acid residue present in the active site of enzyme that is capable of binding to the substrate molecule. For example chymotrypisn are inactive because their zymogen form don’t have accessible catalytic center. alcohol dehydrogenase. etc. a small region of the enzyme where the substrate is bound. For example of several seine proteases like chymostrypsinogen. pyruvate dehydrogenase will also use the C4 homolog of pyruvate etc. They are only small part of the total enzyme and they must be accessible to the substrate. coli will act on C2. The amino acids that are not involved in binding to the substrate do not have any contribution towards the specificity. which removes phosphates from a wide range of molecules.. Lock and Key Model for Enzyme Activity The high specificity and efficiency of enzymes can be explained by the manner that . C3.Some enzymes are absolutely specific. Enzymes must bind the correct substrate and position it correctly relative to catalytically active groups in the active site. Other examples include chymotrypsin.substrate specificity and catalytic power.g. Active site most often contains both polar as well as non-polar amino acid thus facilitates the interaction with both hydrophilic and hydrophobic amino acid. In Elastases substrate are blocked from binding due to presence of two bulky side chain valine and threonine while chymostrypsin and trypsin have no bulky group but instead contains glycine amino acid that facilitates the substrate to approach easily. Sometime enzymes have both catalytic as well as binding site as same site. alcohol (ethanol) dehydrogenase of E. a variety of enzymes from different classes have been shown to function in nonaqueous media.

The Induced Fit Model seems to explain why there is some flexibility in the abilility of the active site to accommodate other molecules and at limited temperature and pH ranges. Furthermore. The two substrates are brought together in the active site by a conformational change.the induced fit. Modification of the lock and key model is necessary to account for the occurrence of pH and temperature ranges of optimum enzyme activity and to explain why other molecules can effectively block the active site. certain molecules can lock into the active site even though the bogus molecules have a different shape compared to the true substrate molecule. An active site is an indentation or cavity whereby a reactant molecule (substrate) is attached to. The occurrence of pH and temperature ranges of optimum enzyme activity does not support the assumptions made by the lock and key model of ridged active site cavities. This tolerance would explain why bogus molecules of slightly different size compared to the true substrate molecule can still be accommodated by the elastic active site. Phosphoglycerate kinase catalyzes the synthesis of ATP. Induced fit example Hexokinase catalyzes the ATP-dependent phosphorylation of glucose. According to this model. This does not seem to support the ridged active site assumption in the lock and key model. Evidence does not support these assumptions. Such enzymes will have a lower value of kcat/KM (a numerical value to compare the catalytic efficiency of enzyme). Induced fit increases KM without increasing kcat. Small changes in temperature would distort the active site conformation but not so much that the active site could not still accommodate the substrate molecular size. small temperature changes and small changes in pH will not result in the enzyme being inhibited from catalyzing its intended reaction. An induced fit at an interface. Induced fit assumes that the active site of an enzyme is not complementary to that of the transition state in the absence of the substrate. because some of the binding energy must be used to support the conformational change in the enzyme. Once the product molecule has been formed the electrical attractions that made the substrate molecule adhere to the active site no longer are present. B.they associate with the reactant molecules called the substrate. Also the assumption is that the active site conformation is ridged. each enzyme molecule may have as few as one active site on the surface of the enzyme molecule itself. This is called the enzyme-substrate complex. and the product molecule can disengage itself from the active site thus freeing the site for another incoming substrate molecule. The binding of glucose to the enzyme induces a major conformational change . The analogy is like a hand fitting into a glove. Induced Fit (Hand and Glove) Model of Enzyme Activity Modification of the lock and key model assumes that the active site has a certain amount of elasticity whereby the active site can expand or contract in a limited way in order to accommodate the substrate molecule. This model assumes that molecules that lock into the active site must form a perfect fit. For example. The glove adjusts in shape and size to fit various sized hands within a certain range. This process occurs in a highly efficient manner hundreds or even thousands of times in a short time span. The polar and non-polar groups of the active site attract compatible groups on the substrate molecule so that the substrate molecule can effectively lock into the cavity and position itself for the necessary collisions and bond breaks and formations that must take place for successful conversion to a product molecule. This has the effect of inhibiting enzyme activity. These enzymes show virtually no activity against water-soluble substrates and only hydrolyze substrates present in the interface between water and lipid - . Lipases are enzymes that hydrolyze esters of long chain fatty acids. PH changes which would also change the active site conformation but not so much that the active site could not flexibly accommodate the substrate molecule. One of the first theories or models that help to explain this phenomenally efficient catalytic efficiency of enzymes is called the "lock and key" model.

They are engaged in the catalytic turn-over of the substrate. Its uniqueness is caused by the sequence and nature of its amino acid side chains. Atoms or ionized groups of the coenzyme take part in the catalytic reaction. In that way. The shape of the holoenzyme (= apoenzyme [protein] + coenzyme) causes the substrate selectivity. caused by the enzyme binding to the interface. Numerous enzymes depend on additional factors that are necessary to perform the catalytic reaction. The binding sets reactants into a state of enhanced reactivity recognizable by the close vicinity and the right orientation towards each other. but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to be altered. one or . compared to most of their substrate molecules. though the probability can be considerably increased by the supply of energy (pressure. induced fit. That explains the specificity of catalysis and the selectivity for a certain substrate (and also that for additional regulatory factors). The part that binds to a substrate molecule is termed the active centre or substrate-binding site and is characterized by a shape complementary to that of the substrate molecule. In other words: the enzyme binds first to a coenzyme like NAD or FAD. Mechanisms of Enzyme Catalysis Enzymes are proteins. This model proposes that the initial interaction between enzyme and substrate is relatively weak. the binding is either reversible (achieved through weak interactions) or irreversible (covalent bonds). grooves. These properties explain.interfacial activation. Furthermore. temperature). This conformational change exposes the active site of the enzyme. It is this structure that allows an enzyme to perform its catalytic activity. After binding takes place. Chapter 4 Enzyme-Substrate Interactions The favored model of enzyme substrate interaction is known as the induced fit model. which again are the basis for the spatial arrangement (conformation) of the molecule and which help to maintain this structure by stabilizing it. the enzyme's surface is structured in a way that the coenzyme is bound to a specifically shaped pocket. pockets. rather large. Enzyme molecules are. Their surface is not evenly structured but displays dent-ins. hollows etc. They form a number of weak interactions. The collision theory states that the event of two molecules meeting in solution in a way that a bond can form between them is rather improbable. The activation of the lipases at the interface is a result of a conformational change. Depending on the type of molecule. why reactions take place at an enzyme's surface that occur in solutions only after a considerable amount of activation energy has been supplied. certain amino acid side chains are exposed at the active centre. Often. the onedimensional information that was stored in the genome as a DNA sequence is first transcribed into mRNA and after translation into an amino acid sequence finally transformed into a three-dimensional structure. Every protein is determined by its amino acid sequence (its primary structure) and its tertiary structure (the three-dimensional folding of the polypeptide chain).

c. General acid–base catalysis needs to be distinguished from specific acid–base catalysis. In addition to inducing strain. e. the rate of the reaction is dependent on the buffer concentration. In solution. –OH or H+ accelerates the reaction. general base catalysis Catalysis by electrostatic effects Covalent catalyis (nucleophilic or electrophilic) Catalysis by strain or distortion a. into conformations that strain. the induced structural rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds. . The general acid–base is best when its pKa is near that of the pH of the solution. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases) • Other mechanisms also contribute significantly to the completion of catalytic events initiated by a strain mechanism. • This is paid for by binding energy. In the second step (collapse of the tetrahedral intermediate).. the buffer aids in stabilizing the transition state via donation or removal of a proton. The possible mechanisms of catalysis are five in number: 1. Catalysis by Bond Strain In this form of catalysis. The new conformation often forces substrate atoms and bulky catalytic groups. • Specific acid-base catalysis is due to H+ or OH. General acid-base catalysis is due to proton donors or proton acceptors. which donate or remove protons to (or from) the transition state intermediate. groups such as aspartate are frequently chemically reactive as well.state complexes and reaction products. as well as the appropriate protonation state.g. Enzymatic reactions take place within the confines of the enzyme active-site wherein the substrate and catalytic groups on the enzyme act as one molecule. Catalysis by approximation General acid. • General acid-base catalysis is a commonly employed mechanism in enzyme reactions. Most hydrolytic enzymes use acid/base catalysis. such as aspartate and glutamate. 3. 4. The reaction rate is dependent on pH only. which more easily attain the transition state. then DG‡=DH‡–tDS‡. there is no loss in translational or rotational energy in going to the transition state. Therefore. and not on buffer concentration. • The formation of a transition state is accompanied by losses in translational entropy as well as rotational entropy. • Specific acid–base catalysis means specifically. • If DG‡ is the change in free energy between the ground state and the transition state. b. in order to have appropriate concentrations of each buffer species. existing substrate bonds. and DS‡ would therefore be negative. Catalysis by Proximity and Orientation • Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another.more mechanisms of catalysis generate transition. and their proximity and orientation toward the substrate thus favors their participation in catalysis. The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity. 2. Therefore. the transition state would be significantly more ordered than the ground state. 5. hydrolysis of ester/ peptide bonds. the use of glutamate as a general acid catalyst (proton donor).ions which temporarly donates proton or accept proton. the leaving group must be protonated. • In General acid–base catalysis. • General acid-base catalysis is involved in a majority of enzymatic reactions. phosphate group reactions. for example.

An acidic group in one part of the active site donates a proton and a basic group in another part of the active site removes a second proton from the reaction intermediate. yet enzymes can do this. a covalent bond is transiently formed between the substrate and the enzyme (or coenzyme). One of the best-known examples of this mechanism is that involving proteolysis by serine proteases. chymotrypsin. acyl group or glycosyl group.addition to carbonyl • groups. or contain unfilled valence electron shells. Covalent Catalysis: • In catalysis that takes place by covalent mechanisms. Also. Cysteine enzymes include papain (a protease). water soluble proteins that function as enzymes. elastase. In addition. Acyl enzymes usually attach the acyl group at an active serine or cysteine residue. Biologically important nucleophiles are negatively charged or contain unshared electrons • Biologically important electrophiles generally are either positively charged.glucose 6 phosphatase. These proteases contain an active site serine whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond. Being enzymes. Tyr and Lys can be involved in general acid-base catalysis. phosphotransferase system. Glu.phosphoglucomutase. proteases can be characterized by their substrate affinity and the catalytic rate of the . In covalent catalysis. histidine reacts very fast (protonation/deprotonation of imidiazole ring has a reaction half-time of less than 10-10 seconds at neutral pH). glyceraldehyde phosphate dehydrogenase and most enzymes using acyl CoA derivatives. Concerted acid-base catalysis Providing both acid and base simultaneously is impossible in free solution.0) can act as either an acidic or basic catalyst depending on whether or not it is protonated. histidine (pK around 7. Histidine type . Amino acids with extra carboxyl or amino groups can obviously act as acids or bases. B is usually some chemical group such as a phosphate group. alkaline phosphatase. Phosphoenzymes usually attach the phosphate to serine or histidine. d. which include both digestive enzymes (trypsin. Most proteases (eg trypsin. the substrate is oriented to active sites on the enzymes in such a way that a covalent intermediate forms between the enzyme or coenzyme and the substrate. thereby causing hydrolysis of the peptide bond. subtilisin are serine enzymes. His. This reaction usually involves a nucleophilic group on the enzyme and an electrophilic group on the substrate Uncatalysed: BX + Y BY + X Catalysed: BX + Enz Enz-B + X Enz-B + Y Enz + BY Where. • General base catalysis involves abstraction of a proton by the catalyst. They catalyze the hydrolyses of peptide bonds in proteins. • General acid catalysis involves donation of a proton by the catalyst. Cys. Serine type . or contain an electronegative atom Covalent Intermediates Here the strategy is to lower the transition state energy "hump" by taking an alternative reaction pathway therefore sometime it is also called as alternative pathway catalysis. • The side chains of Asp. and elastase) and several enzymes of the blood clotting cascade. Histidine is rarely found in proteins except at catalytic sites. etc. The proteases are globular. Sometime nucleophilic catalyst form intermediate more rapidly than normal catalysis.

cleaves the • proenzyme plasminogen. tetrahedral intermediate as transition state. peptide with new N-term released from enzyme taking -H from H57 (originally S195. Notice how their active sites are suited for these tasks. H57 binds the -released H+ from S195 (ES*) 3. covalent bond formation between -C=O and S195 hydroxyl (nucleophilic attack). which we’ve talked about. and proximity/orientation catalysis • The tetrahedral intermediate in chymotrypsin. which consists of the Ser195 adduct before departure of the leaving group. and they all have three important conserved residues–a histidine. Chymotrypsin is a protease that cleaves peptide bonds on the carboxyl side of aromatic or large hydrophobic amino acids – the mechanism of chymotrypsin’s cleavage reaction includes covalent. and cleaves after small neutral amino acids. see step 2) (EP*) 4. Using proteases to study the effects of single amino acid substitutions (mutations) on catalytic rate and substrate affinity demonstrated that these two properties are linked and that this linkage can be explained by analyzing the conformation of the catalytic or active site of the enzyme. an aspartate. Thrombin is crucial in the blood-clotting cascade • Subtilisin is bacterial protease • Plasmin breaks down the fibrin polymers of blood clots • Tissue plasminogen activator (or TPA). 4 families of proteases have been defined: a. Cystein proteases c. • The family includes among many others. acyl-enzyme intermediate and peptide bond hydrolysis. and does not relate to the substrate specificity of the proteases themselves. general acid-base. the water molecule initiates a nucleophilic attack and undergoes a reaction with one -H attaching to H57 and -OH to acyl-enzyme intermediate forming again a tetrahedral transition state of the new C-terminal peptide fragment (EP*) 6. aspartate) or a metal as co-enzyme at the active site of the enzyme. stabilizes S* over S). • All three enzymes are similar in structure. while trypsin cleaves after basic amino acids. yielding plasmin The six step reaction mechanism of chymotrypsin: 1. electrostatic. The name of the protease family refers to an amino acid (e. Serine proteases b. Elastase is fairly nonspecific. Metallo proteases This classification uses the functional group within the enzyme. hydrolyzes peptide bond • The serine proteases are a class of enzymes that degrade proteins in which a serine in the active site plays an important role in catalysis. and Elastase. Substrate attaches to enzyme at the main chain binding site and specificity pocket with the scissile (peptide) bond exposed to the triad (ES formation) 2. Peptide released while H57 donates -H (from water molecule) to S195 (E+P) • Chymotrypsin cleaves after mainly aromatic amino acids. A water molecule placed next to H57 and acyl-enzyme intermediate at S195 (EP*) 5. This analysis showed that four major functional groups are found in the catalytic site of proteases and based on this functional groups. Structural element of active site Function The main chain substrate binding unspecific binding of polypeptide segment Specificity pocket semi-specific binding of side chains. is considered to be the transition . Aspartate proteases d.g. sequence specificity Oxyanion whole stabilizes S* over S in enzyme The catalytic triad forms tetrahedral intermediate (transition state.reaction. Chymotrypsin and trypsin. oxyanion hole stabilization. and a serine.

Their nucleophilic groups (imidazole ring. It inhibits the reaction because it blocks entry of normal substrates. proximity and orientation effects Substrate specificities in proteases are due to active site binding pockets.state intermediate in the chymotrypsin reaction. OH and SH respectively) are good electron donors the intermediates they form are unstable and react easily with the final acceptors. which is H-bonding with Asp 102 (catalytic triad) – the catalytic triad serves to activate Ser 195 through general acid-base catalysis – catalytic triads are common in serine proteases Conformation changes from trigonal to tetrahedral in the peptide upon nucleophilic addition allow the oxyanion formed to move into a hole with two backbone amides – inside the oxyanion hole the transition state anion can form two hydrogen bonds with the amide hydrogens as donors – the enzyme cannot form H-bonds with the amide hydrogens in the oxyanion hole when the peptide is in trigonal conformation • The mechanism of chymotrypsin utilizes stabilization of the transition state. one of which bears a negative charge. It was the diisopropylphosphoryl derivative of serine. and the negatively charged oxygen is better accommodated in the oxyanion hole. • Crystal structures of chymotrypsin reveal the reason for the high reactivity of Ser 195 – Ser 195 is in a H-bonding interaction with His 57. . releasing fluoride anion. This results in a covalent bond between the nucleophile and the inhibitor. serine and cysteine operate by nucleophilic catalysis. S). covalent catalysis. It diffuses into the active. S1 has bulky Val residues Histidine. the resulting carbon-oxygen single bond is longer. acid-base catalysis. • The enzyme-inhibitor adduct is very stable. They loosely hydrogen bond to the carbonyl oxygen under attack. wherein a nucleophilic amino acid attacks the phosphate. electrostatic. S1 has Asp – Elastase: cleaves after small side chains (A. a novel amino acid was isolated. hydrophobic pocket that binds the sidechain of the amino acid N-terminal to the site of bond cleavage (S1) – binding of appropriate sidechains to this pocket positions the adjacent peptide bond into the active site for cleavage Many serine proteases have high similarity to chymotrypsin – trypsin and elastase are approximately 40% identical in primary sequence to chymotrypsin and their overall structures are very close • Substrate specificity of these other proteases compared to chymotrypsin can be attributed to their S1 binding pockets – Trypsin: cleaves after positively charged residues. It is high energy because there is a carbon surrounded by 3 electronegative atoms. • How is it that the enzyme stabilizes this transition state intermediate? The backbone amides of gly193 and ser195 form an oxyanion hole. • Chymotrypsin substrate specificity is due almost entirely to one deep. • Diisopropylflurophosphate is an inhibitor of chymotrypsin. Upon formation of the tetrahedral intermediate. 110°C) and amino acid analysis on the hydrolysate. Upon hydrolysis of the protein (6 N HCl. enzyme covalent intermediates are very difficult to isolate. Since the whole point is that covalent intermediates should rapidly break down to release the reaction product.

• It is found in many body fluids. because it would be cleaved during crystal growth and structure determination. Asp-52 exists in its ionized form.cystein prtoease Renin . such as tears. There are 6 subsites within the crevice.An Aspartyl Protease A Metalloprotease with Substrate Carbonic Anhydrase Mechanism His64 assists in H+ removal HCO3.e. • The best studied lysozymes are from hen egg whites and bacteriophage T4. Glu then acts as a general base to deprotonate water in the transition state. i. it hydrolyzes the glycosidic bond between C-1 of N-acetyl muramic acid and C-4 of Nacetyl glucosamine. H2O enters HO.-) with number 17 (EC 3.released. the conformation of the sugar is distorted in order to make the necessary hydrogen bonding contacts. It would not be possible to determine the X-ray structure in the presence of the true substrate.1. • At what position does water attack the sugar? When the lysozyme reaction is run in the presence of H218O. In particular. while Glu-35 is protonated. This analog binds tightly in the enzyme active site to form the ES complex. From the X-ray structure.2. each of which is where hydrogen bonding contacts with the sugars are made.-).1. • Within the class of hydrolases. Glu can act as a general acid to protonate the leaving group in the transition state. The X-ray crystal structure of lysozyme has been determined in the presence of a nonhydrolyzable substrate analog. 18O ends up at the C-1 hydroxyl group at site D. Look at the many hydrogen bonding contacts between the substrate and enzyme active site that enables the ES complex to form.2.and S-glycosyl (EC 3. In site D. enzymes hydrolyzing O. and is one of the body’s defenses against bacteria. Asp can function to stabilize the positively charged intermediate. • The active site consists of a crevice or depression that runs across the surface of the enzyme.attacks CO2 Methylation prevents H-bonding with DNA substrate • Lysozyme is a small globular protein composed of 129 amino acids. but ES cannot be efficiently converted to EP. bringing the substrate closer to the transition state for hydrolysis.-. particularly those found in the peptidoglycan cell wall of bacteria. This distortion raises the energy of the ground state.2. • It is also an enzyme which hydrolyzes polysaccharide chains. lysozyme was the first enzyme to have its structure determined. Lysozyme reaction is the hydrolysis of the beta (1-4) glycosidic bond between N-acetylglucosamine sugar (NAG) and N-acetylmuramic acid sugar (NAM) and therefore it is possible classify it as Glycosidases. it is known that the C-1 carbon is located between two carboxylate residues of the protein (Glu-35 and Asp-52). Lysozyme belongs to the Glycosylases family (EC 3. • Although crystal structures of other proteins had been determined previously.17) in this group . This suggests that water adds at that carbon in the mechanism.

Fe3+. In carboxypeptidase. • The inhibitor is a naturally occurring transition state analog of a protein substrate. Here is a good • example of transition state stabilization using a bacterial glycosidase.contain tightly bound metal cofactors such as Fe2+. Mg2+. nitrate reductase etc. • Another excellent example that clearly shows how transition state stabilization works is • found in the structure of this transferase. The resulting carbonium ion (+) is stabilized by • the ionized side chain of Asp52 until it can react with water) • Many glycosidases utilize a covalent intermediate in their mechanism. Cu2+. as in cytochromes. The ions are usually Na+. • But the binding of residue 4 in the half-chair conformation is favorable (preferential • transition state binding).Lysozyme Lysozyme catalyzes the hydrolysis of the (1-->4) linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in bacterial cell wall polysaccharides and (1 4)-linked poly NAG (chitin). often the metal ion does not get reduced or oxidized. which bind in the active site and react with • His 57. • The binding of NAM4 in the chair conformation is unfavorable. However. • Chloroketones are inhibitors of serine proteases. • Hydrolysis involves acid-base catalysis (Glu35 serves as a proton donor to the • oxygen of the leaving alcohol. Zn2+ polarizes the C=O of the peptide bond which is about to be broken. • Pancreas contains a small protein (6 kDa) that is a potent inhibitor of trypsin. Mn2+. or Ca2+ Many enzymes have metal ions at the active site. Sometimes these are used as redox centers. Zn2+. In alcohol dehydrogenase Zn2+ polarizes the C=O . K+. The lysozyme mechanism illustrates: • Transition state stabilization • Acid-base catalysis • Several residues in the protein participate in substrate binding. but acts to stabilize negative charges on the reaction intermediate. e) Metal Ion Catalysis Nearly 1/3 of all known enzymes require the presence of metals for catalytic activity • Metalloenzymes . Co2+ • Metal Activated enzymes – only loosely bind the metal ions.

7 to 6-7 when it is coordinated to Zinc or Cobalt. In this case. • The structure of the enzyme can only be complementary to one form of the substrate. then binding increases as the reaction proceeds. K+. becoming first the transition state and then products. Zinc is acting as a Lewis acid. which lowers the activation energy. Mediating oxidation-reduction reactions. Electrostatic catalysis refers to the fact that when a substrate binds to an enzyme.group of acetaldehyde. and making the carbon more electrophilic. Metal-activated enzymes contain loosely bound metal ions: Na+. the pka of water drops from 15. Electrostatic interactions are much stronger in organic solvents than in water due to the dielectric constant of the medium. The interior of enzymes have dielectric constants that are similar to hexane or chloroform. This causes the local dielectric constant to be lower. Electrostatically stabilizing or shielding negative charges. Proximity and orientation effects are important in enzymatic reactions. • If the structure of the active site is complementary to the transition state. • Weak interactions between the enzyme and substrate are optimized in the transition state. ion catalysis is widely used. SUMMARY Metal • • • • Mn+2. inducing charge separation. It coordinates to the non-bonding electrons of the carbonyl. water isusually excluded from the active site. • Binding of the substrate in the active site can orient the substrate for most efficient interaction with these side chains. Binding substrates in the proper orientation. • The three dimensional structure of the enzyme can bring several reactive side chains into close proximity in the active site. Metal ions can also function to make potential nucleophiles (such as water) more nucleophilic. Zn+2. For example. Metal ions that are bound to the protein (prosthetic groups or cofactors) can also aid in catalysis. • The structure of the substrate changes during the reaction. The hydroxide ion is 4 orders of magnitude more nucleophilic than is water. Which enhances charge-charge interactions in the active site. • Having the active site bind the transition most strongly also means that . or more susceptible to nucleophilic attack. so allowing the hydride ion (H–) to be added to the carbon atom. Utilization of enzyme-substrate binding energy in catalysis The maximum binding energy between an enzyme and a substrate will occur when there is maximal complementarity between the structure of the binding site and the structure of the substrate. therefore Go‡ is lowered. • Ca+2. • This means that the enzyme binds the transition state of the reaction more tightly than either the substrate or product. Metalloenzymes contain tightly bound metal ions: Fe+2. Cu+2. Preferential Transition State Binding is probably the most important rate enhancing mechanism available to enzymes. Fe+3. Mg+2.

• When the active site is complementary to the transition state the full substrate binding energy is only realized as the reaction reaches the transition state. • It is possible that the substrate and enzyme interact unfavorably and this unfavorable interaction is relived in the transition state. which lowers the activation energy of the reaction by the magnitude of the binding energy.around 1010 fold. which states that it is not the substrate that is distorted but rather that the transition state makes better contacts with the enzyme than the substrate does. b) Orientation Proximity alone is insufficient. which favors dissociation of the products from the enzyme once the reaction is complete. 109 for alcohol dehydrogenase.around 1010 fold . • We might think that the substrate is under stress meaning that it is subjected to forces but not distorted by them. The reacting groups must be properly oriented. • Transition state stabilization is a more modern concept.products are bound weakly. Factors involved in enzyme rate increases: a) Proximity .g. Since chemical reaction rates are proportional to the concentrations of the reactants a rate enhancement of up to 106 may be generated by local concentration effects. f) Distortion of the substrate . 1016 for alkaline phosphatase).up to 106 fold. so the full binding energy is not achieved until the transition state is reached. Molecular Mechanisms for the Utilization of Binding Energy • Strain is a classic concept in which it was supposed that binding of the substrate to the enzyme somehow caused the substrate to become distorted toward the transition state.up to 108 fold (but largely hypothetical) a) Proximity The enzyme binds the substrate so that the susceptible bond is very close to the catalytic group in the active site or in close proximity in the active site. It increases their concentration and and reduces entropy loss for subsequent formation of a transition state.up to 100 fold c) Covalent enzyme-substrate intermediates around 1010 fold d) General acid-base catalysis . Rate increases by enzymes range from 108 to 1020 relative to the uncatalysed. b)Orientation .binding of the substrate(s) to the enzyme aligns the reactive groups so that the relevant molecular orbitals overlap. as for example in induced fit.e) Metal ion catalysis . This has been called as proximation effect or approximation effect. Therefore it appears that enzyme bound transition state is the single most in determining whether the reaction should proceed. This effect is counteracted to some extent by the fact that the concentration of enzyme active sites is low (the active site is a small portion of a very large molecule. the total protein concentration in cytoplasm is 1 to 10mM at most). Orbital steering hypothesis . Catalytic Power Enzymes lower the energy of the transition state by stabilizing the original reaction intermediate or by providing an alternative reaction pathway. Calculations indicate that the local concentration of substrate in the active site may be as much as 50M whereas its concentration in the cytoplasm may be less than 1mM. This increases . • Strain or stress? It is unlikely that there is enough energy available in substrate binding to actually distort the substrate toward the transition state. spontaneous reaction (e. • It is more likely that the enzyme is strained.

Carrea et al. 4. a variety of enzymes from different classes have been shown to function in nonaqueous media. 3. Other examples include chymotrypsin. Traditionally. 3. Consider the attack on an aryl ester by a carboxylic acid. Some of these are illustrated in overheads for porcine pancreatic lipase (Zaks & Klibanov 1984. The question arises as to how much water is really needed by the enzyme to function. Can use enzymes directly within a chemical process. 1995. rather than the total water content of the system. Therefore. we will link R1 and R2 together. As the five examples show. In addition. 1989): 1. Alexander Klibanov & his colleagues at MIT initiated much of the work in this area. 1995. Research by Klibanov. alcohol dehydrogenase. enzymes are more stable. (Ar = methoxyphenyl group. because enzymes are insoluble in organic solvents. his colleagues and other researchers have opened up novel opportunities in enzyme technology and this field has changed from a mere curiosity to a serious alternative to chemical reactions. Lipases.the probability of forming the transition state. 10. water is required to inactivate enzymes at high temperature. Gupta. new catalytic activity seen with lipases or horse radish peroxidase). Reduction in water-dependent side reactions. 8. 1985). enzyme reactions are carried out in aqueous media. 7. They concluded that it is the water bound to the enzyme that is critical for activity. Importance and relevance of whole cell enzymes in biotechnology Enzymes are biological catalysts. Novel enzyme reactions possible (eg. Immobilization is unnecessary. They found startling changes to enzyme properties as the water content of the reaction mixture decreases. altered substrate specificity and a propensity towards synthetic reactions in non-aqueous media. 1992). Altered activity and affinity (Km) of the enzyme. ie. etc..) f. in the absence of water. Horse radish peroxidase (HRP) which is able to depolymerize lignin in 95% dioxane (Dordick et al. With the exception of ribozymes. Thus the maximum effect of orientation would be 100-fold.e. 9. 6. 2. Enhanced thermo stability of enzymes. This led them to test how enzymes function in various amount of organic solvents. Ease of product recovery from low boiling organic solvents. enzymes are . Such systems are applicable to enzymes of varying reaction types. Shifting thermodynamic equilibria to favor synthesis over hydrolysis. the relative rate gets greater as the reacting groups are brought closer together. and can be recovered by simple filtration. 1986). Glucose isomerase for conversion of glucose to fructose to produce high fructose corn syrup (HFCS). Increased solubility of nonpolar substrates. Now let's put both reacting groups on the same molecule . The functions of a number of enzymes under non-aqueous conditions have been studied. 2. For a typical bimolecular reaction in free solution about 1/100 collisions between molecules of sufficient energy actually leads to a reaction. holding them in the correct orientation also helps. Some examples include: 1. 5. Some advantages to using enzymes in non-aqueous solvents include (Dordick.. 4. Catalysis in organic solvents Another approach that has generated some interest in enzyme technology is catalysis in non-aqueous or nearly anhydrous media (Bell et al.i. which show increased thermostability. Elimination of microbial contamination.

The whole biotechnology field depends on the activities of enzymes. These transformations involve a number of enzymes acting sequentially. They have been used throughout the ages in leather tanning. Abzymes Catalytic antibodies are also known as abzymes. Enzymes catalyze a wide range of reactions. Exploitation of enzymes is not a recent development. Therefore. 13. Sterility problems often arise when one deals with whole cells. the presence of metal ions. 12. or ethanol production from glucose. along with the requirement for co-factor regeneration in several steps. and therefore. etc. are capable of catalyzing specific chemical reactions. 7. ie. 4. On the other hand. interferons. 8. the scope of enzyme technology is very broad. Their main task is to decrease the activation energy (Eact) of a chemical reaction. 9. This may include poor stability with respect to temperature. 17. They are able to perform this task because they have been elicited against antigen known as transition state analogues. etc. This is because the use of enzymes also suffers from some serious drawbacks: 14. Formation of by-products. it is easy to decide whether one should use whole cells or isolated enzymes in a biotechnological process to produce or transform compounds. 3. 5. old ones are replaced by new ones. Most enzymes are water-soluble. Competing side reactions from other enzymes. Enzyme by forming the active site similar to the transition state. pH. enzymes can be superior because their use will eliminate some of the potential problems with using whole cells. Conceptually. all enzymes are subject to turnover. enzyme have not been used widely as compared to chemical catalysis. dehydrogenation. Most isolated enzymes are not stable enough for use under industrial conditions. There may be some drawbacks to using whole cells in catalysis. Despite all these striking characteristics. eg. reduction. either acting alone or in concert. and may also possess lipid or sugar moieties. dehydration. These processes use enzymes in the form of whole cells. enzyme stabilizes or . 11. and the product. dehalogenation. Optimizing 1 or 2 enzyme catalyzed reactions is easier than optimizing the whole pathway in a whole cell. Non-polluting catalysis. 2. hydration. these are molecules that closely resemble in shape and charge the reaction transition state (the highest energy spices in reaction pathway known as activated complex). in cheese-making. 16. These include 1. 15. and hence are difficult to isolate. Less by-products formed. the synthesis of antibiotics. High catalytic rate possible. and this can result in loss of biocatalysts or severely decrease reaction rate depending on the nature and type of enzyme reaction. Less problem with sterility. monoclonal antibodies. with one-step or two-step stereospecific transformations. may be difficult to separate from reactants or products. Some processes require multi-step transformations. Enzyme observed to bind transition complex but not the substrate. In cells.mainly proteins in nature. They are clearly candidates for using whole cells. some with high stereospecificity. This is not surprising considering that most enzymes have evolved to function in a biological milieu where the conditions may differ from those used in industry. 10. Some conditions may lead to cell lysis. Functions in moderate conditions. such as oxidation. This is difficult to do with isolated enzymes in vitro. The advantages of using enzymes include: 6. Many useful enzymes are intracellular. in the preparation of malted barley for beer brewing & the leavening of bread.

Coenzyme. Define Enzyme ? Classify the Enzyme on the basis of enzyme commission? 4. The general strategies behind the production of catalytic antibody is to (i) design and synthesize a molecule whose shape is closely resemble to that of transition state of the reaction one wishes to catalyze. monomeric and oligomeric enzymes. g. Enzyme activity b. Enzyme inhibition. an enzyme that catalysed the hydrolysis of this ester would also bind tightly to phosphonate analogue. In this example. the phosphonate containing molecule analogue is able to reproduce the shape of the transition state as well as the partially negative charged oxygen. cofactor. d. e. (3) elicit an immune response to this conjugate.lowers down the energy of this species. where as the actual transition state exists only fleetingly. Holoenzyme. 5. Define Biocatalyst ? Describe how biocatalyst lower down the activation energy? 3. Define enzyme specific activity? Write the different method of enzyme substrate interaction in brief? Short type 1. Metalloenzymes. 1 Exercises LONG TYPE 1. Cofactor f. Describe the six classes of enzyme with examples? 2. Module II Specificity of enzyme action. In addition. Define holoenzyme ? write the role of apoenzyme. (2) tether this molecule to larger molecule. Enzyme commission. The effect is to accelerate the reaction because the activation barrier is more easily overcome. . When an ester is hydrolysed the central carbonyl group changes from planer sp2 structure to tetrahedral sp3. Antibodies that are elicited against transition state analogue and therefore have the ability to bind them will be chemically and sterically complementary to the transition state and will therefore be potentially capable of catalyzing the reaction. (4) screen the resultant monoclonal antibodies for catalytic activity of the type desired. write short notes on following a. it is chemically stable. This complementarity in shape and charge to the transition state is readily demonstrated by the effectiveness of transition state analogue is a phosphonate containing molecule that mimics the transition state of the hydrolysis of the corresponding acyl derivative. Enzyme specificity c. and metalloenzymes on enzyme activity. Table 7.

Competitive inhibition. The gene products (enzymes) of the different gene loci (pseudoalleles) are called isoenzymes. They may also differ in their rate of substrate turnover.but are not changed by the enzyme. Initial velocity is the change in reactant or product concentration during the linear phase of a reaction. Competitive inhibitors are molecules that bind to the same site as the substrate preventing the substrate from binding as they do so .Chapter-5 ENZYME KINETICS & INHIBITION TERMS Enzymes are protein catalysts of biological origin that. The rate of reactions depends on the order and molceulcarity. Alloenzymes. when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency. Rate refers to the change in total quantity per unit time. Furthermore. Enzyme kinetics The study of the rate at which an enzyme works is called enzyme kinetics. Polypeptides (enzymes) that are encoded by different alleles are called alloenzymes. They may be separated by gel electrophoresis under favourable conditions. Noncompetitive inhibition. speed up the rate of a chemical reaction without being used up in the process. INTRODUCTION Kinetics is the study of the rate of change of reactants to products . The gene product a. is favourable under other conditions (more in the section about evolution). Isoenzymes.Velocity refers to the change in concentration of substrate or product per unit time. two different polypeptide chains will be generated. Study of rate of the catalyzed reaction is called as kinetics. for example. Noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power. By duplication of the genetic material a gene may be present within a haploid genome for two or more times. When enzyme concentration is small and substrate concentration increases then first the reaction occurs rapidly but maximum rate is achieved only when all the space in the active site is filled . This is the important step in understanding the mechanism of catalysis. may be inactive while that of A is fully active. it was shown in numerous examples that A may be favourable under certain environmental conditions while a. If a gene exists in a heterozygous state. They achieve their effect by temporarily binding to the substrate. But all states in between are also possible. like all catalysts. It is not important whether the gene loci are on one chromosome or distributed over several chromosomes. when the substrate and inhibitor compete for binding to the same active site. Therefore. it is necessary to study the factors affecting the rate of catalysis.

brackets define concentration in molarity and the k is known as a rate constant. the reaction in which A is converted to B is written as follows: A B----------------------------1 The rate of this reaction is expressed algebraically as either a decrease in the concentration of reactant A: -[A] = k[B]-----------------------2 or an increase in the concentration of product B: [B] = k[A]--------------------3 In the second equation (of the 3 above) the negative sign signifies a decrease in concentration of A as the reaction progresses.and no more substrate can be filled. not to an actual negative value for the constant. respectively. Reaction rate is always dependent on the concentration of the chemicals involved in the process and on rate constants that are characteristic of the reaction. for the forward reaction: V forward = k+1[A]---------------5 and for the reverse reaction: V reverse = k-1[B]----------------6 In the above equations. the rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into product(s) in a specified time period. To put the relationships of the two equations into words. The negative subscript refers only to a reverse reaction. Thus. For example. Chemical Reactions and Rates According to the conventions of biochemistry. The rate constant for the forward reaction is defined as k+1 and the reverse as k-1. k+1 and k-1 represent rate constants for the forward and reverse reactions. Initially substrate consumes and product is released. This assumption is true for single substrate and single enzyme. Each chemical reaction has characteristic values for its rate constants. Therefore maximum velocity is affected by the occupying all space of the active site by the substrate. rate constants and the equilibrium constant for this simple case. a relationship which is algebraically symbolized as: V forward = v reverse---------------4 Where. This time reaction rate become slower and the product formation starts. Rate in the itegarl form is shown by the rate = dX/ dt. we state that the rate of the forward reaction [v forward] is equal to the product of the forward rate constant k+1 and the molar concentration of A. reaction can be rewritten as an equilibrium expression in order to show the relationship between reaction rates. The rate of the reverse reaction is equal to . At equilibrium the rate (v) of the forward reaction (A B) is by definition is equal to that of the reverse or back reaction (B A). these in turn directly relate to the equilibrium constant for that reaction. Rate constants are simply proportionality constants that provide a quantitative connection between chemical concentrations and reaction rates. Therefore initially the order is first and later on it becomes zero order. If this is occupied by the inhibitor as in competitive inhibition the v max remain same since space is same and inhibitor has similar structure as the substrate.

However. order is easily determined by summing the exponents of each concentration term in the rate equation for a reaction. The catalytic event that converts substrate to product involves the formation of a transition state. high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events.order reactions need not be different chemical species. An example of a second order reaction is the formation of ATP through the condensation of ADP with orthophosphate: ADP + H2PO4 FIGURE 10. The exponent on the substrate concentration in the rate equation for this type of reaction is 1. the rate of the forward reaction is equal to the rate of the reverse reaction leading to the equilibrium constant of the reaction and is expressed by: [B]/[A] = k+1/k-1 = K eq. reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. At equilibrium. Menten in 1913 postulated that enzyme first combine reversibly with the substrate to form an enzyme substrate complex in a relatively fast reversible step and then product is released along with free enzyme. Michaelis and M. A reaction characterized by the conversion of one molecule of A to one molecule of B with no influence from any other reactant or solvent is a first-order reaction. In biochemical reactions. called the catalytic site of the enzyme. As we know at fixed enzyme concentration. each enzyme molecule catalyzes the conversion to product of many reactant molecules.the product of the reverse rate constant k-1 and the molar concentration of B. Empirically. A reaction with two substrates forming two products would a second-order reaction. reactants are commonly known as substrates. 1 FIGURE 10.---------------7 This equation demonstrates that the equilibrium constant for a chemical reaction is not only equal to the equilibrium ratio of product and reactant concentrations. and it occurs most easily at a specific binding site on the enzyme. The ATP + H2O---------8 . The Michaelis derived an equation to explain the fact as Initial velocity Michaelis-Menton constant In typical enzyme-catalyzed reactions. Chemical Reaction Order Order of reaction is refers to the number of molecules involved in forming a reaction complex that is competent to proceed to product(s). Consequently. but is also equal to the ratio of the characteristic rate constants of the reaction. 2 For this reaction the forward reaction rate would be written as: V forward = k1 [ADP][H2PO4]---------------9 L.and higher. This site. has been evolutionarily structured to provide specific. Is increased then initially rate become faster and at maximum velocity where enzyme saturates the rate become steady as shown in figure. the reactants in second. when substrate conc.

here the rate will be proportional to the [s] because the equilibrium of equation will shift for the formation of more ES as S is increased. the amount of substrate bound by the enzyme at any given time is negligible compared to total [S]. At any time the enzyme-catalyzed reaction.(12) Since the second step is slower and therefore it limits the rate of overall reaction. (15) k-1 denotes the rate of ES breakdown= k-1[ES] + k2 [ES]-------------------------(16) ………… . The series of events can be shown thus: E + S ES P-------------------10 and K1 k-1 The ES complex then breaks down in a slower second step to yield the free enzyme and the reaction product P: k2 ES -------. b. Under these state the enzyme is “saturated” with its substrate.complex that forms when substrate(s) and enzyme combine is called the enzyme substrate (ES) complex. It is too difficult to observe because of very short time. so that further increase in [S] have no effect on rate. At low [S]. most of the enzyme will be in uncombined form E.. Pre-steady state:-The duration in which ES formation takes place is called as pre-steady state. which is ES. 11 ES* EP E + VO can be determined by the breakdown of ES to give product. Also. The maximum initial rate is observed when the entire enzyme E0 is present as ES complex and the concentration of E is very small. This is the condition when [S] is sufficiently high that essentially all the free enzyme is converted into the ES form. So free enzyme can be represented by [Et]—[ES].. and the transition state complex must advance to an enzyme product complex (EP). K1 denotes the rate of formation of ES= k1( [Et]—[ES] [S] …. this complex must pass to the transition state (ES*).E+P ……………. Steady state:-Here ES complex is constant over a period of time. Derivation: E+S k-1 k1 k2 ES E+P ………………(13) E+S ES ………. because [S] is greater than Et. overall rate of enzyme-catalyzed reaction must be proportional to the concentration of species that reacts in the second step. the enzyme exists in the combined form E or uncombined form ES. After the ES complex breaks down to yield product P the enzyme is free to catalyze another reaction. It means that. At a minimum S. a. and the reappearance of free enzyme and product. which is determined by the [ES]: V0=k2 [ES]-----------------------------------------------------(14) TOTAL enzyme concentration can be represented by [Et]. Reaction products arise when the ES complex breaks down releasing free enzyme. The latter is finally competent to dissociate into product and free enzyme. Between the binding of substrate to enzyme. an ES complex must be formed. a series of complex events must take place.

. we get Km+[S] = 2[S]. and it is defined as dissociation constant. the higher is the enzyme's affinity for its substrate. k2 is the rate limiting . the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate). Ks for the ES complex.-----------------23 ½= [S]/ km+[S] solving for km. The lower the Km. Putting value of ES in equation 21 from equation 13 gives new final equation that is Vo= V max . The smaller the value of kM . Consequently. enzyme catalyzed reaction and is denoted by the Km. thus k2<<k-1 So Km reduces to Km=k-1/k1. this defines the rate of initial velocity and maximum velocity. 5=6 k1( [Et]—[ES] [S] [ES]-----------------------(17) or k1[Et][S]--. K1[Et][S] [ES] = --------------------------------(20) or [ES] = --------------------------------(21) -----------------k1[S] + k-1+ k2 [Et][S] -----------------[S] +( k-1+ k2)/ k1 The term ( k-1+ k2)/ k1 is called as Michaelis—Menten equation. eq. we finally get K1 [Et][S] k2)[ES]-------------------------(19) Solving for the ES we get. or --------------------24 This represent that Km is equivalent to the substrate concentration at which Vo is one half Vmax. 1. Note that Km has unit of molarity. then Vmax/2= V max [S]/ km+[S].3. This shows that the MichaelisMenten constant equals the substrate concentration at half-maximal reaction velocity. [S] / Km + [S]-------------------22 = (k1 [S]+k-1+ What will happen if the initial velocity is exactly one-half of maximum velocity? So if V0 = Vmax/2. Km can change from one enzyme to other.K1[ES] [S] [ES]----------------------------(18) = = k-1[ES] + k2 (k-1 + k2) To Simplify the equation add the term k1[E] [S] to both side. Km can be very complex in the multistep enzyme catalysis. 2. the rate equation of one substrate. Note :for the Michaelis-Menten reaction. See figure 10. Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. the dimension of kM is Mol.So in the steady state.

Low Km enzyme can efficiently covert very low amount of substrate into the product such enzyme is said to have high affinity of enzyme while. FIGURE 10. See figure 10. best is the enzyme for maximum conversion of substrate into the product. is the maximum number of substrate molecules converted to product per enzyme molecule per unit of time.3. According to M-M model. 3 showing rate of reaction and Km at ½ Vmax Figure 10. one step may be rate limiting that rate limiting step is equivalent to Kcat. Note that two enzymes catalyzing different reaction may have the same Kcat value (turnover number) yet the rate may be different. See from the table below the enzyme having very high value of Kcat have high capacity to convert substrate into the product The turnover number is a measure of catalytic activity. Vo depends on both Et and S.4 & 10.l kcat. Equation 22 can be written as by placing the value of Vmax = Kcat x Et -------------------26 figure 10. K cat is the turn over number of enzyme . From equation 26. 7 . Its advantage is in the favourable graphic depiction of the quantities. On writing the Michaelis Menten constant in reciprocal form 1 / v = (kM / vmax) x 1 / [S]--------------------25 This representation is also known as the Lineweaver-Burk equation.5 1 / kM and 1 / vmax are the points of intersection with the co-ordinates. Km denotes the affinity of enzyme. reciprocal of time. in multistep enzymatic reaction. Note that high the TON of an enzyme. An enzyme that acts on very low concentration of substrate has very low Km. FIGURE 10. This plot has advantage that it allows more accurate determination of Vmax. FIGURE 10. This property is efficiently utilized by the LDH enzyme that has different Km value in the heart and muscle. Catalytic efficiency is denoted by the ratio of Kcat/ Km. 5 showing turn over number of different enzyme. Values of k cat range from less than 1/sec to many millions per sec. 4 Graphical method to find the value of Km by plotting the graph between the 1/Vo verses 1/[S] 10. the turnover number. 4. In muscle LDH have High Km. Km value will also be unsatisfactory. 6 The constant Kcat has unit S-1. k cat = Vmax /Et. 5. Therefore Kcat /Km is the best way to compare the catalytic efficiency of the enzyme. high value of Km denotes low enzyme activity. while in the heart it has low Km essential for the survival. Many enzymes have K cat /Km value in the range 108 to 109 . It also denotes about the reaction between the enzyme and substrate since at low K cat. therefore K cat /Km is a second order rate constant. but it does not tell anything about the nature of enzyme. The kcat is a direct measure of the catalytic production of product under saturating substrate conditions.4 Catalytic efficiency and Turnover number. A double recessive depiction yields a straight line with a gradient of kM / vmax. other enzyme that requires large amount of substances for their activation and conversion into product is said to have as High Km value but low affinity.

and maximum possible velocity. The second point is that the rate of the reaction should be equal to formation of products.. then you can rearrange the equation to look like this: Now group all of the rate constants together We can define this collection of rate constants as something called the Michaelis Constant. k+2 and k-1. and free substrate. The first thing we need to realize is that the total concentration of enzyme in all of its forms should be equal to the original free enzyme concentration: 2. or Km. we wil work within the approximation that we can neglect the backreaction. Before making this approximation. so the initial velocity. and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states. Now we have an even simpler expression: . E.The kinetics of simple reactions like that above was first characterized by biochemists Michaelis and Menten. and to further simplify our treatment. Now. if the steady-state approximation holds. One is to form products. from free enzyme. before making the steady-state approximation. Therefore we can write and from the steady-state approximation. k-2. vo. It is also useful to write down two other definitions and assumptions before we continue: 1. ENZYME KINETICS We start with a very simple expression for formation of the enzyme-substrate complex. can be written as Now. The rate constants for formation and breakdown back to reactants are k+1 and k-1. We can therefore write a simple kinetic scheme as follows: We will work within an approximation called the steady-state approximation. obtained when all the enzyme is substrate-bound. The rate of formation is dependent upon the rate constant k+2 and the enzyme and substrate complexes. i. and is therefore zero. respectively. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today.e. First. Note that this is not an equilibrium constant. we can look at the kinetic scheme above and deduce that the rate of change in the concentration of the enzyme-substrate complex should be equal to the rate of formation of the complex minus the rate of decay of the complex. S. this has to equal zero. The rate constant for formation of the product is k+2. ES. in which the rate of formation of the enzyme-substrate complex is equal to its rate of decay. Therefore the rate of decay of the enzyme-substrate complex will be the sum of two rate constants. The rate of decay involves two possible pathways. that equation is insoluble. you can solve it fairly simply (despite what I did in class). or Vmax. and the other is to reform the reactants. however.

[S].speed up. Competitive Inhibition occurs when a molecule that is close enough to the shape of the true substrate will fit into the active site. Many toxic substances owe their toxic properties to their ability to act as inhibitors (slow down the rate of reactions by different mechanism) to important enzymes responsible for catalyzing important biochemical processes. . cyanide poisoning is due to the cyanide ion competitively inhibiting the active site of the cytochromases enzymes responsible for catalyzing the Oxidation and Reduction processes of the Electron Transport System which is responsible for cellular respiration. Once locked into position. coma or even death of the organism. vo. Temperature pH Inhibitors The rate at which an enzyme works is influenced by several factors. The temperature. To get the equation into that form. 3. The concentration of substrate is designated [S] and is expressed in unit of molarity. Other inhibitors latch themselves not to the active site itself but to some portion of the enzyme molecule close to the active site which results in the .and hence collisions between enzyme and substrate . Then we can substitute that into our previously simplified equation to obtain Solve for ES Since initial velocity V0 = K2 [ES] and max velocity Vmax = K2 [E total] Chapter-6 Factor affecting the rate of catalysis. As the temperature rises.. molecular motion .We are almost done. the blocker molecule prevents the true substrate molecule from getting into position. Concentration of substrate molecules (the more of them available. we need two things from above. which in turn is the same as the starting concentration of enzyme. The molecule competes for the active site with the true substrate molecule which is concentration dependent. we want an expression in terms of constants and measurable quantities such as the substrate concentration. 2. But as enzymes are proteins. 1. Once the enzyme is inhibited the process cannot take place. and a toxicological symptom occurs that often leads to paralysis. If the concentration of substrate becomes more than the inhibitor they may replace the inhibitor later on. For example.e. the quicker the enzyme molecules collide and bind with them). This is the reason why ethyl alcohol is given to person that has consumed the toxic alcohol (methyl alcohol) so that methyl alcohol can be competitively replaced by ethyl alcohol. there is an upper limit beyond which the enzyme becomes denatured and ineffective. The first is our statement that the total enzyme concentration is equal to the concentration of enzyme in all of its forms. However. 1234Substrate concentration. This effectively blocks the active site. The study of the rate at which an enzyme works is called enzyme kinetics.E total = Eo = [E] + [ES] or conveniently [E] = [Eo] – [ES] more. and the initial velocity. The presence of inhibitors. i.

this causes a change in the secondary and tertiary levels of protein structure. Toxicology is the study of how toxicological substances can interfere with life sustaining enzymes via inhibition. A is the Arrhenius constant. will give almost a linear graph.e. ΔG* =EA is the standard free energy of activation (kJ M-1) which depends on entropic and enthalpy factors. 10. FIGURE 10. If the substrate concentration is.5 for every 10°C rise in temperature. All in all the use of inhibitors can be used for the benefit of mankind or its destruction. 9 10. See the graph 10. causing .2 .changing of the shape of the active site. because active site contains amino acid residues inside the pocket. But if the substrate concentration is high enough then we will obtain the graph that will be parabolic for some time and then straight in term of velocity.2. rise with increase in temperature in accordance with the Arrhenius equation. This occurs because as the temperature changes this supplies enough energy to break some of the intramolecular attractions between polar groups (Hydrogen bonding. Of all most important factor is the active site. R is the gas law constant and T is the absolute temperature. Most enzymes (and there are hundreds within the human organism) within the human cells will shut down at a body temperature below a certain value which varies according to each individual. Q10 is within the range 1. Biological warfare owes its success to enzyme inhibition but so does the life giving chemotherapeutic treatment of cancerous tumor growths with agents that inhibit important cancel cell enzymes. dipole-dipole attractions) as well as the Hydrophobic forces between non-polar groups within the protein structure. including those catalyzed by enzymes. At some temperature activity becomes zero and at some temperature enzyme efficiency of conversion of substrate into product becomes high. and Chromium will function as non-competitive inhibitors. Outside that temperature range the enzyme is rendered inactive and is said to be totally inhibited.70 kJ M-1) give rise to increases in rate by factors between 1. When these forces are disturbed and changed. low enough then the graph between the velocity of reaction and substrate utilization. This is referred to as non-competitive inhibition or mixed inhibition.9. Many heavy metals like Lead.2 Effect of temperature and pressure Every enzyme has a temperature range of optimum activity. This factor for the increase in the rate of reaction for every 10°C rise in temperature is commonly denoted by the term Q10 (i. also known as the frequency factor. and the active site is altered in its conformation beyond its ability to accommodate the substrate molecules it was intended to catalyze.5).3 RT where k is the kinetic rate constant for the reaction. All the rate constants contributing to the catalytic mechanism will vary independently.4.1 Substrate concentration As we know that substrate react with the enzyme and it is converted into the product. and the ability of these residue to interact with the substrate functional group and capacity to breaking and making bonds is certainly going to be effected by the .2 and 2. 8 FIGURE 10. Typical standard free energies of activation (15 . Mercury. This can happen if body temperature gets too low (hypothermia) or too high (hyperthermia).4 . in this case. K = A e –ΔG*/RT --------------27 OR log K = log A –EA /2. Rates of all reactions. The pesticide and herbicide industries make use of competitive and Non-Competitive Inhibitors. Temperature is an important factor in the regulation of enzyme activity.

however. E1 may have higher or lower activity than E) whereas A2 is normally very small or zero. the reverse reaction (having higher activation energy) increases more rapidly with temperature than the forward reaction.long incubation period. —— 60°C. This. —— 55°C. In general. above a critical temperature. at the beginning of the reaction: [E] = [E]0 -----------28 and: [E1] = [E2] = 0 ----29 At time t.changes in both Km and Vmax. The reverse holds for endothermic reactions such as that of glucose isomerase where the ratio of fructose to glucose. E is the native enzyme which may.e. be an equilibrium mixture of a number of species. under these circumstances.7). increases from 1.6). tyrosinase) and the somewhat commoner cases involving immobilised enzyme where there is a small initial activation or period of grace involving negligible discernible loss of activity during short incubation periods but prior to later deactivation. it would be preferable to use enzymes at high temperatures in order to make use of this increased rate of reaction plus the protection it affords against microbial contamination.. ________________________________________ FIGURE 10. in an exothermic reaction.17 at 80°C. This model allows for the rare cases involving free enzyme (e.g. conformational alteration entailing a loss of biological activity) at temperatures above those to which they are ordinarily exposed in their natural environment. it may be seen that it is prudent to err on the low temperature side. —— short incubation period.1 Where kd1 and kd2 are the first-order deactivation rate coefficients. there is a rapid rate of loss of activity (Figure 8. and E1 and E2 are enzyme molecules of average specific activity relative to E of A1 and A2. at equilibrium. —— 65°C. The optimum productivity is seen to vary with the process time. but also reduces the optimum temperature for maximum conversion as the reaction progresses.e. The actual loss of activity is the product of this rate and the duration of incubation (Figure 8. are proteins and undergo essentially irreversible denaturation (i. Enzymes. It is often difficult to get precise control of the temperature of an enzyme catalyzed process and. Note that the temperature at which there appears to be maximum activity varies with the incubation time. ----. not only alters the equilibrium constant.300 kJ mole-1 (Q10 in the range 6 . ________________________________________ ________________________________________ FIGURE 10.g. .5). It may be due to covalent changes such as the deamination of asparagine residues or non-covalent changes such as the rearrangement of the protein chain. which may be determined by other additional factors (e. Inactivation by heat denaturation has a profound effect on the enzymes productivity (Figure 8. These denaturing reactions have standard free energies of activation of about 200 . distinct in structure or activity.00 at 55°C to 1. 11 A schematic diagram showing the effect of the temperature on the activity of an enzyme catalyzed reaction. A1 may be greater or less than unity (i. overhead costs). The thermal denaturation of an enzyme may be modeled by the following serial deactivation scheme: --------27. Assuming. It follows that.36) which means that. or may not. 12 A schematic diagram showing the effect of the temperature on the productivity of an enzyme catalyzed reaction. 10 FIGURE 10.

appear to follow this seriestype deactivation scheme. such as the presence of thiols anti-oxidants. It has been found that the heat denaturation of enzymes is primarily due to the proteins' interactions with the aqueous environment. which prevent freezing. Alternatively it may be due to quaternary structure equilibrium or the presence of distinct genetic variants. equations 38 and 39 remain quite useful and the theory possesses the definite advantage of simplicity. The practical effect of this is that usually kd1 is apparently much larger than kd2 and A1 is less than unity.1]. They are generally more . be assumed to be rather error-prone. Most enzymes are stable for months if refrigerated (0 . Af = 0. It is not surprising that such data can be made to fit a model involving four determined parameters (A1. both free and immobilised. gives: Af = [E] + A1 [E1] + A2 ([E0]-[E]-[E1]) /[E0]-------------37 therefore: ------38 When both A1 and A2 are zero. These may have been formed during the past history of the enzyme during preparation and storage due to a number of minor reactions. limited proteolysis or disulphide interchange. If the enzyme inactivation obeys equation 39. t1/2 = 0. inactivation data should. in general. Many enzyme preparations. Freezing enzyme solutions is best avoided as it often causes denaturation due to the stress and pH variation caused by ice-crystal formation.e (-Kd2t) ) -----------35 If the term 'fractional activity' (Af ) is introduced where. glycerol). enzymeinhibitor and enzyme-product complexes which helps to explain the substantial stabilizing effects of suitable ligand. [S] >> Km).e. Despite this possible reservation. the half-life of the enzyme is shown to be inversely proportional to the rate of denaturation. In any case.693 / Kd1 --------------41 In this simple case.d[E1] / dt =Kd2[E1]-Kd1 [E] ---------------33 Substituting for [E] from equation 32 . The first order deactivation constants are often significantly lower in the case of enzyme-substrate. A2. Cooling below 0°C in the presence of additives (e. the larger the variability the more apparent will be the series-type inactivation kinetics.g. the half-life may be simply derived. Ln (1/2) = =Kd1 t 1/2 -----------------------40 Therefore. even moderate temperatures should be avoided. substituting for [E2] from equation 30. the simple first order deactivation rate expression results Af = e (-Kdt) -----------39 The half-life (t1/2) of an enzyme is the time it takes for the activity to reduce to a half of the original activity (i. Other factors. . especially at concentrations where little free enzyme exists (e.g.d[E] / dt =Kd1[E] -----------------31 Integrating equation 31 using the boundary condition in equation 28 gives: [E] = [E]0 e (-Kdt) = ---------------32 From the reaction scheme . kd1 and kd2). Af = [E] + A1 [E1] + A2 [E2] /[E0]---------36 then.5).d[E1] / dt =Kd2[E1]-Kd1 [E]0 e (-Kdt) -------------34 Integrating equation 33 using the boundary condition in equation 29 gives: [E1]=Kd1 [E]0 / Kd2-Kd1 = (e (-Kd1t) . where the enzyme preparation consists of a heterogeneous mixture of a large number of closely related structural forms. However because reliable and reproducible data is difficult to obtain. may improve the thermal stability in particular cases.[E] = [E1] = [E2] = [E0] ----------30 It follows from the reaction scheme [27. In order to minimise loss of activity on storage. such as de amidation of one or two asparagine or glutamine residues.4°C). In some cases the series-type deactivation may be due to structural micro heterogeneity. can generally increase this storage stability even further.

4. Every enzyme has an optimum pH range outside of which the enzyme is inhibited. These effects are especially important in the neighborhood of the active sites. Buffers are a substance or mixtures of substances that resist any change in the pH. The equilibrium position of the reaction will also be shifted due to any difference in molar volumes between the reactants and products. This change in the pH will affect the polar and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme and the active site as well to the point where the substrate molecule could no longer fit. the changes in charges with pH affect the activity. they remain active for considerable periods even at temperatures above 100°C. with the pH of their environment (Table 1. and the chemical change would be inhibited from taking place as efficiently or not at all. Pressure changes will also affect enzyme catalyzed reactions. may lead to a doubling of the kcat. There are many buffer systems found in the body to adjust the pH so that enzymes might continue to catalyze their reactions. and/or a halving in the Km for a 1000 fold increase in pressure. rather than dilute.3 Effect of pH on Enzyme Enzymes are amphoteric molecules containing a large number of acid and basic groups. Other enzymes like alpha amylase found in the saliva of the mouth operate most effectively at near neutrality. However an additional. in turn. This. 10. these conditions do not take place because of the highly efficient buffers found in the blood that restrict the pH of the blood to a very narrow range. They act slowly on the low pH or high pH. The charges on these groups will vary. This is because of change in the ionization pattern of the amino acid residue inside the active site of the enzyme. Normally. So an optimum pH is needed for its full activity. The enzyme is said to be irreversably denatured. Figure 10. influence is due to the volume changes which occur during enzymic binding and catalysis.1). This would alter the electrical attractions between polar groups. If the pH climbs to an unacceptably high value called alkalosis then enzymes ceases to function effectively. structural stability and solubility of the enzyme. In a dry or predominantly dehydrated state. Some enzyme-reactant mixtures may undergo reductions in volume amounting to up to 50 ml mole-1 during reaction due to conformational restrictions and changes in their hydration. 13 Figure showing optimum pH for two separate enzymes. in addition to the reactivity of the catalytically active groups. Changes in the pH or acidity of the environment can take place that would alter or totally inhibit the enzyme from catalyzing a reaction. Correcting pH or temperature imbalances will usually allow the enzyme to resume its original shape or conformation. The relative effects on kcat and Km depend upon the relative volume changes during binding and the formation of the reaction transition states. If the environment was too basic the acid groups would be deprotonated. Some substances when added to the system will irreversibly break bonds disrupting the primary structure so that the enzyme is inhibited permanently.stable in concentrated. if rather small. Many toxic substances will break covalent bonds and cause the unraveling of the protein . mainly situated on their surface.g. oxygenases and decarboxylases) will be particularly affected by the increased gas solubility at high pressures. If the pH drops in the blood called acidosis then enzymes in the blood will be inhibited outside their optimal pH range. Still other enzymes like the lipases will function most effectively at basic pH values. Either proton is removed or added. Some enzymes like many of the hydrolytic enzymes in the stomach such as Pepsin and Chymotrypsin effective operate at a very low acidic pH. Clearly any reaction involving dissolved gases (e. according to their acid dissociation constants. This property has great technological significance and is currently being exploited by the use of organic solvents. solutions. Therefore all enzymes are affected by the change of pH. Taken together. In an acid solution any basic groups such as the Nitrogen groups in the protein would be protonated. This will affect the total net charge of the enzymes and the distribution of charge on their exterior surfaces.

a number of other factors may mean that the optimum pH in the Vmax-pH diagram may not be the pH of choice in a .7. however.0 Lipase (stomach) 4.0 . The variation of activity with pH.0 Lipase (castor oil) 4.enzyme. the catalytic efficiency and the amount of active enzyme.5 Maltase 6. may occur.5. The temperature also has a marked effect on ionizations. the charge and charge distribution on the substrate(s). in acid solutions (pH < 4).is the only active form of the enzyme.1. as pKa's are based on logarithms with base 10.7 Urease 7. is normally a reversible process. may be reflected in changes in the binding of the substrate.303) is the natural logarithm of 10. characteristic of each enzyme. The importance of the knowledge concerning the variation of activity with pH cannot be over-emphasized.5. sometimes found next to aspartic acid residues. at high hydroxyl concentrations. denaturation. Decreasing hydrogen ion concentration. Extremes of pH will. hydrolysis of the labile peptide bonds. there may be partial destruction of cystine residues due to base catalysed b-elimination reactions whereas.5 . the extent of which depends on the heats of ionization of the particular groups concerned. additionally.8. at which the net charge on the molecule is zero. generally true.8 . Other toxic substances will precipitate enzymes effectively removing them from the solution thus preventing them from catalyzing the reaction.0 Invertase 4. Both Vmax and Km will be affected due to the resultant modifications to the kinetic rate constants k+1.6. plus any consequent structural alterations.7 . and the variation in the concentration of active enzyme. This is called the isoelectric point (pI). assume EH. their complete removal from the enzyme. Δ H is the heat of ionisation and the numeric constant (2. increase the successful competition of hydrogen ions for any metal cationic binding sites on the enzyme. reducing the bound metal cation concentration.6 Trypsin 7. within a range of 2-3 units each side of the pI. In a similar manner to the effect on enzymes. This variation is sufficient to shift the pI of enzymes by up to one unit towards lower pH on increasing the temperature by 50°C. where T is the absolute temperature (K). The effect of pH on the Vmax of an enzyme catalysed reaction may be explained using the. k-1 and kcat (k+2 in the Michaelis-Menten mechanism).6 . product(s) and coenzymes (where applicable) will also be affected by pH changes. Increasing hydrogen ion concentration will. on the other hand. However. assumption that only one charged form of the enzyme is optimally catalytic and therefore the maximum concentration of the enzyme-substrate intermediate cannot be greater than the concentration of this species.0 Amylase (malt) 4. leads to increasing hydroxyl ion concentration which competes against the enzymes' ligand for divalent and trivalent cations causing their conversion to hydroxides and. The relationship between the change in the pKa and the change in temperature is given by a derivative of the Gibbs-Helmholtz equation. In alkaline solution (pH > 8). at which the enzyme generally has minimum solubility in aqueous solutions. R is the gas law constant (8. This is also called denaturation.314 J M-1 K1).8 Amylase (pancreas) 6.0 There will be a pH. These charge variations. In simple terms.2 Catalase 7. Table II pH for Optimum Activity Enzyme pH Optimum Lipase (pancreas) 8.7 Pepsin 1. cause a timeand temperature-dependent. essentially irreversible.1 .

dioxan.g. reducing the dielectric constant of an aqueous solution by the addition of a co-solvent of low polarity (e. Thus.2 x 12) = 0. This process is encouraged within solutions of higher polarity and reduced by less polar solutions.1 M solution of CaCl2 . the variation must be determined under the industrial process conditions.g. only likely to exert a small influence on the enzyme's isoelectric point. The ionisation of the carboxylic acids involves the separation of the released groups of opposite charge. . Generally.e. i. If the charges are opposite then there is a decrease in the reaction rate with increasing ionic strength whereas if the charges are identical. Changes in the ionic strength (I) of the solution may also have some effect. I=0. The ionic strength is defined as half of the total sum of the concentration (ci) of every ionic species (i) in the solution times the square of its charge (zi). the rate controlling step in the catalytic mechanism of chymotrypsin involves the approach of two positively charged groups. This is especially noticeable where catalysis depends on the movement of charged molecules relative to each other. by coupling to ethylene diamine by means of a carbodiimide the profile can be shifted about a pH unit towards higher or lower pH. At higher solution ionic strength. is 0. have lowered pKa) by solutions of lower polarity. The pKa of basic groups are not similarly affected as there is no separation of charges when basic groups ionise.e. The important parameter derived from these influences is the productivity of the enzyme (i. 57histidine+ and 145arginine+ causing a significant increase in kcat on increasing the ionic strength of the solution). an increase in the reaction rate will occur (e. respectively. extensive as they may be. It is found that a single change in charge has little effect on the pH-activity profile. or by immobilisation. ethanol). Thus both the binding of charged substrates to enzymes and the movement of charged groups within the catalytic 'active' site will be influenced by the ionic composition of the medium. changes in the position of equilibrium for a reaction. and the reduction in susceptibility to oxidation or microbial contamination. increases the pKa of carboxylic acid groups. the ionic strength of a 0. These include the variation of solubility of substrate(s) and product(s). This relationship is further complicated by the variation in the effect of the pH with both the duration of the process and the temperature or temperature-time profile. unless it is at the active site. It is possible to alter the pH-activity profiles of enzymes. Chemical derivatisation methods are available for converting surface charges from positive to negative and vice-versa. The variation of productivity with pH may be similar to that of the Vmax-pH relationship but changes in the substrate stream composition and contact time may also make some contribution. This method is sometimes useful but not generally applicable to enzyme catalysed reactions as it may cause a drastic change on an enzyme's productivity due to denaturation (. have little effect on the overall charge on the enzyme molecule at neutral pH and are. it is clearly important to control the ionic strength of solutions in parallel with the control of pH.e. therefore.For example. The ionic strength of the solution is an important parameter affecting enzyme activity. However if all lysines are converted to carboxylates (e. These changes.g. Even if a more complex relationship between the rate constants and the ionic strength holds. and the effects are most noticeable at low ionic strength. suppression of the ionization of a product to facilitate its partition and recovery into an organic solvent.technological process involving enzymes. protonated basic groups which are stabilised by neighbouring negatively charged groups will be stabilised (i. The major such factor is the effect of pH on enzyme stability.g.1 x 22 + 0. charge separation is encouraged with a concomitant lowering of the carboxylic acid pKas.3 M.5 x (0. The cause of these shifts is primarily the stabilisation or destabilisation of the charges at the active site during the reaction. However.5Σ(CiZi2) . how much substrate it is capable of converting to product). by reaction with succinic anhydride) or if all the carboxylates are converted to amines (e.

These are generally discussed in terms of a simple extension to the Michaelis-Menten reaction scheme. where the loss of activity is time dependent and cannot be recovered during the timescale of interest. 000 o From: Daniel Koshland..There may be time-dependent changes in both Km and Vmax. 14 showing molecule activation at high and low temperature Enzyme Substrate Product Rate without Enzyme µmoles/L per min Rate with Enzyme µmoles/L per min Acceleration due to Enzyme Hexokinase Glucose Glucose 6-Phosphate <. 1956. Others.g. Jr. Heavy metal ions (e. --------------------42 where I represents the reversible inhibitor and the inhibitory (dissociation) constants Ki and Ki' are given by --------------------43 and. involving incomplete inactivation. Loss of activity may be either reversible. irreversible inhibition behaves as a time-dependent loss of enzyme concentration (i. but makes no net contribution to the rate equation as it must be equivalent to the equilibrium established through: ------45 Binding of inhibitors may change with the pH of the solution.000006 2700 > 450 million Creatine Kinase Creatine Creatine Phosphate <. Equilibrium between EI and ESI is allowed. and result in the independent variation of both Ki and Ki' with pH. urea) are non-specific protein denaturants. mercury and lead) should generally be prevented from coming into contact with enzymes as they usually cause such irreversible inhibition by binding strongly to the amino acid backbone. or irreversible. which generally act in a fairly specific manner.0000001 1300 > 13 billion Phosphorylase <. as discussed earlier for substrate binding. lower Vmax).g. Journal of Cellular & Comparative Physiology 47: 217-234. Some of these (e.000000005 1600 > 320 billion Alcohol Dehydrogenase Ethanol Acetaldehyde <.FIGURE 10.003 40 > 13. --------------------------44 For the present purposes.e. . are known as inhibitors. it is assumed that neither EI nor ESI may react to form product. in other cases. More important for most enzyme-catalysed processes is the effect of reversible inhibitors. Molecular geometry in enzyme action. Chapter-7 Enzyme inhibition A number of substances may cause a reduction in the rate of an enzyme catalysed reaction. where activity may be restored by the removal of the inhibitor. If the inhibited enzyme is totally inactive.

e. and often is a reaction product (product inhibition. The rate equation for product inhibition is derived from equations (52) and (53). The rate equation is given by: ---------------------52 where Kmapp is the apparent Km for the reaction. The inhibition is most noticeable at low substrate concentrations but can be overcome at sufficiently high substrate concentrations as the Vmax remains unaffected (Figure 10. A. Competitive inhibition. quite a common state of .In order to simplify the analysis substantially. Uncompetitive inhibition. e. ( 44) and (46). A number of simplified cases exist that are reversible.19b). -----54 Figure 11. A similar effect is observed with competing substrates. Competitive inhibition This occurs when both the substrate and inhibitor compete for binding to the active site of the enzyme.19a). Normal Vmax can be observed when substrate is sufficiently present. Therefore: -----------46 also ----------------------------47 where: -----------48 therefore ---------------49 Substituting from equations (43). 2. 3 showing effect of concentration of inhibitor on enzyme activity. it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. then: --------------------52 This is the equation used generally for mixed inhibition involving both EI and ESI complexes (Figure 10. and is given by -----------------53 Normally the competitive inhibitor bears some structural similarity to the substrate. which may cause a substantial loss of productivity when high degrees of conversion are required. 1 showing effect of concentration of inhibitor on enzyme activity on different time interval. the Km (apparent Km) will increase in presence of inhinitor. inhibition of lactase by galactose). Figure 11. note that on increasing inhibitor concentration 1/Vmax remain unchanged while Km value changes. 1. gives: --------------50 therefore --------------51 If the total enzyme concentration is much less than the total inhibitor concentration (i. In double reciprocal plot this can be obsereved. Noncompetitive inhibition 3. one-half Vmax requires a higher [S] than before and thus Km is larger. Ki' is much greater than the total inhibitor concentration and the ESI complex is not formed but EI is formed. Figure 11.g. [E]0<< [I]0). So while Vmax can still be reached if sufficient substrate is available. it is necessary that the rate of product formation (k+2) is slow relative to the establishment of the equilibria between the species. But at [S] at which Vo=1/Vmax. In the presence of a competitive inhibitor. 2 figure showing competitive inhibition 1/Vo (1/ M/min) verses 1/[S] (1/ mM). followed by simplification.

lower Vmaxapp (= 0. all with different kinetic parameters.g.affairs in industrial conversions. If the rates of product formation are much slower than attainment of the equilibria (i. Uncompetitive inhibition This occurs when the inhibitor binds to a site which only becomes available after the substrate (S1) has bound to the active site of the enzyme. and especially relevant to macromolecular hydrolyses where a number of different substrates may coexist. The relative rates of reaction are in the ratio of their specificity constants. The rate equation is: ----------------------62 where Vmaxapp and Kmapp are the apparent Vmax and Km given by --------------------63 and --------------------64 Figure 11. Vmaxapp unchanged (= Vmax). 5 figure showing case of uncompetitive inhibition In this case the specificity constant remains unaffected by the inhibition. S1).e. ________________________________________ . (b) —— competitive inhibition ([I] = Ki). --------61 The inhibition is most noticeable at high substrate concentrations (i.g. their binding is mutually exclusive and they behave as competitive inhibitors of each others reactions. ________________________________________ figure 11. where the reaction scheme may be represented by.67 Vmax). lower Vmaxapp (= 0.g. ---------------55 Both substrates compete for the same catalytic site and. the rate of formation of P1 is given by: ---------56 and the rate of formation of P2 is given by ---------------57 If the substrate concentrations are both small relative to their Km values: --------------------58 Therefore. lower Vmaxapp (= 0. unchanged Kmapp (= Km). (c) —— uncompetitive inhibition ([I] = Ki').e.5 Vmax) and Kmapp (= 0. therefore. —— no inhibition. again. (d) —— noncompetitive inhibition ([I] = Ki = Ki'). some hydrolyses): ---------------60 therefore --------------------------61 B. Ki is much greater than the total inhibitor concentration and the EI complex is not formed. Normally the uncompetitive inhibitor also bears some structural similarity to one of the substrates and. This inhibition is most commonly encountered in multi-substrate reactions where the inhibitor is competitive with respect to one substrate (e. k+2 and k+4 are very much less than k-1 and k-3 respectively). 4 Figure 11.5 Vmax). in a competitive situation using the same enzyme and with both substrates at the same concentration: --------------59 Where and > in this simplified case. S1 in the scheme above) and cannot be overcome as both the Vmax and Km are equally reduced (Figure 1.8c). If both reactions produce the same product (e. S2) but uncompetitive with respect to another (e.6 A schematic diagram showing the effect of reversible inhibitors on the rate of enzyme-catalysed reactions. The reaction involving two co-substrates may be modelled by the scheme.5 Ki'). higher Kmapp (= 2 Km).5 Km). higher Kmapp (= 2 Km). is often a reaction product. (a) —— mixed inhibition ([I] = Ki = 0.

It follows from equation (52) that: -------------------------67 Even quite high values for KS lead to a leveling off of the rate of reaction at high substrate concentrations. —— KS/Km = 10. Km and kcat/Km should. Both the EI and ESI complexes are formed equally well (i. There are two approaches to this problem using either the reaction progress curve (integral . it is unlikely that KS/Km < 1.e. Figure 11. often by different parts of the substrate molecules binding to different sites within the substrate binding site. If the resultant complex is inactive this type of inhibition causes a reduction in the rate of reaction. Noncompetitive inhibition This occurs when the inhibitor binds at a site away from the substrate binding site. 6 (b) Determination of Vmax and Km It is important to have as thorough knowledge as is possible of the performance characteristics of enzymes.A special case of uncompetitive inhibition is substrate inhibition which occurs at high substrate concentrations in about 20% of all known enzymes (e.20). if they are to be used most efficiently. —— KS/Km = 100. The fractional inhibition is identical at all substrate concentrations and cannot be overcome by increasing substrate concentration due to the reduction in Vmax . described earlier. and lower KS values cause substantial inhibition (Figure 10. —— KS/Km = 1. may be considered as a special case of noncompetitive inhibition. 8 figure showing Noncompetative inhibition slope is Km /Vmax ---------------------------68 where Vmaxapp is given by: --------------------------------69 The diminution in the rate of reaction with pH. The inhibitor binding causes inactivation of enzyme due to which apparent Vmax (Vmax = Kcat [Et] ) lower down whether or not substrate is bound. invertase is inhibited by sucrose). causing a reduction in the catalytic rate. therefore. enzyme rate (velocity) is reduced for all values of [S]. By the nature of the binding causing this inhibition. ________________________________________ Figure 11. ________________________________________ C. at high substrate concentrations. —— no inhibition. the inhibitor being the hydrogen ion on the acid side of the optimum or the hydroxide ion on the alkaline side. be determined. A comparison is made between the inhibition caused by increasing KS relative to Km.g. KS/Km >> 100. The kinetic parameters Vmax. It is quite rarely found as a special case of mixed inhibition. Ki equals Ki'). The rate equation is given by: Figure 11. 9 Figure 11. including Vmax and one-half Vmax but Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged Therefore there is no effect on Km. It may be modeled by the following scheme ---------------65 where ---------------------66 The assumption is made that ESS may not react to form product. It is primarily caused by more than one substrate molecule binding to an active site meant for just one. 7 The effect of substrate inhibition on the rate of an enzymecatalysed reaction.

81.25). 4.method) or the initial rates of reaction (differential method). 1.70. 1.03. 1.87.94. This is a powerful non-parametric statistical method which depends upon the assumption that any errors in the experimentally derived data are as likely to be positive (i.87.96.16.70 mM) and 18th (2.65.96.[P]). It is common practice to show the data obtained by the above statistical methods on one of three linearised plots. each intersection forming a separate estimate of Km and Vmax. These estimates are determined from the intersections of lines passing through the (x.14.21.38. In this example there are 7 data points and.45. therefore. 4. 21 estimates for both Km and Vmax. The list of the estimates for Vmax ( M.98. the double reciprocal plot is preferred to test for the qualitative correctness of a proposed mechanism.05. with a . on integration. 2. 3. 4. 4. ------------------70 which. 1. 4.59.99. the predetermined and accurately known substrate concentration at the start of the reaction. 1. 1.25).18. The Km must lie between the 4th (1. Equation (70) can be utilised directly using a computer program. 3. The error in these estimates can be simply determined from sub-ranges of these estimates. 3.29. 1. using the boundary condition that the product is absent at time zero and by substituting [S] by ([S]0 . 3. Use of either method depends on prior knowledge of the mechanism for the reaction and. 3. Its use also ensures that there is no effect of reaction reversibility or product inhibition which may affect the integral method based on equation (73).v). derived from equation (70) (Figure 10. The intersections are separately ranked in order of increasing value of both Km and Vmax and the median values taken as the best estimates for these parameters.89. with a median value of 1.01. 1. involving a weighted least-squares fit.89. where the parameters for determining the hyperbolic relationship between the initial rate of reaction and initial substrate concentration (i. Alternatively the direct linear plot may be used (Figure 10.25. 3. if not.85.min-1) is ranked separately as 3.35. ________________________________________ Figure 11.40. and the Eadie-Hofstee plot is preferred for discovering deviations from linearity.e.98. becomes ------------71 If the fractional conversion (X) is introduced. 3.98. If the mechanism is known and complex then the data must be reconciled to the appropriate model (hypothesis). Many such computer programs are currently available and. If the mechanism is not known. 2. 2.06. too low). A plot of the initial rate of reaction against the initial substrate concentration also showing the way estimates can be directly made of the Km and Vmax. 1978). 4. at least approximately. n(n-1)/2 estimates from n data points with differing [S]0).e. 4. 2. Of these.91. 2.68.25 mM) estimate at a confidence level of 97% (Cornish-Bowden et al. usually by use of a computer-aided analysis involving a weighted least-squares fit. 4.94. the optimum conditions for the reaction. and the assumption is made that the errors inherent in the practically determined data are normally distributed about their mean (error-free) value.0) and (0.1.13.03.98 mM. 1.96.e.12. 1. the programming skill involved is usually fairly low. the width of the subrange dependent on the accuracy required for the error and the number of data points in the analysis. 2. 7 The direct linear plot.e. where -----------------------72 then equation (72) may be simplified to give: ----------73 Use of equation (70) involves the determination of the initial rate of reaction over a wide range of substrate concentrations. The ranked list of the estimates for Km (mM) is 0.26. Every pair of data points may be utilised to give a separate estimate of these parameters (i. 4. 3. 2.85. 3. initial attempts are usually made to fit the data to the Michaelis-Menten kinetic model. Km and Vmax) are chosen in order to minimise the errors between the data and the model. 2. too high) as negative (i.y) points (-[S]0.80.. The initial rates are used. 1. so that [S] = [S]0..91. 3.

this procedure may even be used in cases where there is competitive inhibition by the product. or where the reaction is reversible.min-1. Table -2 .median value of 3.18 M. The Vmax must lie between the 4th (3. possibly only one progress curve is necessary. in a closed system. 11 FIGURE 10. ________________________________________ Three ways in which the hyperbolic relationship between the initial rate of reaction and the initial substrate concentration can be rearranged to give linear plots (a) Lineweaver-Burk (double-reciprocal) plot of 1/v against 1/[S]0 giving intercepts at 1/Vmax and -1/Km --------------74 Figure 11.98 M. is utilised in the analysis. 10 c Hanes-Woolf (half-reciprocal) plot of [S]0/v against [S]0 giving intercepts at Km/Vmax and Km. and sometimes the initial rate of reaction is rather difficult to determine due to its rapid decline. ----------76 The progress curve of the reaction (Figure 10. ________________________________________ Figure 11. The specificity constant may be determined by a weighted least-squared fit of the data to the relationship given by equation (73). This has the advantage over the use of the initial rates (above) in that fewer determinations need to be made. or its derivative. A schematic plot showing the amount of product formed (productivity) against the time of reaction.85 M.28) can be used to determine the specificity constant (kcat/Km) by making use of the relationship between time of reaction and fractional conversion (see equation (73). 9 (b) Eadie-Hofstee plot of v against v/[S]0 giving intercepts at Vmax and Vmax/Km (75) Figure 11. This is a major advantage over the least-squared statistical procedures where rogue data points cause heavily biased effects. 15 . If only the early part of the progress curve.min-1) estimate at a confidence level of 97%. 8 (a) Lineweaver-Burk (double-reciprocal) plot of 1/v against 1/[S]0 giving intercepts at 1/Vmax and -1/Km Figure 11. It can be seen that outlying estimates have little or no influence on the results.min-1) and 18th (4.

e. Linear plots. Alberty equation. Inhibitors and activators.can be ordered or random . Michaelis Menten equation. . and then a second reactant interacts with the modification. while ping pong has products off before all substrates on . one substrate is obligated to bind to the enzyme before a second substrate. In random sequential mechanisms there is no preference. Sigmoidal kinetics and Allosteric enzymes Chapter-8 Multisubstrate enzymes Many Enzymes react with two or more substrates simultaneously (includes cofactors : ATP. • In practice. vary the concentration of the second substrate and repeat.: ________________________________________ • Adenylate kinase displays sequential kinetics.g. FAD etc) Michaelis Menten kinetics is observed when only one substrate is varied with the other(s) held constant (usually the cofactors) Common observed Mechanisms: Sequential – All substrates are bound by enzyme before the any products are formed and released Ordered : Substrates bind and products released in specific order Random : no order Ping Pong – When one or more product(s) are released before all substrates are bound by enzyme (acyl/phosphoryl enzyme intermediate) sequential means all substrates on before any products off. • This is distinguished from what is termed ping-pong kinetics. NADP. • In ordered sequential reactions. • Lineweaver-Burk (double-reciprocal) analysis should yield a family of lines that intersect at the left of the y-axis of the graph. • Within the realm of sequential reactions lies ordered sequential and random sequential at the extreme ends. Then. ping-pong mechanism. King-Altman’s method. • Sequential kinetics can be distinguished from ping-pong kinetics by initial rate studies.Module III Enzyme Kinetics: Single substrate steady state kinetics. there is usually some degree of order in binding. FADH. NADPH. measure initial rates as a function of one substrate while holding the other constant. Multisubstrate systems. In practice. in which both substrates must be bound before any product is released. in which one reactant modifies the enzyme.

distinguishing between seemingly identical groups.. • Ping-pong mechanisms • Ping-pong reactions are ones in which at least one product is released before all the substrates have bound. • To clarify these distinctions we'll look at each of these mechanisms in turn using a typical bi bi enzyme: • A + B P + Q • We'll start by looking at an ordered sequential reaction.e. o Chemical specificity: functional groups. Types of specificity: o Geometric specificity: shape Chiral specificity: most chirally specific enzymes are absolutely stereospecific. sequential and ping-pong (also known as double displacement) mechanisms. in which the reactants and products are bound and released in an obligatory sequence. • Sequential mechanisms • Sequential reactions are ones in which all reactants bind to the enzyme before the first product is released. which is perhaps the simplest in kinetic terms. enzyme molecules) are available • The kinetic mechanism of an enzyme is simply the sequence in which substrates bind to. Kinetic mechanisms can be broadly divided into two main types. Prochirality. . and random reactions. because of their own chiral nature enzymes can often hold substrates in such a way that on one chiral product is made. • Example enzymes to demonstrate these mechanisms in enzyme catalysis. They can be further subdivided into ordered reactions.DG= refers to the difference in free energy between the transition state and the substrate Enzymes work by decreasing DG= to facilitate more rapid formation of P Think of it as enzymes helping the chemical reaction get over the “hump” in order to form product (P) • ES complex demonstrated in a variety of ways including X-ray crystallography. and spectrophotometry • Enzymes derive much of their catalytic power by bringing in a substrate molecule at a “favorable” orientation (this is how enzymes reduce the free energy required to convert S into P) • Leonor Michaelis (1913): noticed that at constant [enzyme] the rate of a reaction increases with increasing [S] until a “maximal velocity” (Vmax) is achieved • This “saturation effect” is an important distinction versus uncatalyzed reactions • Interpretation of data: ES complexes formed until substrate saturation occurs at which point no more substrate binding sites (i. Enzyme Catalysis We will look at catalysis in two types of systems: • Model systems in organic chemistry to elucidate probable mechanisms of chemical catalysis. in which there is no obligatory binding sequence. types of chemical reaction. It should not be confused with the chemical mechanism which is concerned with the chemical interactions between the enzyme and its substrate(s) which results in the creation of product(s). electron microscopy. and products are released from the enzyme.

one-substrate enzymes then. • For S P assume • • And for initial reaction conditions [P] = 0 & therefore k4 = 0. important in enzyme catalysis. Generally we will be looking at three ways to increase rates 1. S P get rectangular hyperbola type plot for vi vs [S] (text Figure 6-11). increase the concentrations of intermediates 3. • • • Let's look at a mathematical model and attempt to generate curve. have Michaelis-Menten Equation as a model for enzyme activity. Thus if we can find a way to stabilize the transition state (lower Ea) then the reaction rate will be enhanced. For simple. This was first done by Michaelis and Menten for an equilibrium model.in water) o General (Bronsted acid definition: proton donor/acceptor pairs. Types of Catalysis: • Acid/Base o Specific (H+ & OH. • Assume steady state (steady state assumption: d[ES]/dt= 0): • d[ES]/dt= 0. so have • • Now vi = d[P]/dt = k3[ES] (Note that kcat is often used instead of k3). which in turn depends only on [R3CCl] • Second order: r = k[A][B] for A + B P. buffers.k2[ES] . 0 order also only occurs above a minimum [A]. Better is the steady state model of Haldane and Briggs (more general). stabilize transition states 2. & r =-d[S]/dt = d[P]/dt. • Higher order reactions occur. Which is known as the Michaelis-Menten Equation.SN2 from organic chemistry: A + B C + D as 2nd order reaction.(reverse of figure above) are involved in slow step.) • Covalent o nucleophilic o electrophilic • Proximity/orientation • Stabilization of Transition State Conformation (Strain/distortion.SN1 from organic chemistry: A + B C + D as 1st order reaction. [S] for 0 . Charge neutralization) • Metal/Metal ion. . but uncommon. as in the hydroxylation of primary alkylchlorides: Now 2nd order because both RH2CCl and OH. use a different reaction pathway. which we will derive. or can have r = k[A]2 . So. So how does catalysis work? Recall that the slow step of a reaction is reaching the transition state. o Example . similar to Mb binding curve. o Example .3rd order • First Order: r = k[S] for S P. Thus: 0 = d[ES]/dt= k1[E][S] .k3[ES]. one-substrate enzymes: For simple enzyme. • Zero order: r = k: Only occurs with catalysts. o • Plots of vi = d[P]/dt vs.Mechanisms of Chemical Catalysis Look at some examples of catalysis in model systems (organic chemistry) and how they might operate in enzymes. as in the hydroxylation of t-alkylchloride: First order because rate depends only on the formation of the carbocation.

the substrate concentration at half-saturation. kcat (k3). while low precision points [low concentration] are more spread out. Three major types of inhibition: • Competitive inhibition • Noncompetitive inhibition • Uncompetitive inhibition Competitive Inhibition: S & I are mutually exclusive. In addition the value of KM is obtained from the slope. Plots: (text Box 6-2 figure 1) We can model this inhibition with chemical equations. Note that the data points are distributed much more evenly over the plot giving better statistics for the slope. giving better precision. • Note consequences for a plot: start off with approximately linear slope with y = kx. FYI . E can bind to one OR the other: (text Figure 6-15a) Model: . overlapping sites for S & I. with a strong influence on the slope and the KM intercept ¬ this is not as much of a concern now with computer statistical packages. steric hindrance between S & I in different sites. o Understanding drugs and toxins to counter etc. then . 3.Note predicted consequences of model: • [S] >> KM. Partial sharing of sites. Note that this is best determined under saturating conditions. The rate constant (First order) for the breakdown of the [ES] complex. then vi = Vmax/2 This is definition of KM. the formula for a rectangular hyperbola: (text Figure 6-12) Turnover Number. best quality points [high concentration] have least influence on slope. but you still have to understand the statistics). This is exactly what we expect if we look at the general form of the equation: . Then at the limit of high concentrations have a horizontal line. . E can bind to one OR the other. and: . and are better in terms of statistics (L-B one of worst. is also known as the turnover number. (text Box 6-01 figure 6-1) Other linear plots are also available. so take reciprocals of both sides and have . (text Table 6-08) At very low concentrations of [S] can find the second-order rate constant for the conversion of E + S E + P: vo = (kcat / KM)([E][S]. Classically assume binding to same site. that is the maximum number of substrate molecules processed/active site (moles substrate/mole active site): kcat=Vmax / [E]total. and get First order (r = k [S]) • [S] = KM.The Eadie-Hofstee Plot One common plot is shown below. We will see others in the laboratory discussion. Linear plots for enzyme kinetic studies Double Reciprocal or Lineweaver-Burke Plot: Need in form: y = ax + b . but other possibilities also. 2. and have a large moment. ENZYME KINETICS AND INHIBITION What's exciting about enzyme inhibition? • Potential to tell us about enzyme. where . then vi = Vmax and get Zero order (r = k) • [S] << KM. • Potential uses as drugs and toxins. 1. keeping in mind that S & I are mutually exclusive.

P or Q may be released last. • Multi-substrate Enzymes Look at three common and easily understood types. Cunningham. but is found in multi-substrate systems. two off). Plots for classic. ed. "Substrate Inhibition: in Contemporary Enzyme Kinetics and Mechanism. Note: A must bind first.5): Mixed: Like non-competitive above. New york (1983)) Kinetic Mechanism Variable Substrate Product Type of Inhibition Ordered Sequential Bi Bi Ordered Sequential Bi Bi A B Q Noncompetitive A P Noncompetitive B P Noncompetitive Random Sequential Random Sequential B Q A P B P Ping pong Bi Bi Ping pong Bi Bi B Q A P B P Bi Bi Bi Bi A Q Noncompetitive Noncompetitive Noncompetitive Q Competitive Noncompetitive A Q Competitive Noncompetitive Noncompetitive Competitive Note that in each case we can predict/explain the pattern of inhibition on the . For double reciprocal plots get parallel lines! (text Box 6-2 figure 2) This is not generally found for single substrate enzymes. Noncompetitive: the inhibitor can bind to either E or ES. they may be the same. one on. McGraw-Hill Book Company. and . or could be different. Biochemistry: Mechanisms of Metabolism. • Ping Pong Bi Bi (one on. with a L-B plot with an intersection on the plot above the 1/vo axis. one off). New York (1978). Two-substrate Enzyme Product Inhibition Patterns (Based on: E. two off). We will use Cleland Nomenclature and "Kinetic mechanism diagrams. leading to more complex behavior. S & I do not bind to the same sites! (text Figure 6-15c) Model: . Note: have some sort of modified enzyme intermediate (often covalent intermediate) • Random Sequential Bi Bi (two on." • Ordered Sequential Bi Bi mechanism (two on.) Academic Press. Q is released last.4. one off. Purich. Conformational change of enzyme with binding of either such that other can not bind. and but with different values of Ki. (Daniel L. and W. simple situation (overhead MvH 11. Note: A or B may bind first. This is in fact the more common situation. Model: . Model: . as in the equation above. Note that will have two inhibitor binding constants. B. and . Cleland. (text Box 6-2 figure 3) Uncompetitive: In uncompetitive inhibition the inhibitor binds ONLY to the ES complex (text Figure 6-15b).

Similarly for the Ping pong mechanism the first substrate and last product should be competitive as the both bind the free enzyme. enzyme F. The Random Sequential mechanism is a bit more subtle. Here we see across the board noncompetitive since in each case the substrates (and products) can each bind to more than one substrate form. What would happen though if you repeated the experiment with an increased concentration of the fixed substrate. The next reaction is the key to the whole process: (EA) (FP) In this reaction a part of the substrate has been removed from substrate A. The removed section has become covalently bound to the enzyme to create a new form of the enzyme. let's say a typical bi bi enzyme: A + B P + Q we can carry out exactly the same experiment. The results would be exactly the same as you'd expect in a single substrate system and you could use any of the methods that we've studied to calculate the kinetic parameters. for instance the serine proteases (trypsin. To start with we'll examine the effects of changes in substrate concentration on multisubstrate reactions. Since you're increasing the concentration of a substrate you would expect the velocity to rise and in fact the ." Thus for the Ordered Sequential mechanism only the first substrate and last product bind to the same form. Now that we're familiar with the principal types of kinetic mechanism we need to think about experimental techniques to distinguish between them. in this case the free enzyme. which is then released: (FB) (EQ) (EQ) E + Q The animation should help to clarify this.) The ping-pong (double displacement) mechanism ________________________________________ The distinguishing feature of these enzymes is that at least one product is released from the enzyme before all of the substrates have bound. The Cleland plot for a ping-pong enzyme is quite distinctive: as the first upward arrow (product P) is to the left of the first down arrow (substrate B). chymotrypsin. but it's actually easily explained and quite a common mechanism.basis of the substrate and inhibitor binding to the same "enzyme form. so competitive inhibition will not be possible! (Think of the product as competing with one order of binding but not the other. This might seem slightly unlikely at a first glance. Some very familiar enzymes. Effects of Substrate Concentration in Multisubstrate Systems ________________________________________ With a multisubstrate enzyme. We simply need to keep one substrate (the fixed substrate) at a constant concentration in all our assays. The active site has no room for the second substrate so it is full. etc. The first product of the reaction is now released and the second substrate binds: (FP) F + P F + B (FB) Now the stored section of the first substrate is transferred to the second substrate to create the second product. while we vary the concentration of the other substrate (the variable substrate).) and the amino transferases work in this way. converting it to product P. since they both bind to the E-X complex. The active site is full as this substrate will be converted to product before the second substrate can bind. The process starts by binding of the enzyme to the first substrate in the usual way: E + A (EA) Notice that I've used parentheses around the EA complex to indicate that it is a central complex. In this case we also see a competitive inhibition between the second substrate and the first product.

A change in slope indicates the effect of a change in concentration of the fixed substrate on the speed at very low concentrations of the variable substrate. In discussing graphs of this type we'll be considering changes in the Vmax and slope of the line. We'll start by considering the expected results of this experiment when carried out with a sequential enzyme. As we're looking at effects at low A concentrations this would be seen as a change in the slope of the Lineweaver-Burk plot. and therefore of the overall reaction. If you repeated this at a variety of concentrations of the fixed substrate you would get a series of lines. An increase in B would increase the speed of either of these as a reactant in the second reaction and by the knock on effect of generating more EAB for the central reacton to occur. Substrate concentration assays with sequential enzymes ________________________________________ Consider a bi bi ordered sequential reaction: A + B P + Q The Cleland plot for such a reaction would be: We'll discuss the results of a set of enzyme assays in which substrate A is used as the variable substrate and substrate B as the fixed substrate. You might like to demonstrate that yourself as an exercise. A typical Lineweaver-Burk plot obtained as a result of this type of experiment can be seen below. At very low A concentrations the rate limiting step of the reaction would be the binding of A to the enzyme as the substrate is in very short supply. or EA + B EAB at low B levels. enables us to distinguish between sequential and ping-pong enzymes.reaction would be faster at any given concentration of the variable substrate than it was in the previous experiment. Remember in our discussion of enzyme inhibition we found that the slope was the rate constant at low substrate concentrations. So the increase in B has increased the speed of the rate limiting step. A change in Vmax indicates the effect that a change in the concentration of the fixed substrate on the reaction speed at very high concentrations of the variable substrate. The kinetic parameters would change to reflect the change in velocity. as we'll see in the next couple of pages. At very high A concentrations the rate limiting step would be the EAB EPQ. Reducing the concentration of the product of the E + A EA reaction will pull it to the right by the Law of Mass Action. for an ordered sequential reaction. In summary then. which is the inherently slowest reaction. The actual pattern of lines obtained will vary according to the way in which the enzyme interacts with the two substrates and. So you'd end up with a second set of data which you could use in your chosen plot. Substrate A was used as the variable substrate and substrate B as the fixed substrate. This would be seen as a change in Vmax as we are considering effects at high A concentration. a change in the concentration of the fixed substrate would bring about a change in both the slope and intercept of the Lineweaver-Burk plot and the graph would be similar to that suggested on the previous page. A similar result would be obtained if the assay was reversed and B used as the variable substrate or if the enzyme used a random sequential mechanism. Remember that Vmax is the velocity of the reaction under those conditions. An increase in the concentration of B would reduce the concentration of the EA complex in the reaction mixture by binding to it to form EAB complex. .

Dye –ligand chromatography. GPC gel permeation chromatography.( power full method ) Source of enzyme . [iv] Based on change in dielectric strength by adding organic solvent.Chapter-9 ISOLATION AND PURIFICATION OF ENZYME INTRODUCTION ENZYME is isolated and purified to obtain maximum desired product because they are not utilized during the product formation. Covalent chromatography. Often during purification enzyme yield is decreases many folds. [iii] Based on changes in solubility ( by change of pH.) 4. Electrophoresis (Examining enzyme composed of non identical subunit) 3. change in ionic strength (Salting in or salting out). Immuno-adsorption chromatography. Purifying enzyme is much labor intensive process and main aim is to separate other protein that are not the part of the enzyme. isoelectric focusing. PURIFIED enzyme is needed for laboratory work and less purified enzyme is needed for commercial purpose. Mass –spectrometry. Ultrapurification (for impurities < 5%) 2. source of enzyme Method of enzyme isolation (cell lysis by osmotic. [i] Based on size and mass ( centrifugation. Isoelectric –focusing ( very sensitive method) 6. 1 strategies for enzyme purification. enzyme. FIGURE 8. Therefore for the diagnostic purpose much pure enzyme is needed. Purification of enzyme is aimed to get maximum turn over number that is to increase the activity of enzyme in other world to increase its efficiency of conversion of per mole substrate into the product. electrophoresis. Affinity chromatography. Dialysis and ultracentrifugation). test of purity b). Other need of the enzyme purification is the study of kinetics that is rate of enzyme and its specificity towards the substrate. Capillary Electrophoresis 5. (4). [v] Based on specific binding site. or homogenization method) Method of separation. Test of catalytic activity c). Test of purification a). its regulation mechanism. hydrophobic interaction chromatography). And also isolation of enzyme gives the maximum understanding of the behavior of the enzyme in complex system. sonication. During the disturbance in the metabolic pathway some enzyme may not form and it results in the formation of unwanted product like in the phenylketonuria disease homigenistic acid secretion in the urine results. Active site titration Test of purity is done by following method 1. [ii] Based on polarity (Ion –exchange. Inhibition kinetics helps in functional study of the enzyme. It helps in increasing the sensitivity of the diagnosis test in the clinics. Affinity elution. SDS-PAGE ( good method for detecting impurities and for detecting damage of non-identical subunit.

1.Biologically active enzymes may be extracted from any living organism.4.22.23. Enzyme a EC number b Source Intra/extra -cellular c Scale of production d Industrial use Animal enzymes Catalase 1.4. 2.2.1.4. Non-microbial sources provide a larger proportion of these. including phenolic compounds (from plants).1.5 Bacillus I ++ Fructose syrup Penicillin amidase 3. 5.2. Attempts are being made to overcome some of these difficulties by the use of animal and plant cell culture.1.1 Bacillus E +++ Starch -Amylase 3. A very much larger number of enzymes find use in chemical analysis and clinical diagnosis.5. ________________________________________ Table 2.4.22.2. they are generally cheaper to produce. plant and animal tissues contain more potentially harmful materials than microbes.4 Pineapple latex E Brewing Glucanaseg 3. Of the hundred or so enzymes being used industrially.1. their enzyme contents are more predictable and controllable.2.3 Fig latex E Food Lipoxygenase 1.21.22.1 Aspergillus E ++ Baking .11. endogenous enzyme inhibitors and proteases.13.2.21.12 Soybeans I Food Papain 3.21.4 Pancreas E Leather Plant enzymes Actinidin 3. from spinach to snake venom.1 Malted barley E +++ Brewing -Amylase 3. over a half are from fungi and yeast and over a third are from bacteria with the remainder divided between animal (8%) and plant (4%) sources (Table 8.1.4.1.2 Malted barley E +++ Brewing Bromelain 3.1. 3. and 4.1.14 Bacillus E +++ Detergent Pullulanasej 3.3 Pancreas E Food Rennetf 3.3.1.1.2 Pawpaw latex E ++ Meat Bacterial enzymes Amylase 3.1. reliable supplies of raw material of constant composition are more easily arranged.4.1.1 Pancreas E Leather Lipasee 3.4 Abomasum E + Cheese Trypsin 3.5.11 Bacillus I Pharmaceutical Proteasei 3.4.2.1). Some important industrial enzymes and their sources.4.2 Bacillus E + Starch Asparaginase 3.1 Escherichia coli I Health Glucose isomeraseh 5.22.2.1.14 Kiwi fruit E Food Amylase 3. at the present time.41 Klebsiella E Starch Fungal enzymes Amylase 3. A very wide range of sources are used for commercial enzyme production from Actinoplanes to Zymomonas. Microbes are preferred to plants and animals as sources of enzymes because: 1.6 Liver I Food Chymotrypsin 3.6 Malted barley E ++ Brewing Ficin 3.11.

3.1.4. h xylose isomerase.15 Aspergillus E ++ Drinks Pectin lyase 4. i subtilisin.22 Saccharomyces I Food a The names in common usage are given.1. the great majority of microbial enzymes come from a very limited number of genera. j dextrin endo-1.4. + > 1 ton year-1. e triacylglycerol lipase.1. Bacillus species and Kluyveromyces (also called Saccharomyces) species predominate.6 Mucor miehei E ++ Cheese Pectinasen 3.3 Aspergillus E +++ Starch Catalase 1.2.2.extracellular enzyme.1. There are very few examples of the industrial use of enzymes having been developed for one task.1.4 Trichoderma E Waste Dextranase 3.-glucanase.1.-glucosidase.3 Rhizopus E Food Rennetm 3. . .6.11.1.22 Mortierella I Food Yeast enzymes Invertasep 3.2. n polygalacturonase.3(4). k glucan 1.23.5.10 Aspergillus E Drinks Proteasem 3. of which Aspergillus species.1. b The EC number of the principal component. As most industrial enzymes consist of mixtures of enzymes.< 1 ton year-1. the recommended names of this principal component is given below.6 Aspergillus E + Baking Raffinaseo 3. ++ > 10 ton year-1. Most of the strains used have either been employed by the food industry for many years or have been derived from such strains by mutation and selection.2. d +++ > 100 ton year-1.intracellular enzyme.2. In practice.1. m microbial aspartic proteinase. these names may vary from the recommended names of their principal component.11 Penicillium E Food Glucose oxidase 1.2.4. g Endo-1.6 Aspergillus I Food Cellulase 3. Where appropriate.1. f chymosin.2.23 Kluyveromyces I/E Dairy Lipasee 3. l -galactosidase.2.1.2.2.-glucosidase. o -galactosidase.1.Aminoacylase 3.4 Aspergillus I Food Lactasel 3.1.23.1. E .3 Candida E Food Raffinaseo 3.26 Saccharomyces I/E Confectionery Lactasel 3. c I .1.23 Aspergillus E Dairy Lipasee 3. Shining examples of such developments are the production of high fructose syrup using glucose isomerase and the use of pullulanase in starch hydrolysis.1.2.14 Aspergillus I Pharmaceutical Glucoamylasek 3. p -fructofuranosidase.

If such variability is likely to significantly reduce the efficiency of the standard methodology. sometimes refined but often simply as ground cereal grains. The chances of a susceptible peptide bond in a structured . Clearly defined media are usually out of the question for large scale use on cost grounds but may be perfectly acceptable when enzymes are to be produced for high value uses. 10% w/w of the protein) in order to ease the downstream processing task. minerals. This may be achieved by developing the fermentation conditions or. proteolysis.Media for enzyme production Detailed description of the development and use of fermentors for the large-scale cultivation of microorganisms for enzyme production is outside the scope of this volume but mention of media use is appropriate because this has a bearing on the cost of the enzyme and because media components often find their way into commercial enzyme preparations. Enzyme inactivation can be caused by heat. Soybean meal and ammonium salts are frequently used sources of additional nitrogen. often more dramatically. only other enzymes and material likely to interfere with the process which the enzyme is to catalyze. Of this heat inactivation. Thus molasses. In contrast. oxidation. frequently below 10% of the activity of the original material (Table 2. for instance. It is also the major reason for enzyme inactivation by microbial contamination. Most of these materials will vary in quality and composition from batch to batch causing changes in enzyme productivity. Waste materials and byproducts from the food and agricultural industries are often major ingredients. Also some components of media may be changed from batch to batch as availability and cost of. carbohydrate feedstock change. enzymes have highly structured domains which are resistant to attack by proteases because many of the peptide bonds are mechanically inaccessible and because many proteases are highly specific.2). distillers soluble and wheat bran is important components of fermentation media providing carbohydrate. It may well be economically viable to spend some time cloning extra copies of the required gene together with a powerful promoter back into the producing organism in order to get 'over-producers' (see Chapter protein engineering). nitrogen and some vitamins. by genetic engineering. irreversible inhibitors and loss of cofactors or coenzymes. but the problem is less when preparing thermophilic enzymes. Details of components used in industrial scale fermentation broths for enzyme production are not readily obtained. Extra carbohydrate is usually supplied as starch. sub-optimal pH. The content of the required enzyme should be as high as possible (e. plus additives to stabilize the enzyme's activity. The effects of changing feedstock must be considered in relation to downstream processing. It is important that the maximum activity is retained during the preparation of enzymes. Proteolysis is most likely to occur in the early stages of extraction and purification when the proteases responsible for protein turnover in living cells are still present. It is likely to occur during enzyme extraction and purification if insufficient cooling is available. This is not unexpected as manufacturers have no wish to reveal information that may be of technical or commercial value to their competitors. Less-defined complex media are composed of ingredients selected on the basis of cost and availability as well as composition. will be removed. corn steep liquor. manpower and loss of enzyme activity. In their native conformations. As a result. Unnecessary purification will be avoided as each additional stage is costly in terms of equipment. it may be economical to use a more expensive defined medium of easily reproducible composition. Such changes reveal themselves in often quite profound differences in appearance from batch to batch of a single enzyme from a single producer. this together with associated pH effects is probably the most significant. Often the enzyme may be purified several hundred-fold but the yield of the enzyme may be very poor. industrial enzymes will be purified as little as possible. some commercial enzyme preparations consist essentially of concentrated fermentation broth. such as analysis or medical therapy where very pure preparations are essential.g. denaturants.

animal cells and some gram-negative bacteria such as Azotobacter species).8) the following equation for cell breakage is obtained (2. the whole polypeptide chain may be available for proteolysis and the same. Cell breakage by osmotic shock Various intracellular enzymes are used in significant quantities and must be released from cells and purified (Table 2. It should have maximum possible purity. Consequently a variety of cell disruption techniques have been developed involving solid or liquid shear or cell lysis.3. Homogenization 4. protease may destroy it.domain being available for protease attack are low. Clearly the best way of preventing proteolysis is to rapidly remove. The amount of energy that must be put into the breakage of cells depends much on the type of organism and to some extent on the physiology of the organism. Solid/liquid separation is generally required for the initial separation of cell mass. in general. Cell can be break down byfollowing methods 1. green algae. are heating . centrifugation or aqueous biphasic partition. or inhibit. This can be achieved by filtration. whilst others are highly resistant to breakage. (2. Figure 2. filtration or aqueous biphasic systems are used to remove unwanted cells or cell debris whereas centrifugation is the preferred method for the collection of required solid material.1). Some intracellular enzymes are used commercially without isolation and purification but the majority of commercial enzymes is either produced extracellularly by the microbe or plant or must be released from the cells into solution and further processed (Figure 2. protease activity.g. weight for weight. Ultracentrifugation. however. Some types of cell are broken readily by gentle treatment such as osmotic shock (e.5) where P represents the protein content remaining associated with the cells. The particular hazards to enzyme activity relevant to cell breakage are summarized in Table 2.1). of reinforced concrete.9) It is most important in choosing cell disruption strategies to avoid damaging the enzymes. Single 'nicks' by proteases in these circumstances may have little immediate effect on protein conformation and. The rate of protein released by mechanical cell disruption is usually found to be proportional to the amount of releasable protein. Enzymic lysis. specific. Integrating from P = Pm (maximum possible protein releasable) at time zero to P = Pt at time t gives (2. t is the time and k is a release constant dependent on the system. therefore.1. Before this can be achieved it is important to keep enzyme preparations cold to maintain their native conformation and slow any protease action that may occur. The main objective of the purification is To obtained maximum possible yield. In general. It should have maximum possible activity. 3. Flow diagram for the preparation of enzymes. the removal of cell debris after cell breakage and the collection of precipitates. The effect. If the domain is unfolded under these changed conditions.7) As the protein released from the cells (Pr) is given by (2. fungal mycelia and some gram-positive bacteria which have cell wall and membrane structures capable of resisting internal osmotic pressures of around 20 atmospheres (2 MPa) and therefore have the strength. Osmotic shock 2. may severely reduce the conformational stability of the enzyme to heat or pH variation so greatly reducing its operational stability.6) (2. These include yeasts. A. activity. The most significant of these.

in particular yeasts and Bacillus species. Lysozyme-rich.g. but it can be used on a very large scale if necessary. Autolysis is a slow process compared with mechanical methods. This problem may be overcome by the use of adsorbents. mercaptoethanol and dithiothreitol) may be necessary. Where costs are reduced by the use of the relatively inexpensive. Where applicable. B. Such action may be minimised by increased speed of processing with as much cooling as possible. Polyphenolics derived from plants are potent inhibitors of enzymes. The presence of substrates. substrate analogues or polyols may also help stabilise the enzyme. Shear Shear forces are needed to disrupt cells and may damage enzymes. such as EDTA. Proteases Disruption of cells will release degradative enzymes which may cause serious loss of enzyme activity. This may be improved by the presence of an excess of alternative substrates (e. Some types of cell may be caused to autolyse. from hen egg-white.and shear. Each method has its drawbacks but may be particularly useful under certain specific circumstances. and microbial contamination is a potential hazard. The presence of substrates. desiccation. Chemical Some enzymes may suffer conformational changes in the presence of detergent and/or solvents.g. gentle and convenient method of releasing enzymes but has not apparently been used on a large scale. ________________________________________ Table 2. Media will usually be buffered at a pH value which has been determined to give the maximum stability of the enzyme to be extracted. may be very cheap. ascorbic acid.3. do not subject the enzyme to heat or shear. generating heat. The chief objection to its use on a large scale is its cost. copper and nickel) may be introduced by leaching from the homogenisation apparatus.000 U Kg-1 (dry weight). Heat All mechanical methods require a large input of energy. Yeast-lytic enzymes from Cytophaga species . Enzymic lysis using added enzymes has been used widely on the laboratory scale but is less popular for industrial purposes. Other components will combat other hazards to the enzyme. The rate of drying is very important in these cases. a major separation problem may be introduced. substrate analogues or polyols may also help stabilise the enzyme. freezing followed by thawing. and are quiet to the user. Enzymes may be protected from irreversible inactivation by the use of chelating reagents. cold shock.g. This would be a cheap. is the only lytic enzyme available on a commercial scale. primarily factors causing denaturation (Table 2. Yeast invertase preparations employed in the industrial manufacture of invert sugars are produced in this manner. Certain types of cell can be caused to lyse by osmotic shock. dried egg white.3). Foaming The gas-liquid phase interfaces present in foams may disrupt enzyme conformation. such as polyvinylpyrrolidone. slow methods being preferred to rapid ones like lyophilisation. ________________________________________ Media for enzyme extraction will be selected on the basis of cost-effectiveness so will include as few components as possible. desiccation may be very useful in the preparation of enzymes on a large scale. inexpensive protein) or inhibitors in the extraction medium. iron. particularly in the presence of heavy metal ions and/or an air interface. pH Buffered solutions may be necessary. Hazards likely to damage enzymes during cell disruption. enzymic lysis and chemical lysis. Cooling is essential for most enzymes. Oxidation Reducing agents (e. The methods that are available include osmotic shock. It has often used to lyse Gram positive bacteria in an hour at about 50. Lysozyme. and by the use of ascorbic acid to reduce polyphenol oxidase action. Use of enzymic lytic methods The breakage of cells using non-mechanical methods is attractive in that it offers the prospects of releasing enzymes under conditions that are gentle. Heavy-metal toxicity Heavy metal ions (e.

Use of radical scavengers (e. Ultrasonic cell disruption The treatment of microbial cells in suspension with inaudible ultrasound (greater than about 12 kHz) results in their inactivation and disruption.g. small displacements (less than about 50 m). The constant (k) is independent of cell concentrations up to high levels and approximately proportional to the input acoustic power above the threshold power necessary for cavitation. very fine cell debris particles may be produced which can hinder further processing. useful and simple small-scale method for cell disruption. Detergents. This consists of a positive displacement pump which draws cell suspension (about 12% w/v) through a check valve into the pump cylinder and forces it. The amount of protein released by sonication has been shown to follow Equation 2.9. through an adjustable discharge valve which has a restricted orifice (Figure 2. Disintegration is independent of the sonication frequency except insofar as the cavitation threshold frequency depends on the frequency. steep transverse velocity gradients (up to 4. As with most cell breakage methods.000 L hr-1.000 g). However. such as Triton X-100.5). such as guanidine HCl. Cell lysis by High pressure homogenisers Various types of high pressure homogeniser are available for use in the food and chemicals industries but the design which has been very extensively used for cell disruption is the Manton-Gaulin APV type homogeniser. If significant markets for lytic enzymes are identified. at high pressures of up to 150 MPa (10 tons per square inch) and flow rates of up to 10. the scale of their production will increase and their cost is likely to decrease.have been studied in some detail and other lytic enzymes are under development. Reasons for this may be the conformational lability of some (perhaps most) enzymes to sonication and the damage that they may realize though oxidation by the free radicals.1 MHz). Cells are subjected to impact. such materials are costly and may be difficult to remove from the final product. Ultrasonication utilises the rapid sinusoidal movement of a probe within the liquid. used alone or in combination with certain chaotropic agents. provided that the enzymes are sufficiently robust. a popular. The collapse of the bubbles converts sonic energy into mechanical energy in the form of shock waves equivalent to several thousand atmospheres (300 MPa) pressure. Ultrasonication produces cavitation phenomena when acoustic power inputs are sufficiently high to allow the multiple production of microbubbles at nucleation sites in the fluid. This causes the impact and shear stress which are proportional to the operating pressure. This energy imparts motions to parts of cells which disintegrate when their kinetic energy content exceeds the wall strength.Lysis by acid. Sonication remains. surfactants and solvents can be effective in releasing enzymes. singlet oxygen and hydrogen peroxide that may be concomitantly produced. d. shear and a severe pressure drop across the valve but the precise mechanism of cell disruption is not clear. alkali. moderate velocities (a few m s-1). An additional factor which increases cell breakage is the microstreaming (very high velocity gradients causing shear stress) which occur near radially vibrating bubbles of gas caused by the ultrasound. a violent shock wave passes through the medium. It is characterised by high frequency (12 kHz . Equipment for the large-scale continuous use of ultrasonic has been available for many years and is widely used by the chemical industry but has not yet found extensive use in enzyme production. are effective in releasing membrane-bound enzymes. .000 s-1) and very high acceleration (up to about 20. c. then are collapsed during the compression phase. On collapse. The whole process of gas bubble nucleation. however. Much of the energy absorbed by cell suspensions is converted to heat so effective cooling is essential. The main disruptive factor is the pressure applied and consequent pressure drop across the valve. The bubbles grow during the rarefying phase of the sound wave. growth and collapse due to the action of intense sound waves is called cavitation. N2O) have been shown to reduce this inactivation.

the release constant (k) has been found to be proportional to the pressure raised to an exponent dependent on the organism and its growth history (e. a large number of small beads will be more effective than a relatively small number of larger beads because of the increased likelihood of collisions between beads and cells.9 in Saccharomyces cerevesiae and k=k'P2.10) In the commonly-used operating range with pressures below about 75 MPa. The rate and effectiveness of enzyme release can be modified by changing the rates of agitation and the size of the beads. this advantage cannot be used since the temperature rise due to adiabatic compression is very significant so samples must be pre-cooled and cooled again between multiple passes. the higher the operating pressure.0 mm diameter) they are broken by the high liquid shear gradients and collision with the beads. both of which consist of a cylindrical vessel containing a motor-driven central shaft equipped with impellers of different types. Both can be operated continuously. where P represents the operating pressure and k' is a rate constant). Multiple passes are undesirable because. the size . C.Figure 2. In addition to the fragility of the cells. The bead mills that have been studied in most detail are the Dyno-Mill and the NetschMolinex agitator. k=k'P2. Different growth media may be selected to give rise to cells of different cell wall strength. In practice. they decrease the throughput productivity rate and because the further passage of already broken cells results in fine debris which is excessively difficult to remove further downstream. the more efficient is the disruption process. the temperature rise each pass is about 12 deg. as well as the dimensions of the equipment. the larger sized cells will be broken more readily than small bacteria. Glass Ballotini or stainless steel balls are used. Consequently. in general. The cell suspension is pumped at high pressure through the valve impinging on it and the impact ring. At an operating pressure of 50 MPa. the location of an enzyme within the cells can influence the conditions of use of an homogeniser. filamentous or unicellular. homogenisers will be used at the highest pressures compatible with the reliability and safety of the equipment and the temperature stability of the enzyme(s) released. may be disrupted by bead milling but.2 in Escherichia coli. The protein release rate constant (k) is temperature dependent.g.2 -. Clearly. The valve unit is prone to erosion and must be precision made and well maintained. ________________________________________ As narrow orifices which are vulnerable to blockage are key parts of this type of homogeniser. The model depicted is the 'CD Valve' from APV Gaulin. being equipped with devices which retain the beads within the milling chamber. A cross-section through the Manton-Gaulin homogeniser valve. The release of proteins can be described by Equation 2. of course. Use of bead mills for cell lysis When cell suspensions are agitated in the presence of small steel or glass beads (usually 0. disruption being more rapid at higher temperatures. High pressure homogenisers are acceptably good for the disruption of unicellular organisms provided the enzymes needed are not heat labile.9 but normally a similar relationship is used where the time variable is replaced by the number of passes (N) through the homogeniser. (2. Unbound intracellular enzymes may be released by a single pass whereas membrane bound enzymes require several passes for reasonable yields to be obtained. it is unsuitable for the disruption of mycelial organisms but has been used extensively for the disruption of unicellular organisms. The shear forces produced are not capable of damaging enzymes free in solution.1. showing the flow of material.2. For the same volume of beads. The shape of the exit nozzle from the valve seat varies between models and appears to be a critical determinant of the homogenisation efficiency. Bead mills are available in various sizes and configurations from the Mickle shaker which has a maximum volume of about 40 ml to continuous process equipment capable of handling up to 200 Kg wet yeast or 20 Kg wet bacteria each hour. e. Any type of biomass.

Unfortunately. There will be an optimum impeller tip speed at which the increases in disruption are balanced by increases in backmixing. They are related with the study of macromolecular structure rather than the collection of particle fractions. the protein release rate constant (k) is a function of temperature. bead loading. to release membrane-bound enzymes from bacteria. the chamber is surrounded by the refrigerated. Centrifugation B. In general. subcellular organelles.6 Method of separation of enzyme from lysed cell. shape. particularly on a large (e.e. plasma membranes.000 g. Impeller speeds can be increased with advantage until bead breakage becomes significant but heat generation will also increase. The technique can be divided into two types: 1 preparative centrifugation: for separation of the whole cells. However. Smaller vessels may be cooled adequately through cooling jackets around the bead chamber but larger mills require cooling through the agitator shaft and impellers.9 at low (near zero) values of i.9 with respect to the time (t) that a particle spends in the mill. Heat production is the major problem in the use of bead mills for enzyme release. sealed and evacuated to minimized excess rise in temperature. The kinetics of protein release from bead mills follows the relationship given by Equation 2. For safety purpose ultracentrifuge are always enclosed in heavy armour plating. It may be counteracted by designing the bead mill to encourage plug flow characteristics. 2. ULTRACENTRIFUGATION This technique is used to investigate the different parameter of the molecule such as molecular mass. At a constant impeller speed. nucleic acids. ribosome. when continuously operated there will be a significant amount of backmixing which reduces the efficiency of the protein released with respect to the average residence time (τ) . This is more noticeable at low flow rates (high average residence times) and when the proportion of protein released is high.11 reduces to give the simplified relationship of Equation 2. however well designed these mills are. This is type of preparative . Analytical centrifugation: it is devoted to study of pure or virtually pure or macromolecular or particles. for a longer period. the efficiency of the equipment declines with throughput as the degree of backmixing increases.11) where i represents the degree of backmixing (i.25 mm diameter beads must be used. lipoprotein and viruses 2. increased bead loading increases the rate of protein release but also increases the production of heat and the power consumption. the basis of centrifugation separation techniques therefore is to exert the lager force than does the gravitational earth. Under these circumstances the relationship can be shown to be (2. 20 liters) scale. Therefore. if cooling is effective there is little damage to the enzymes released. In this process gravity plays most important role. Equation 2.range being selected for most effective release of the enzyme required. Note that there must be equal loading of sample. and density. Thus 1 mm diameter beads are satisfactory for the rapid release of periplasmic enzymes from yeast but 0. Principles: Rate depends on the centrifugal field G directed radially outward (angular velocity) G = ωr 2 ω =2π/60 revolution min-1 ultracentrifuge have maximum speed of 600. chromatin. i = 0 under ideal plug flow conditions and i = 1 for ideal complete backmixing). impeller rotational speed and cell loading. polysomes.g. thus increasing the rate at which the particle settles. In addition to bead size.

05 for 1%. sub cellular organelles. Optical system records the change in refractive index. So .5. Light of suitable wavelength is passed through moving analytical cell containing the solution under analysis e. A. and density of the particle. 3%. separation of similar particle by the rate zonal technique is based mainly upon differences in their sizes and can not be separated easily like mitochondria. peroxisomes. After the centrifugation. 12% and 20% solids volume fraction respectively. protein or nucleic acid and intensity of light is measured on the photographic plate. pellet and supernatant are separated. Differential centrifugation This method is based upon the difference in the sedimentation rate of particles of different size and density. 0. shape. Only material which reaches a surface during the flow through continuous centrifuges will be removed from the centrifuge . the resulting supernatant then being centrifuged at higher speed to separate medium sized particle and so on. The rate zonal technique and the isopycnic (equal density) Isopycnic centrifugation depends solely upon the buoyant density of the particles and not its shape or size and is independent of time the size of the particle affecting only the rate at which it reaches its isopycnic position in the gradient. Sedimentation of material in a centrifugal field may be described by (2.000 g). η is the kinematic viscosity. approximately equaling 1. Another type of ultracentrifuge is analytical type. d is the particle diameter. In differentiated centrifugation. --1. rs is the particle density. Fs is a correction factor for particle interaction during hindered settling and θ is a shape factor (=1 for spherical particles). the denser particle is sedimented first and less dense will sediment later. The particle having the same mass but different density. Particle having similar density can be separated by the differential centrifugation or the rate zonal method.11) where v is the rate of sedimentation. It can also measure the refractive index. which makes it useful in analysis in the locating in boundaries in sedimentation velocity measurements. better for the deproteinisation of physiological fluids for amino acid analysis. ribosomal subunits. lysosome. The technique is useful in separating the particle of same size but differing in density. so pellet will not be homogeneous but will contain a mixture of all the sediment components.000 rev per min (500. pellet is washed several time and again centrifugation is done. Particle separation by the rate zonal technique is based on differences in the size. Most of the biological particles having same size are having narrower density range. the particle to be separated is divided centrifugally into a number of fractions by increasing the applied centrifugal field. In centrifugation the larger particle are sedimented first. and is equipped with the ultraviolet light absorption system. since particles of varying sizes and densities were distributed homogeneously at the start of centrifugation. Fs depends on the volume fraction of the solids present. rl is the solution density is the angular velocity in radians s1. However. Centrifugation is continued long enough to pellet all the largest class of particles. The technique has been used for the separation of enzymes hormones and RNA and DNA hybrids. 0. its fabrication is of same type.1 and 0. It has speed 70. There are two type of density gradient centrifugation. The particle moves against respective sedimentation rates. r is the radius of rotation.g. 1Svedberg = 10-13 second Centrifugation separates on the basis of the particle size and density difference between the liquid and solid phases.ultracentrifuge. The Schleiren optical system plots the refractive index gradient against distance along the analytical cell.

where g is the gravitational constant. sometimes near-impossible.1 Kg of wet deposit whereas large models. is minimised. with consequent enzymic inactivation. The capacities of these centrifuges are only moderate. centrifuges are long and thin enabling rapid acceleration and deceleration. continuous centrifuges of various types are employed (Figure 2.000 g. due to the large effective surface area. however. semicontinuous or continuous removal of solids.000 g) so they are suitable only for the collection of comparatively large particles. primarily because the mechanical stress on the centrifuge head increases with the square of the radius. it is essential that .12) The efficiency of the process is seen to depend on the solids volume fraction. 921 cm s-2. The maximum throughput of a centrifuge for efficient use is given by (2. the effective clarifying surface (V/D) and the acceleration factor (ω2r/g. These allow the continuous addition of feedstock. They may be operated either semi-continuously or. Although many types of centrifuge are available. Centrifugation is the generally preferred method for the collection of enzyme-containing solids as it does not present a great hazard to most enzymes so long as foam production. is restricted giving a maximum acceleration factor of about 2.2.g. These can only be used at low acceleration (about 3. of course. Coagulation is caused by the removal of electrostatic charges (e. by using a centripetal pressurising pump within the centrifuge bowl which forces the sludge out through a valve. the efficiency of centrifugation can be improved if the particle diameter (d) is increased.2). Clearly from Equation 2. Flocculation and coagulation are cheap and effective aids to precipitating or otherwise harvesting whole cells. Flocculation is achieved by adding small amounts of high-molecular-weight charged materials which bridge oppositely-charged particles to produce a loose aggregate which may be readily removed by centrifugation or filtration. The product of these factors (ω2rV/gD) is called the sigma factor Σ and is used to compare centrifuges and to assist scale-up. continuously. the efficiency depending on the residence time within the centrifuge and the distance necessary for sedimentation (D). contain multiple coned discs in a stack which are spun and on which the precipitate collects. the efficient precipitation of small particles of cell debris can be difficult. This residence time will equal the volumetric throughput (Ф) divide by the volume of the centrifuge (V).feedstock. Where discontinuous or semicontinuous removal of precipitate occurs. This can be done either by coagulating or flocculating particles.000 g. This type of design cannot be scaled-up safely. which must increase with increasing capacity. designed to accumulate up to 5 Kg of deposit. minimizing the down-time required for the removal of the sedimented solids. cell debris or soluble proteins but. A different design which is rather similar in principle is the solid bowl scroll centrifuge in which an Archimedes' screw collects the precipitate so that fluid and solids leave at opposite ends of the apparatus. Low acceleration factors of about 1 500 g may be used for harvesting cells whereas much higher acceleration factors are needed to collect enzyme efficiently. originally designed (by Westfalia and Alpha-Laval) for cream separation. accumulating 0. The capacity and radius of such devices are large and the thickness of liquid is very small.000 g. the continuous removal of supernatant and the discontinuous. are restricted to 16. small models can be used at acceleration factors up to 50. Here the radius and effective liquid thickness are both small allowing a high angular velocity and hence high centrifugal force. the precipitate is flushed out by automatic discharge systems which cause its dilution with water or medium and may be a problem if the precipitate is required for further treatment. by pH change) and allowing particles to adhere to each other. For large-scale use.Multichamber discstack centrifuges. Laboratory centrifuges using tubes in swing-out or angle head rotors have high angular velocity ω and radius of rotation (r) but small capacity (V) and substantial sedimentation distance (D). The angular velocity. a rotor of radius 25 cm spinning at 1 rev s-1 has an acceleration factor of approximately 1 G).

These filters are generally rather messy and difficult to contain making them generally unsuitable for use in the production of toxic or recombinant DNA products.4.1 µm diameter (cf. Problems associated with the build-up of the filter cake may also be avoided by high tangential flow of the feedstock across the surface of the filter. Its efficiency is limited by the shape and compressibility of the particles. . A comparatively recent introduction designed for the removal of cell debris is a moderately hydrophobic product in which cellulose is lightly derivatised with diethylaminoethyl functional groups. There have been recent developments that improve their suitability. such as celite. such as the Disposable Rotary Drum Filter.g.13) Where. Bacillus diameter of about 2µ m). The volumetric throughput of a filter is proportional to the pressure (P) and filter area (AF) and inversely proportional to the filter cake thickness (DF) and the dynamic viscosity (2. Cell debris is collected on a cloth with. ________________________________________ Figure 2.3). a process known as crossflow microfiltration (Figure 2. k is a proportionality constant dependent on the size and nature of the particles. is mixed with the feedstock to improve the mechanical stability of the filter cake. The basic design of the rotary vacuum filter. For very small particles k depends on the fourth power of their diameter.the agents used must not inhibit the target enzymes. Principles of (a) dead-end filtration and (b) cross-flow filtration. however.4). plant material) can be removed satisfactorily. However. ________________________________________ Figure 2. or without. safety decrees that the angular velocity must be low and so only large particles (e. filter aid and can be skimmed off when necessary using a suitable blade. This material (Whatman CDR. Large-scale simple filtration employs filter cloths and filter aids in a plate and frame press configuration. Most flocculants have very definite optimum concentrations above which further addition may be counter-effective. in rotary vacuum filters or centrifugal filters (Figure 2.3. ________________________________________ A simple and familiar filtration apparatus is the perforate bowl centrifuge or basket centrifuge.7 Filtration of enzyme after separation Filtration separates simply on the basis of particle size.3 to describe the system.1 . binds to unwanted negatively charged cell constituents. Filter aids are generally used only where the liquid phase is required as they cause substantial problems in the recovery of solids. It is often difficult for a process development manager to decide whether to attempt to recover enzyme trapped in this way. Some flocculants can be rapidly ruined by shear. Filtration of particles that are easily compressed leads to filter blockage and the failure of Equation 2. the viscosity of the liquid phase and the maximum allowable pressures. allowing high volumes. Such centrifugal filters have a large radius and effective liquid depth. This produces a filter cake which is removed with a blade. in effect a spin drier. The suspension is sucked through a filter cloth on a rotating drum. cell debris remover) is inexpensive (essential as it is not reusable). Under these circumstances a filter aid. It is important to note that the choice of flocculants is determined by the pH and ionic strength of the solution and the nature of the particles. This method dispenses with filter aids and uses special symmetric microporous membrane assemblies capable of retaining particles down to 0. The filter cake may be rinsed during its rotation. acts as a filter aid and may be incinerated to dispose of hazardous 2. They also may cause loss of enzyme activity from the solution due to physical hold-up in the filter cake.

In dead-end filtration the flow causes the build-up of the filter cake, which may prevent efficient operation. This is avoided in cross-flow filtration where the flow sweeps the membrane surface clean. ________________________________________ Chapter-10 Separation in Aqueous biphasic systems The 'incompatibility' of certain polymers in aqueous solution was first noted by Beijerinck in 1296. In this case two phases were formed when agar was mixed with soluble starch or gelatin. Since then, many two phase aqueous systems have been found; the most thoroughly investigated being the aqueous dextran-polyethylene glycol system (e.g. 10% polyethylene glycol 4000/2% dextran T500), where dextran forms the more hydrophilic, denser, lower phase and polyethylene glycol the more hydrophobic, less dense, upper phase. Aqueous three phase systems are also known. Phases form when limiting concentrations of the polymers are exceeded. Both phases contain mainly water and are enriched in one of the polymers. The limiting concentrations depend on the type and molecular weight of the polymers and on the pH, ionic strength and temperature of the solution. Some polymers form the upper hydrophobic phase in the presence of fairly concentrated solutions of phosphates or sulphate (e.g. 10% polyethylene glycol 4000/12.5% potassium phosphate buffer). A drawback to the useful dextran/polyethylene glycol system is the high cost of the purified Dextran used. This has been alleviated by the use of crude unfractionated dextran preparations, much cheaper hydroxypropyl starch derivatives and salt-containing biphasic systems. Aqueous biphasic systems are of considerable value to biotechnology. They provide the opportunity for the rapid separation of biological materials with little probability of denaturation. The interfacial tension between the phases is very low (i.e. about 400-fold less than that between water and an immiscible organic solvent), allowing small droplet size, large interfacial areas, efficient mixing under very gentle stirring and rapid partition. The polymers have a stabilizing influence on most proteins. A great variety of separations have been achieved, by far the most important being the separation of enzymes from broken crude cell material. Separation may be achieved in a few minutes, minimizing the harmful action of endogenous proteases. The systems have also been used successfully for the separation of different types of cell membranes and organelles, the purification of enzymes and for extractive bioconversions. Continuous liquid twophase separation is easier than continuous solid/liquid separation using equipment familiar from immiscible solvent systems, for example disc-stack centrifuges and counter-current separators. Such systems are readily amenable to scale-up and may be employed in continuous enzyme extraction processes involving some recycling of the phases. Cells, cell debris proteins and other material distribute themselves between the two phases in a manner described by the partition coefficient (P) defined as. --------------------(2.14) Where Ct and Cb represent the concentrations in the top and bottom phases respectively. The yield and efficiency of the separation is determined by the relative amounts of material in the two phases and therefore depends on the volume ratio (Vt/Vb). The partition coefficient is exponentially related to the surface area (and hence molecular weight) and surface charge of the particles in addition to the difference in the electrical potential and hydrophobicity of the phases. It is not generally very sensitive to temperature changes. This means that proteins and larger particles are normally partitioned into one phase whereas smaller molecules are distributed more evenly between phases. A partition coefficient of greater than 3 is required if usable yields are to be achieved by a single extraction process. Typical partition coefficients for proteins are 0.01-100 whereas the partition coefficients for cells and cell debris are effectively zero. The influence of pH and salts on protein partition is complex, particularly when phosphate buffers are present. A given protein distributes differently between the phases at different pH's and ionic strength but the presence of phosphate ions

affect the partition coefficient in an anomalous fashion because these ions distribute themselves unequally resulting in electrostatic potential (and pH) differences. This means that systems may be 'tuned' to enrich an enzyme in one phase, ideally the upper phase with cell debris and unwanted enzymes in the lower phase. An enzyme may be extracted from the upper (polyethylene glycol) phase by the addition of salts or further polymer, generating a new biphasic system. This stage may be used to further purify the enzyme. A powerful modification of this technique is to combine phase partitioning and affinity partitioning. Affinity ligand (e.g. triazine dyes) may be coupled to either polymer in an aqueous biphasic system and thus greatly increase the specificity of the extraction. Phases form when limiting concentrations of the polymers are exceeded. Both phases contain mainly water (typically 70-90% w/w water) and are enriched in one of the polymers. The limiting concentrations depend on the type and molecular weight of the polymers and on the pH, ionic strength and temperature of the solution. Some polymers form a two-phase system by themselves; PEG forming the upper morehydrophobic phase in the presence of fairly concentrated solutions of citrates, phosphates or sulfates or at higher temperatures (see below). Such aqueous liquidliquid two-phase systems are finding increasing use in the extractive separation of labile biomolecules such as proteins, offering mild conditions due to the low interfacial tension between the phases (i.e. about 400-fold less than that between water and an immiscible organic solvent) allowing small droplet size, large interfacial areas, efficient mixing under very gentle stirring and rapid partition. The polymers also have a stabilizing influence on most proteins. A great variety of separations have been achieved, by far the most important being the separation of enzymes from broken crude cell material. Separation may be achieved in a few minutes, minimizing the harmful action of endogenous proteases. The systems have also been used successfully for the separation of different types of cell membranes, organelles and actinide ions, the purification of enzymes, extractive bioconversions. Although sometimes perceived as due to polymer incompatibilities, the properties of these biphasic systems can be mainly attributed to incompatibility between aqueous pools of low and higher density water. Each phase may be considered as a different, although aqueous, solvent with properties determined by its structuring. PEG usually has a far higher concentration in the upper (low-density) phase of such solutions in spite of its inherent density being greater than water. These, together with the properties of this PEG phase encourage the belief that it creates a predominantly low-density water environment due to its partially hydrophobic character, in turn mainly determined by the methylene groups. Further proof of this may be seen by use of microwave dielectric measurements, which show the water surrounding PEG to be ordered, whereas that surrounding more hydrophilic polymers is disordered [332]. Also, the dissolution of PEG is exothermic (and increasingly exothermic with PEG size), in line with a shift in the ES CS equilibrium towards the more ordered ES structure. It is interesting and perhaps not simply fortuitous that the diameter (4.9 Å) of the favored PEG helix (formed by trans, gauche, trans links across the C-O-C-C, O-C-C-O, C-C-O-C bonds) is the same as the diameter of the spines of the ES water cluster (4.7 Å) formed by pentagonal boxes, the ether (O-C-C-O) distances (2.22 Å) are close to the O•••O distances (2.24 Å) in water and the next ether (O-C-C-O-C-C-O) (5.6 Å) distances are close to the next vertex distance on opposite sides of the pentagonal boxes (5.4 Å).a Model building shows that optimum hydrogen bonding would tend to distort this PEG helix, however. The strongly-held hydration, as determined by viscosity, increases from two molecules of water per PEG monomer at very low polymerization (tetramer) to 5 molecules of water per PEG monomer for 45-mer , showing that the extent of water clustering increases with PEG size. The partitioning of proteins into the hydrophobic PEG phase shows great sensitivity to the protein's surface hydrophobicity (partition increasing with surface hydrophobicity) and also depends on the PEG size; increasing with PEG molecular mass , in line with the extent of

water clustering. Increasing PEG size and concentration both increase the proteins' effective hydration as the PEG is excluded from the proteins' surface. However, when the PEG phase becomes too ordered (e.g. at higher PEG size) partitioned proteins are excluded due to the reduced available water content. An interesting and revealing phenomenon occurs in PEG solutions as the temperature is raised; the solution at low temperatures separates into two phases (PEG-rich and PEG-poor) at higher temperature (separating at the cloud-point) and reverts to a single phase at even higher temperatures. This may be explained as the PEG creating a low-density water environment with decreased entropy. At low temperatures a solution is formed due to the enthalpy of hydrogen bonding between the PEG and the water more than compensating for the entropy lost in forming the low-density water. This entropy loss is required, due to the hydrophobicity of the methylene groups, but is not great as the water is somewhat ordered already at lower temperatures. At the cloud point, the entropy cost is greater as the water is no longer naturally as structured, and two phases develop. The stronger hydrogen bonding in D2O, relative to H2O, is expected to raise this cloudpoint. At higher temperatures still, the water possesses excess energy and cannot be structured by the PEG. This reduces the entropic cost, so allowing a solution to form once more. Anions have a distinct effect on the cloud point in line with the Hofmeister Series (cloud point lowering: SCN- < I- < Br- < Cl- < F- < OH- < SO42- < HPO42- < CO32- < PO43-); the greater lowering of the cloud point is in line with greater surface charge density , stronger hydration, greater tendency to avoid low-density water and the greater destruction of the natural structuring of the water. A oppositely-ordered compensating effect on the cloud point has been recognized due to binding of the anions to the polymer surface. This tends to raise the cloudpoint at lower salt concentrations as the bound salt increases the polymer net charge and, hence, solubility. The relative effect of the ions is the reverse of the Hofmeister series just given with weakly hydrated ions binding best, i.e. SCN- having the greatest effect and ionic kosmotropes below Cl- having negligible effect. Cations have a lesser but opposite effect to anions with chaotropes (e.g. NH4+) tending to lower the cloud point but kosmotropes (e.g. Li+) raising it. Exceptionally, however, some di- and trivalent cations such as Mg2+ and Zn2+ act counter to their normal Hofmeister behavior, due presumably to their specific chelation to oxygen atoms in the PEG molecules. Anions and cations distribute themselves differently between the phases depending on their affinity for low or higher density water but with the requirements that the phases be electrically neutral and iso-osmotic, so producing an interfacial potential difference, which may aid the partitioning of charged biomolecules. Thus sulfate and phosphate ions prefer the bottom phase and, as a consequence, negatively charged proteins are partitioned into the upper PEG phase, so allowing more sulfate or phosphate ions to partition into their preferred lower phase. Preference for the PEG-rich or PEG-poor phase is related to the Hofmeister Series for the structuring ability of the salts, particularly the anions (e.g. preference for PEG-rich phase: I- > Br- > Cl- > F- > SO42-; Cs+ > Na+ > Ba2+ > Ca2+; preference for PEG-poor phase: SO42- > F- > Cl- > Br- > I-). A similar Hofmeister Series effect is noticed intensifying the incompatibility between two polymers such as polyethylenimine-PEG, or dextran-PEG, by increasing the concentration of strongly hydrated (CS-forming) anions, such as sulfate. a Note that the polymers formed with either one (-O-C-O-C-O-) or three (-O-C-C-CO-C-C-C-O-) methylene groups between the oxygen atoms are both insoluble in water. The reason however is not so much that the O•••O distances (2.12 Å and 4.79 Å respectively) fit less well with the water cluster spacing but rather that the molecules form almost-linear extended (rather than helical) chains with a pronounced hydrophobic character that have strong intra-molecular attraction. 2.9 Preparation of enzymes from clarified solution; Ultrafiltration In many cases, especially when extracellular enzymes are being prepared for sale,

the clarified solution is simply concentrated, preservative materials added, and sold as a solution or as a dried preparation. The concentration process chosen will be the cheapest which is compatible with the retention of enzyme activity. For some enzymes rotary evaporation can be considered, followed if necessary by spray drying. The most popular method, though, is ultrafiltration, whereby water and low molecular weight materials are removed by passage through a membrane under pressure, enzyme being retained. Ultrafiltration differs from conventional filtration and microfiltration with respect to the size of particles being retained (< 50 nm diameter). It uses asymmetric microporous membranes with a relatively dense but thin skin, containing pores, supported by a coarse strong substructure. Membranes possessing molecular weight cut-offs from 1000 to 100,000 and usable at pressure up to 2 MPa are available. There are number types of apparatus available. Stirred cells represent the simplest configuration of ultrafiltration cell. The membrane rests on a rigid support at the base of a cylindrical vessel which is equipped with a magnetic stirrer to combat concentration polarization. It is not suitable for large scale use but is useful for preliminary studies and for the concentration of laboratory column eluates. Various large-scale units are available in which membranes are formed into wide diameter tubes (1 - 2 cm diameter) and the tubes grouped into cartridges. These are not as compact as capillary systems (area/volume about 25 m1) and are very expensive but are less liable to blockage by stray large particles in the feedstream. Cheaper thin-channel systems are available (area/volume about 500 m-1) which use flat membrane sandwiches in filter press arrangements of various designs chosen to produce laminar flow across the membrane and minimise concentration polarization. Capillary membranes represent a relatively cheap and increasingly popular type of ultrafiltration system which uses micro-tubular membranes 0.2 - 1.1 mm diameter and provides large membrane areas within a small unit volume (area/volume about 1000 m-1). Membranes are usually mounted into modules for convenient manipulation. This configuration of membranes can be scaled up with ease. Commercial models are available that give ultrafiltration rates of up to 600 L hr-1. The steady improvement in the performance, durability and reliability of membranes has been a boon to enzyme technologists, encouraging wide use of the various ultrafiltration configurations. Problems with membrane blockage and fouling can usually be overcome by treatment of membranes with detergents, proteases or, with care, acids or alkalis. The initial cost of membranes remains considerable but modern membranes are durable and cost-effective. Ultrafiltration, done efficiently, results in little loss of enzyme activity. However, some configurations of apparatus, particularly in which solutions are recycled, can produce sufficient shear to damage some enzymes.

Chapter-10 Concentration and purification of enzyme Concentration by precipitation Precipitation of enzymes is a useful method of concentration and is ideal as an initial step in their purification. It can be used on a large scale and is less affected by the presence of interfering materials than any of the chromatographic methods described later. There are method of salting in and salting out. Salting in is the method in which ammonium sulphate is used for dissolving the protein. Note that protein dissolves least at its isoelectric point. On increasing the ammonium sulphate concentration further proteins starts precipitating this phenomenon is called as salting out. All this process must be done in the ice cold solution so that no protein can denature. Salting out of proteins is done by use of ammonium sulphate, is one of the best known and used methods of purifying and concentrating enzymes, particularly at the laboratory scale. Increases in the ionic strength of the solution cause a reduction in the repulsive effect of like charges between identical molecules of a protein. It also reduces the forces holding the salvations shell around the protein molecules. When these forces are sufficiently reduced, the protein will precipitate; hydrophobic proteins precipitating at lower salt concentrations than hydrophilic proteins. Ammonium sulphate is convenient and effective because of its high solubility, cheapness, lack of toxicity to most enzymes and its stabilizing effect on some enzymes (see Table 2.4). Its large-scale use, however, is limited as it is corrosive except with stainless steel, it forms dense solutions presenting problems to the collection of the precipitate by centrifugation, and it may release gaseous ammonia, particularly at alkaline pH. The practice of using ammonium sulphate precipitation is more straightforward than the theory. Reproducible results can only be obtained provided the protein concentration, temperature and pH are kept constant. The concentration of the salt needed to precipitate an enzyme will vary with the concentration of the enzyme. However, fractionation of protein mixtures by the stepwise increase in the ionic strength can be a very effective way of partly purifying enzymes. The solubility of an enzyme can be described by the equation (2.11) where S is the enzyme solubility, Kintercept is the intercept constant, Ksalt is the salting out constant and T is the ionic strength which is proportional to the concentration of a precipitating salt. Kintercept is independent of the salt used but depends on the pH, temperature, enzyme and the other components in the solution. Ksalt depends on both the enzyme required and the salt used but is largely independent of other factors. This equation (2.11) may also be used to give the minimum salt concentration necessary before enzyme will start to

precipitate; the concentration change necessary to precipitate the enzyme varying according to the magnitude of the salting out constant. Some enzymes do not survive ammonium sulphate precipitation. Other salts may be substituted but the more favored alternative is to use organic solvents such as methanol, ethanol, propan-2-ol and acetone. These act by reducing the dielectric of the medium and consequently reducing the solubility of proteins by favoring protein-protein rather than protein-solvent interactions. Organic solvents are not widely used on a large scale because of their cost, their flammability, and the tendency of proteins to undergo rapid denaturation by these solvents if the temperature is allowed to raise much above 0°C. On safety grounds when organic solvents are used, special flameproof laboratory areas are used and temperatures maintained below their flashpoints. Except when enzymes are presented for sale as ammonium sulphate precipitates, the precipitating salt or solvent must be removed. This may be done by dialysis, Ultrafiltration or by using a desalting column of, for instance, Sephadex G-25. A. Nucleic acid removal Intracellular enzyme preparations contain nucleic acids which can give rise to increased viscosity interfering with enzyme purification procedures, in particular ultrafiltration. Some organisms contain sufficient nuclease activity to eliminate this problem but, otherwise, the nucleic acids must be removed by precipitation or degraded by the addition of exogenous nucleases. Ammonium sulphate precipitation can be effective in removing nucleic acids but will remove some protein at the same time. Various more specific precipitants have been used, usually positivelycharged materials which form complexes with the negatively-charged phosphate residues of the nucleic acids. These include, in order of roughly decreasing effectiveness, polyethyleneimine, the cationic detergent cetyltrimethyl ammonium bromide, streptomycin sulphate and protamine sulphate. All of these are expensive and possibly toxic, particularly streptomycin sulphate. Also, they may complex undesirably with certain enzymes. They may be necessary, however, where possible contamination of the enzyme product must be avoided, such as in the preparation of restriction endonucleases. Otherwise, treatment with bovine pancreatic nucleases is probably the most cost-effective method of nucleic acid removal. B. Heat treatment In many cases, unwanted enzyme activities may be removed by heat treatment. Different enzymes have differing susceptibility to heat denaturation and precipitation. Where the enzyme required is relatively heat-stable this allows its easy and rapid purification in terms of enzymic activity. For such enzymes heattreatment is always considered as an option at an early stage in their purification. This method has been particularly successfully applied to the production of glucose isomerase, where a short incubation at a relatively high temperature is used (e.g. 60 - 25°C for 10 min). No interfering activity remains after this treatment and the heat-treated, and hence leaky, cells may be immobilised and used directly. 1. Enzyme purification by Chromatography For identification and separation of biochemical compound chromatography is one of the most effective techniques. The method was developed in 1906 by the Russian botanist Mikhail Tswett. Chromatography involves the principle of partition or distribution coefficient ( Kd ). Kd describes that how a substance distributes itself between different type of immiscible phases. Enzyme preparations that have been clarified and concentrated are now in a suitable state for further purification by chromatography. For enzyme purification there are three principal types of chromatography utilizing the ion-exchange, affinity and gel exclusion properties of the enzyme, usually in that order. Ionexchange and affinity chromatographic methods can both rapidly handle large quantities of crude enzyme but ion-exchange materials are generally cheaper and, therefore, preferred at an earlier stage in the purification where the scale of

operation is somewhat greater. Gel exclusion chromatography (also sometimes called 'gel filtration' or just 'gel chromatography' although it does not separate by a filtering mechanism, larger molecules passing more rapidly through the matrix than smaller molecules) is relatively slow and has the least capacity and resolution. It is generally left until last as an important final purification step and also as a method of changing the solution buffer before concentration, finishing and sale. Where sufficient information has been gathered regarding the size and variation of charge with pH of the required enzyme and its major contaminants, a rational purification scheme can be devised. A relatively quick analytical method for obtaining such data utilises a two-dimensional electrophoresis whereby electrophoresis occurs in one direction and a range of pH is produced in the other; movement in the electric field determined by the size and sign of a protein's charge, which both depend on the pH. As the sample is applied across the range of pH, this method produces titration curves (i.e. charge versus pH) for all proteins present. A large effort has been applied to the development of chromatographic matrices suitable for the separation of proteins. The main problem that has had to be overcome is that of ensuring the matrix has sufficiently large surface area available to molecules as large as proteins (i.e. they are macroporous) whilst remaining rigid and incompressible under rapid elution conditions. In addition, matrices must generally be hydrophilic and inert. Although the standard bead diameters of most of these matrices are non-uniform and fairly large (50 – 150 µm), many are now supplied as uniform-sized small beads (e.g. 4 - 6µm diameter) which allows their use in very efficient separation processes (high performance liquid chromatography, HPLC), but at exponentially increasing cost with decreasing bead size. Relatively high pressures are needed to operate such columns necessitating specialized equipment and considerable additional expense. They are used only for the small-scale production of expensive enzymes, where a high degree of purity is required (e.g. restriction endonucleases and therapeutic enzymes). Column manufacturers now supply equipment for monitoring and controlling chromatography systems so that it is possible to have automated apparatus which loads the sample, collects fractions and regenerates the column. Such equipment must, of course, have fail-safe devices to protect both column and product. a. Ion-exchange chromatography Enzymes possess a net charge in solution, dependent upon the pH and their structure and isoelectric point enzyme can be separated. Note that In solutions of pH below their isoelectric point amino acid will be positively charged and bind to cation exchangers whereas in solutions of pH above their isoelectric point amino acid will be negatively charged and can bind to anion exchangers. Therefore pH chosen must be sufficient to maintain a high, but opposite, charge on both protein and ion-exchanger and the ionic strength must be sufficient to maintain the solubility of the protein without the salt being able to successfully compete with the protein for ion-exchange sites. The binding is predominantly reversible and its strength is determined by the pH and ionic strength of the solution and the structures of the enzyme and ion-exchanger. Normally the pH is kept constant and enzymes are eluted by increasing the solution ionic strength. A very wide range of ion-exchange resins, cellulose derivatives and large-pore gels are available for chromatographic use. Ion-exchange materials are generally water insoluble polymers containing cationic or anionic groups. Cation exchange matrices have anionic functional groups such as -SO3-, -OPO3- and -COO- and anion exchange matrices usually contain the cationic tertiary and quaternary ammonium groups, with general formulae -NHR2+ and -NR3+. Proteins become bound by exchange with the associated counter-ions. Ion-exchange polystyrene resins are eminently suitable for large-scale chromatographic use but have low capacities for proteins due to their small pore size. Binding is often strong, due to the resin hydrophobicity, and the conditions needed to elute proteins are generally severe and may be denaturing. Nevertheless such resins are a potential means of concentrating or purifying enzymes.

Ion-exchange cellulose and large pore gels are much more generally suitable for enzyme purification and, indeed, many were designed for that task. A variety of charged groups, anionic or cationic, may be introduced. The practical level of substitution of cellulose is limited as derivatisation above one mole per kilogram may lead to dissolution of the cellulose. Consequently, proteins may be eluted from them under mild conditions. Ion-exchange cellulose can be used in both batch and column processes but on a large scale they are used mainly batch wise. This is because the increased speed of large-scale batch wise processing and the avoidance of the deep-bed filtering characteristics of columns outweigh any advantage due to the increase in resolution on columns. Careful preparation before use and regeneration after use is essential for their effective use. Batch wise operations involve stirring the pretreated and equilibrated ionexchanger with the enzyme solution in a suitable cooled vessel. Adsorption to the exchangers is usually rapid (e.g. less than 30 minutes) but some proteins can take far longer to adsorb completely. Stirring is essential but care must be taken not to generate fine particles (fines). Unadsorbed material may be removed in a variety of manners. Basket centrifuges are a particularly convenient means of hastening the removal of the initial supernatant and the elution of the adsorbed material. This is usually done using stepwise increases in ionic strength and/or changes in pH but it is possible to place the exchangers, plus adsorbed material, in a column and elute using a suitable gradient. However, whilst ion-exchange cellulose are widely used for column chromatography on the laboratory scale, their compressibility causes difficulty when attempts are made to use large scale columns. Some of the problems with derivatised cellulose may be overcome using more recently introduced materials. Derivatives of cross-linked agarose (Sepharose CL6B) and of the synthetic polymer Trisacryl have high capacities (up to 150 mg protein ml-1) yet are not significantly compressible. In addition, they do not change volume with pH and ionic strength which allows them to be regenerated without removal from the chromatographic column. More detail description is given in chapter 9. 2 Affinity chromatography This is a term which is based on specific interaction between the enzyme and the immobilised ligand. In its most specific form, the immobilised ligand is a substrate or competitive inhibitor of the enzyme. Ideally it should be possible to purify an enzyme from a complex mixture in a single step and, indeed, purification factors of up to several thousand-fold has been achieved. An alternative, equally specific approach is to use an antibody (they are specific in binding because they are raised against specific antigen inside an animal) to the enzyme as the ligand. Such specific matrices, though, are very expensive and cannot be generally employed on a large scale. Additionally, they often do not perform as well as might be expected due to non-specific binding effects. In general, affinity chromatography achieves a higher purification factor (with a median value in reported purifications of about ten fold) than ion-exchange chromatography (with a median performance of about three fold), in spite of it generally being used at a later stage in the purification when there is less purification possible. A less specific approach, suitable for many enzymes, is to use analogues of coenzymes, such as NAD+, as the ligand. This method has been used successfully but has now been superceded by the employment of a series of water soluble dye as ligand. These are much cheaper and, usually by trial and error, have been found to have surprising degrees of specificity for a wide range of enzymes. This dyeaffinity chromatography was allegedly discovered by accident, certain enzymes being found to bind to the blue-dyed dextran used, as a molecular weight standard, to calibrate gel exclusion columns. More detail description is given in chapter 9. .3 Hydrophobic interaction chromatography (HIC) or affinity elution HIC is found to be very useful when it was noted that certain proteins were unexpectedly retained on affinity columns containing hydrophobic spacer arms.

Table 2. some are much more effective at their destabilization by binding to them and disrupting the localized structure of water (the chaotropic effect. Samples are applied to the matrix in a concentrated (over 50% saturated. 20% NaCl). A recent introduction is cellulose derivatised to introduce even more hydroxyl groups. More detail account is given in chapter 9.11 Maintaining Enzyme Activity The key to maintaining enzyme activity is maintenance of conformation. Effect of ions on enzyme stabilization.g. Table 2. because enzyme will stop its movement only after a situation where pH of enzyme will be equal to the pI of the buffer. In addition ions can offer some protection against oxidation to groups such as thiols by salting-out the dissolved oxygen from solution. increased chaotropic effect . From this it can be seen why ammonium sulphate and potassium hydrogen phosphate are a powerful enzyme stabilizers whereas sodium thiosulphate and calcium chloride destabilize enzymes. osmotic effect. This may result in the interactions between an enzyme's hydrophobic areas being strengthened causing the enzyme molecules to compress and making them more resistant to thermal unfolding reactions. Affinity chromatography is not used extensively in the large-scale manufacture of enzymes.Hydrophobic adsorbents now available include octyl or phenyl groups. Not all salts are equally effective in stabilizing hydrophobic interactions. Particles should retain good flow and porosity properties after attachment of the ligand and should not be capable of the non-specific adsorption of proteins. enzyme A 1 forming separate bands in compare to the Enzyme A2). enzyme manufacturers will make increased use of these very powerful techniques. Elution is achieved by changing the pH or ionic strength or by modifying the dielectric constant of the eluent using. the controlled use of covalent modification. This introduces more water which competes with protein for the hydrogen bonding sites. for instance. CHOICE OF MATRIX Careful choice of matrices for affinity chromatography is necessary. Proteins are eluted by diluting the ammonium sulphate. In general. for example Ca2+ stabilises αamylases and Co2+ stabilises glucose isomerases. Isoelectric focusing When any enzyme mixture is placed in the gel having solution of different pH then after passing the current a potential difference is established which helps in development of a pH gradient. and 3. so preventing unfolding. Three approaches are possible: 1. aggregation and changes in the covalent structure. This pH gradient decides the final movement of the enzyme in the gel.4. > 2M) solution of ammonium sulphate. 2. primarily because of cost. ethanediol. (See figure below. At high concentrations (e.4). Electrophoresis This method was developed by Arne Tiselius in 1937 and is based on principle that any charged molecule will move towards the opposite pole. Many enzymes are specifically stabilized by low concentrations of cations which may or may not form part of the active site. Hydrophobic interactions are strong at high solution ionic strength so samples need not be desalted before application to the adsorbent. Doubtless as the relative costs of materials are lowered. 2. This material (Whatman HB1) is designed to interact with proteins by hydrogen bonding. and experience in handling these materials is gained. Agarose beads fulfill these criteria and are readily available as ligand supports . The selectivity of both of these methods is similar to that of fractional precipitation using ammonium sulphate but their resolution may be somewhat improved by their use in chromatographic columns rather than batch wise. proteins are stabilized by increasing their concentration and the ionic strength of their environment. Enzyme immobilization. Neutral salts compete with proteins for water and bind to charged groups or dipoles. salt discourages microbial growth due to its. use of additives.

g. It should be noted that enzymes stabilised by making them more rigid usually show lower activity (i. HPO42-. thiols to create a reducing environment. NH4+. I-. sorbitol and mannitol) are also useful for stabilizing enzymes. Chapter-12 Techniques used in Enzyme characterization 2. Cl-. glycerol. more commonly.g. A useful example of this is the derivatisation of lysine side chains in proteases with N-carboxyamino acid anhydrides. by repressing microbial growth. Additives of these types must. They may also act by stabilizing the hydrophobic effect within the enzymes. be compatible with the final use of the enzyme's product. . This observation. lactose. and by the formation of protective shells which prevent unfolding processes. In the meantime it remains possible to convert lysine residues to arginine-like groups by reaction with activated urea. carboxymethylcellulose and other poly-electrolytes which protect the enzyme during a cheaper spray-drying stage. These form polyaminoacylated enzymes with various degrees of substitution and length of amide-linked side chains. Vmax) than the 'natural' enzyme.12 HOW TO KNOW THE PROPERTY OF ENZYME 1. This derivatisation is sufficient to disguise the proteinaceous nature of the protease and prevent autolysis. among others. polyvinyl alcohol. resulting in more rigid structures. has given hope that site-specific mutagenesis may lead to enzymes with significantly improved stability . may not) result in stabilization.Cations Al3+. Calculate the molecular weight by following method Ultracentrifugation. Consequently. Solid enzyme preparations sometimes consist of freeze-dried protein. More usually they are bulked out with inert materials such as starch. Br-. Ca2+. Li+. Many specific chemical modifications of amino acid side chains are possible which may (or. This may be possibly explained by noting that arginine is bidentate and has a higher pKa than lysine or histidine (see Table 2. Na+. polyvinylpyrrolidone and hydroxypropylcelluloses) stabilise enzymes by a process of compartmentalisation whereby the enzyme-enzyme and enzyme-water interactions are somewhat replaced by less potentially denaturing enzyme-polymer interactions. Glycerol may be used to protect enzymes against denaturation due to ice-crystal formation at sub-zero temperatures. Other materials which are added to enzymes before sale may consist of substrates. Mg2+. inhibitors of contaminating enzyme activities and chelating agents. benzoic acid esters as preservatives for liquid enzyme preparations. (CH3)4N+ Anions SCN-. of course. K+. due to the reduction in the water activity. Some hydrophilic polymers (e. Important lessons about the molecular basis of thermostability have been learned by comparison of enzymes from mesophilic and thermophilic organisms. antibiotics. ClO4-. A frequently found difference is the increase in the proportion of arginine residues at the expense of lysine and histidine residues. citrate3increased stabilization ________________________________________ Low molecular weight polyols (e. SO42-. Gel filtration.e. it forms stronger salt links with bidentate aspartate and glutamate side chains. Enzymes are more stable in the dry state than in solution.1).

Presence of amide or peptide bond can be calculated by UV-IR spectroscopy. Many specialized technique that use them are: --Basically. liquid. all type of chromatography consists of two type of phase Stationary phase: the phase that can not move and is prepared by dissolving some solid . The basis of all type of chromatography is the partition or distribution coefficient (Kd). Analyte are separated due to their varying degree of adsorption onto the solid surfaces. which can have very different physisorption characteristics due to steric effects in the molecules. This may be solid. liquid or other material that can be packed inside the column and may provide the support for movement the mobile phase. Two immiscible phases are formed after the distribution of compound in the matrix and is denoted by Kd= concentration of compound in phase A / concentration of compound in phase B. 2. The main advantage of adsorption chromatography is in separating isomers. and the cation exchanger is the CMC carboxymethyl cellulose. adsorption chromatography are of following type . c) Affinity chromatography. Basically. Example Thin layer chromatography. Mobile phase is poured in the gel and get separated due to attachment with the stationary phase. Introduction After salting in and salting out procedure. or solid liquid mixture. Stationary phase in paper chromatography is the paper. Direct structure can be determined by the X-ray crystallography. Chromatography involves the principle of partition or distribution coefficient (Kd). Dowex-1 both are anion exchanger. gel.SDS –page. Kd describes that how a substance distributes itself between different type of immiscible phases. Mass spectrometry. Amino acid can be determined by Ninhydrin reaction and taking OD at 570 nm. In this chapter we will focus mainly on choice of these techniques and their broad details of the application and principles.Chromatography. there is several type of chromatographic technique based on following property Adsorption. d) High performance liquid chromatography (HPLC) Based on adsorption of solute in the matrix. Mobile phase is used to isolate the biomolecules because most of the biomolecules are present in the solution. Mobile phase: This is chosen according to the nature of the biomolecules. enzymes are further purified by two main common techniques 1. DEAE and sephadex. 1. Amino acid sequencing can be done to know the order of amino acid. Chromatography Principles of chromatography For identification and separation of biochemical compound chromatography is one of the most effective techniques. while in ion-exchange chromatography is the ion-exchanger like Dowex -50 . 2-Ion-exchange. The method was developed in 1906 by the Russian botanist Mikhail Tswett. 2. Column chromatography is of following type a) GPC or gel permeation chromatography b) Ion –Exchange chromatography. 3-Molecular-sieve Adsorption Chromatography The stationary phase in adsorption chromatography is silica or alumina particles. A.Gel electrophoresis.

If the analyte is large then kd =0. a material called Sephadex. Smaller molecules will be distributed between the mobile phases inside and outside the molecular sieve. charge. Size exclusion chromatography/ gel filtration (permeation) chromatography History Since the discovery in the 1940s that certain porous material can retain molecules of certain sizes and let others elute. Here separation of molecule is done on the basis of their molecular size and shape with the help of gel that have pores formed during the gel formation. Hence they appear last in the effluent. the stationary phase chemically interacts through adsorption. Principle Size Exclusion Chromatography differs from conventional chromatography in the behavior of the stationary phase. or various other actions.a. which has led to the development of other divinylbenzene polymers for size exclusion chromatography. although the surface is often modified by adding organic compounds to resist adsorption. developed for zone electrophoresis. there have been many discoveries that have made size exclusion chromatography practical. the General principle is simple. There are various materials that are porous in nature. denotes the distribution of an analyte (that we have to separate) in a column of a gel is determined solely by the total mobile phase. was applied to chromatography. The most commonly used material is organic gel and they form different type of pores on polymerizations. This is the reason why molecules are eluted from the column in order of decreasing size or if the shape is relatively constant. Today. both gel filtration and gel permeation chromatography are generally referred to as size exclusion chromatography. In size exclusion chromatography. b. The amount of cross-linking (controlled by the availability of divinylbenzene) determines pore size. The larger size molecule are separated out or excluded from the pores (see figure). Silica has also been used. When using nonpolar organic solvents. This initial use of size exclusion was limited to aqueous solvents and called gel filtration chromatography (at Tiselius’s suggestion). it led to further interest. So Kd =1. . Note that in the column there is always an inert substance. Size exclusion chromatography was discovered using starch as a packing material. Paper chromatography. whereas if the analyte is sufficiently small to gain complete access to the inner mobile phase. Soon. Although starch was not a very durable material. the processes are still used extensively in polymer chemistry but have gained new uses in biochemistry. Therefore. using molecular sieve. Sephadex is a cross-linked styrene divinylbenzene copolymer. The process developed using Sephadex was used extensively in characterizing polymers and polymer molecular weight distributions. Term gel filtration or exclusion or permeation chromatography is used to describe the separation of molecule of different molecular size utilizing gel material. Since gel have pores after polymerization and it can be used for the separation of the molecule of fixed size. Thin layer chromatography. According to availability of the mobile phase kd varies between 0 to 1. For given type of gel Kd depends on the molecular size of the analyte. COLUMN CHROMATOGRAPHY In the column chromatography column is filled with the stationary phase attached to suitable matrix and mobile phase passed through the column either with the gravity or by applied pressure. but still they can pass though the spaces around the pore called as void space and appear in the effluent first. researchers prefer modified divinylbenzene and polyacrylamide as packing materials. They will pass through the column at slower rate. Kd. the stationary phase simply acts as a sieve to filter molecules based on size. this innovation made size exclusion chromatography realistic. The gel is filled in the column that is equilibrium with the mobile phase. In most forms of chromatography. allowing packing materials to be easily customized for different applications. both inside and outside the gel particle.

N’. It is marketed in the name of Bio-Gel –P. Void volume. agarose. and mean radii. They can not be used for molecules that have size more than 600. and are uncharged. The pore size is usually much larger than sephadex. In contrast. Beads can be prepared with defined degrees of porosity. The pore size of the bead will determine whether a protein of s Moreover. The coarser beads are unable to hold the fluid. Note. It forms a gel held together with crosslink of H-bonds. so poorer resolution results. however.the fractionation range. since proteins have roughly constant density (mass per volume).Agarose is a linear polymer of Dgalactose. we will make the assumption that many proteins have a roughly spherical shape (often called globular proteins. as opposed to fiber proteins). do not bind or react with the material that is being analyzed.methylene bis acrylamide. Access to included volume (volume that hydrates the inside of the bed) depends on the size of the sphere (the hydrodynamic radius) that each protein occupies.to high resolution chromatographic (polishing) technique. and polyacrylamide. etc. In general. These excluded molecules elute in the "void volume" (Vo) of the column and are not fractionated. gel permeation chromatography is found to separate globular proteins according to mass. the size of this sphere is directly related to the mass of the globular protein. The radii of the beads are more important for determining the capacity of the column to separate molecules of similar size. For chromatography beads of a given porosity. molecules below a certain size for beads of a given porosity will completely penetrate the pore structure and will elute with the . The gels currently in use are of three types: ---dextran. the alkyl dextran is called as N. the porosity of the beads determines the size range of molecules that can be effectively separated. that large deviations from spherical shape can cause a protein to migrate anomalously during gel permeation chromatography that is with regard to migration versus mass.Polyacrylamide gels are produced by cross-linking acrylamide and N. They are used in the aqueous solution. Mechanism of separation by size in gel permeation chromatography For our discussion of the theory of gel permeation chromatography. The space within the gel is filled with liquid occupies most of the gel volume.For two substances of different relative molecular mass and kd value for example Kd’ and kd’’ is the difference in their elution volumes. The gel is a three dimensional network whose structure is usually random. Mixed gel of polyacrylamide and agarose is known as Ultra-Gel. molecules above a certain size will be completely excluded from the pore structure of the beads. superfine beads are used. N’methylene-bis acrylamide. DNA. This is the reason why they can be used for the separation of the large macromolecular substance like protein. They made strong beads.Kd’’) V1------------------------------------1 The resolution depends on the beads. Thus. The product is called as Sephacryl S-300. average radii. Gel permeation chromatography is normally considered to be a medium. Vs. can be derived from the equation and shown to be: VS = (K’d.Dextran is a polysaccharide composed of glucose residues. It is commercially available in the trade name “Sephadex”. For maximum resolution. The gels acts as molecular sieves consist of cross linked polymers that are generally inert. 000.

acid. Table:1 MATERIAL: POLYMER 1. AGAROSE 10-4000 SEPHAROSE 60---20. such as with salt.7 SEPHADEX 1."included volume" (Vi). gel permeation chromatography provides an estimate of mass for the oligomeric holoprotein.0-5 1. Gel permeation chromatography is a common and often excellent polishing step during any protein purification. affinity.DEXTRAN < 0. the molecular mass of an unknown protein can be estimated. These can be characterized as either bulk or polishing steps. both of which are typical of the starting material to be used in a protein purification. Common bulk purification methods are centrifugation. as described above). filtration.. high abundance of the target protein in the starting material). Common polishing purification methods are ion exchange. Polishing purification steps are generally useful for samples of higher quality and smaller volume. typical of partially purified fractions obtained from bulk purification steps. or polyethylene glycol. and gel permeation chromatography. By comparing the elution volumes of protein standards of known molecular mass.g. such as a cellular extract. and the elution volumes (Ve) of the proteins of interest.5—3.0 4--150 5---600 SEPHYCRYL 5---250 10---1500 20—8000 2. Only molecules with masses (hydrodynamic radii) that allow them to penetrate the pore structure of the beads only partially fall within the fractionation range and are separated according to their molecular size (approximately. however. The combination of denaturing gel electrophoresis and gel permeation chromatography is thus exceedingly powerful for elucidating basic features of protein structures. it would not be used as a first step in purification except in unusual circumstances (e.The use of a gel permeation column as an analytical tool requires the determination of Vo. Bulk purification steps are generally useful for samples of low purity and large volume. In contrast to denaturing gel electrophoresis.000 4B 2 B G S 200 200 S 300 G 50 G 100 TRADE NAME FRACTIONATION G 10 G 25 RANGE S 400 . and ion-exchange chromatography. Thus. precipitation. solvent. Vi. which gives subunit molecular masses for an oligomeric protein. these molecules are not fractionated either. Use of gel permeation chromatography as part of a purification scheme Most protein purification schemes involve several steps.

see below) is applied onto the top of the column packing (beads) and allowed to flow into them.0--100. the protein sample migrates through the column. With such a curve. it is collected in fractions until all the components of the original sample have passed through the column. As buffer is eluted from the column.8 1. a protein sample (often a partially purified fraction from a previous purification step.1---1. As additional buffer is applied to the top of the column and allowed to flow through the column.6B 70—40.000 3. not M. POLYACRYLAMIDE 0. Note that the independent variable is plotted on the y axis by convention. For . you are to select a representative sampling of points. the protein molecules are separated. It is customary to plot log(M). Vo. Note that all the large M’s come out at nearly the same volume.000 A 5m 10---5000 15.0—6 BIO-GEL P2 P6 BIO-GEL A 50 A 15m 40--- P100 5. So they all elute together at the void volume. Due to the differential partitioning of molecules into the pore structure of the beads (as described above).000 100---50. Vo. This is because none of the very large polymers ever enter a pore.0 Overview of a typical gel permeation chromatography experiment In a typical gel permeation experiment.

In addition. Application of GPC or gel permeation chromatography Size exclusion chromatography is used primarily in two areas of chemistry: polymer chemistry and biochemistry. is . that can be processed only at temperatures above 135 °C (1999). The elution volume is related in a simple manner to molecular weight. O’Donohue and E.3-hexafluoro-2-propanol may not be a good solvent for size exclusion chromatography as polar portions of macromolecules (such as polyesters and polyamides) may be solvated and extended. it is not necessary to actually know dn/dc in simple GPC. because size exclusion chromatography does not destroy or alter the sample. J.3. or a protein of unknown size. Anke van Rijk and colleagues used size exclusion chromatography to isolate a small protein called alpha-A-crystalline to study the insertion of hamster alpha-Acrystalline into mice (1999). size exclusion chromatography allows the researchers to isolate the biomacromolecule based on its relative size without destroying the sample. Starting at VeA read up until and then read left to get MA from the left y-axis. S. Ve trend Obtain DRIA similarly from wish! The DRI response is DRI c ( in g/mL) indicated by the cross.3. pH. In biochemistry. the right ordinate.1. but in both polymer chemistry and biochemistry. Antonio Moroni and Trevor Havard discuss the use of solvents in the analysis of engineering thermoplastics. This extension or enlargement causes them to be eluded at later times than expected.e. For example.. In polymer chemistry. Their experimentation leads to the suggestion that 1. One can also obtain the number average molecular weight: Mn = = = = ADVANTAGE OF GEL –CHROMATOGRAPHY In this chromatography the material used is inert one so there is no effect of temp. making these molecules seem larger than they actually are. Meehan discuss the use of different solvents in size exclusion chromatography and molecular weight detectors (light scattering and viscometry) at high temperatures to characterize polymers. However. You cannot measure concentrations in an isorefractive solvent (i. They can be used for very labile material like enzyme.example. in a genetics study.1. consider point A. such as o-chloronaphthalene. size exclusion chromatography can separate polymers with different numbers of monomer units. One can obtain average molecular weights without it. the constant of proportionality factors out of the numerator and denominator identically. If a polymer chain of unknown size. ionic strength. particularly polyesters and polyamides (1999). Repeat for as many points as you proportional to the concentration of polymer: The constant of proportionality is dn/dc. the fractions of polymer can be further tested. giving the polymer’s producer information about the length of the chains produced. size exclusion chromatography is useful in separating highmolecular-weight molecules from other molecules. For example. you hit the M vs. Since there is no adsorption by the inert material so labile material are not affected. the same specific refractive index increment needed in light scattering. Biochemical separations may seem to make this more of a separation technique. size and molecular weight estimations can be made. For example: Mw = = Since. one in which dn/dc = 0).

This Rf value is fixed and is generally less than one. The equilibrium constant for this reaction is: [-SO3. ketone. the mobile phase--determined by their partition coefficient--measured as an Rf value. it is usually a mixture of Solutes partition between H2O in the stationary phase and the solvent of the mobile phase. using the conditions that make them more stable and tight binding. The mobile phase solvent will be less polar than H2O. First –the substances to be separated are bound to the exchanger. proteins. (s) indicates the solid or stationary phase. Ion –exchange chromatography This type of chromatography is used for many biological materials like amino cid and proteins.e. Vs= volume of stationary phase or = Vt---Vo. where Ve= elution volume. aldehyde etc. For cation separation the cation-exchange resin is usually a sulfonic or carboxylic acid. The principle of ion exchange chromatography is that charged molecule adsorb to ion exchangers reversibly so that molecule can be bound or eluted by changing the ionic environment. The bound H2O is the Stationary Phase. For cation-exchange with a sulfonic acid group the reaction is: -SO3. In study of protein –binding study Estimation of the molecular weight: Gel 1 gel 2 Log M THE plot is between K verses log M (molecular weight). they have either negative or positive charge. drawn by capillary action Solutes move as spots with a rate depending upon how much time they spend in the stationary phase vs.Mx+(s) + H+(aq) where Mx+ is a cation of charge x. hormones. Ion exchange separation is carried out mainly in column packed with an ion –exchangers. then when the compound in question eludes. adjust the composition). antibodies. Paper Chromatography is the most common form of cellulose chromatography in which solute is "spotted" on "dry" paper (still contains H2O) encircle by the pencil at a line mark and chromatograph is "developed by dipping one end in the mobile phase.Mx+]s [H+]aq Keq = ------------------------------------------ . it can be compared with the molecules that it eluded closest to determine an approximate molecular weight. which have ionisable groups. In purification of Macromolecule: GPC can be used to separate the viruses. It yields a straight line except for very small and very large molecule.analyzed along with molecules of known size or molecular weight. The parameter K = Ve---Vo / Vs. enzyme. Vt is the total volume of the column PAPER CHROMATOGRAPHY Cellulose has many hydroxyl groups which are polar and bind H2O. nucleic acid and polysaccharide. and for anion separation the anion-exchange resin is usually a quaternary ammonium group. and (aq) indicates the aqueous or mobile phase. H2O run across the cellulose paper due to capillary action. There are two modes of chromatography1: Ascending and 2-Descending. One can change the characteristics of separation by changing the polarity of the mobile phase (i. Separation by ion exchange is usually involving two step. Maximum distance covered by the solvent is noted and the distance covered by the solute is noted.) and possibly water.e. i. Vo= void volume. Mobile Phase is mixture of organic solvents (alcohols.H+(s) + Mx+(aq) -SO3.The solvent moves through the paper. The column packing for ion chromatography consist of ion-exchange resins bonded to inert polymeric particles (typically 10 µm diameter). The ratio of two gives Rf value. The name ion exchange is given because of exchanging ions for ion in aqueous solution.

cellulose and copolymer of styrene and vinyl benzene. The porosity of the matrix is very important factor because of charged group are present both outside and inside of the matrix. peptides etc. If an H+ is bound to group. Because the cross links in these materials maintain the insolubility of the matrix even cross link in these materials maintain the insolubility of the matrix even if the matrix is highly polar. strong cation exchangers: Sulfonic Acid (or derivatives) RSO3. Strong Ion Exchangers: based upon strong acids or bases and are charged over a wide range of pH a. amino acids. Finer the mesh the slower will be the flow rate. Ion exchanger come in variety of particle size called as mesh size. The available capacity is the capacity under particular conditions like pH. ionic strength.[-SO3. tertiary amines b. . agarose.weak cation exchangers: carboxy methyl (CM).cation or anion exchanger -. It can exchange one H+ for one Na+ or two H+ for one Ca++. If stable below isoelectric point then a cation exchanger can be used. Since pore also act like molecular sieve. For materials that have either single charge the choice of exchanger is not difficult. The sulphonic acid groups are called as a strongly acidic cation exchanger. Strong Exchanger is used for more stable molecules such as nucleotides. CM-Sephadex. The extent to which an ion exchanger is charged depends upon the pH. Cationic exchanger posses negative charge. Finer mesh means an increased surface to volume ratio and therefore increased capacity and decrease time for exchange to occur for given volume of exchanger. a weak exchanger can be used. the exchanger is said to be in the acid form. DEAE-cellulose. This will also depend upon pH. weak anion exchangers: Diethyl-amino-ethyl (DEAE).strong anion exchangers: quaternary ammonium salts R-N (CH3)+ Weak Ion Exchangers: based upon weak acids or bases which are charged only over a limited pH range a. DEAE-Sepharose. These may be bonded to a variety of supports: e.The matrix is used of various materials like dextran.depends upon the charge of the molecules to be separated. The more highly charged molecule to be exchanged.H+]s [Mx+]aq Different cations have different values of Keq and are therefore retained on the column for different lengths of time. The time at which a given cation elutes from the column can be controlled by adjusting the pH ([H+]aq). Weak Exchanger is useful for labile molecules such as proteins.g. phosphoryl. As we know above the isoelectric point if the pH is stable one then an ion exchanger can be used. If the substance is labile. It is measured in term of milliequivalent of exchangeable group permiligram of dry weight. DEAE-Sephadex. Other weakly acidic cation exchangers are phenolic hydroxyl group and carboxylic group. A typical group used in ion exchange is the sulphonic group. There are two type of ion exchanger 1) cationic exchanger 2) Anion exchanger. and a programmable pump that can change the pH of the mobile phase during the separation. SO3 -. CMCellulose. But in case of material that have both negative and positive charge the choice depends upon the charge that is stable. CM-Sepharose Choosing an Ion Exchanger Charge -. The sephadex and bio-gel exchanger offers a particular advantage for macromolecules that are unstable in low ionic strength. Most ion-chromatography instruments use two mobile phase reservoirs containing buffers of different pH.b. The total capacity of ion exchanger is measure of its ability to take up exchangeable ion. while anionic exchanger posses positive charge. The negative charge ion exchangers bind to positive charge molecule. Large molecules may be unable to penetrate the pore so the capacity will decrease with increase in molecular weight. the tighter it binds to the exchanger and less readily it is displaced by other ion.

it is important to consider how eluting fluid will affect the assay for the material. or glass beads .larger diameter column is packed with an inert support -commonly diatomaceous earth. r is the distance between the groups. polyethylene glycol) but the column is run at higher temperature where the coating melts. For example to separate the sugar mixed with the polar and nonpolar solvent. This hydrocarbon chains is bound to an inert matrix to provide hydrophobicity. (Most common method): F = q1 q2 / D r2 q1and q2 are the charges on two groups.g. For example if spectral analysis is to be done then eluting fluid should not absorb in the required wavelength. and D is the dielectric constant of the solvent which is increased with higher ionic strength thus weakening the force between the solute and the ion exchanger. For selection of buffer following rule is always adopted. Reversed Phase Chromatography In reverse phase chromatography stationary Phase is apolar (hydrophobic) and is reversed with respect to cellulose chromatography.molecules usually adsorb tightly in the column so. Gas / Liquid Chromatography In gas chromatography mobile Phase is usually a gas -. Simply because. bound material can be eluted by changing pH. the binding strength. Commonly use a .) which give mass spectrum that can be used to identify and quantitate samples as them come off the column.usually inert (He. and anionic buffer is used with the cationic exchanger. the solution is applied to the column. Note that sample must be somewhat volatile and stable at higher temperature. Ion exchanger has been found to be an effective way to separate weakly polar substances by using the exchanger as the matrix for partition chromatography. A related resin called a mixed bed resin is used to prepare deionized water. For best ionic resolution ionic condition should be same as the eluting condition. teflon powder.changes the charges on the molecules being separated. This is called as reverse phase chromatography. Other phase is mobile Phase that depends upon hydrophobicity of stationary phase. Detectors present are of various types. The cellulose ion exchanger proved to be best for the macromolecule like protein and the nucleic acid. must be weakened. Hydrophobicity can be varied by changing the hydrocarbon chain length or by aromatic groups. GC Can provide very high resolution by making very long columns. Gel chromatography is an effective means of removing ion from solutions of macromolecules. It is because of larger available capacity.These changes can be made stepwise by changing the buffer reservoir (step gradient) or as gradient -. Ar. N2) and Stationary Phase is a liquid coating on an inert solid support. thus altering the charge of the material. this interaction.by mixing two buffers The basic principle of ion exchange chromatography is that the affinity of substance for the exchanger depends on both the electrical properties of the material and the relative affinity of other charged substances in the solvent. Small mesh sizes improve the resolution but decrease flow rate. Hence. also can change the charge of a weak ion exchanger . Most powerful detector is a mass spectrometer (GC/Mass Spec. One important use of on exchange is in desalting. which increases the zone spreading and decrease resolution. The polar solvent binds to the matrix forming the polar stationary phase and sugar partition between this phase and the mobile weakly phase. There is open tube or capillary operation and other a coat the inside surface of a long thin tube (30 . depends on the ionic factor. In deciding the eluting condition. The cationic buffer is used with the anionic exchanger.100 m long) Packed column -.Macromolecular separation always needs larger pore size while micro molecule can be separated by the small pore. Coating may be solid at room temp (e. Eluting Ion Exchange Columns -. Another way to look at this is that other ions in the buffer compete for the ion exchanger binding site.change pH -. Sample usually injected as a liquid which is then heated to vaporize it.

a sample of 0.01 to 0. the flow rate of a column drops as the particle size decreases and this allow sufficient time for significant diffusional spreading to occur. Short. Usually the resolving power of a column increases with the length of the column and the number of theoretical plates per unit. which are placed before an analytical column to trap junk and extend the lifetime of the Stationary Phases. and guard columns.g. such as methanol. as the mobile phase. Reverse-phase partition chromatography uses a relatively nonpolar stationary phase and a polar mobile phase. such as n-hexane. Also it requires the less amount of test material. Partition Chromatography In partition chromatography the stationary phase is bonded to inert particles of 3-10 µm of diameter. This can be done by establishing a pressure difference across the top and bottom of the column to force the liquid through the bed. with a typical column having a diameter of 3—6mm and a length of 10-20 cm operating at 50-200 pounds per square inches [ 5—20 kg/ cm2 ] . Analyte separate as they travel through the column due to the differences in their partitioning between the mobile phase and the stationary phase. and phenyl groups. 3-5 µm. At present time this procedure is applied principally with the ion-exchange and adsorption chromatography of small molecule.1 ml. acetonitrile. Normal-phase partition chromatography uses a polar stationary phase and a nonpolar organic solvent. with the smaller sizes. propanol. The most common functional groups in order of increasing polarity are: cyano: -C2H4CN diol: -C3H6OCH2CHOHCH2OH amino: -C3H6NH2 dimethylamino: -C3H6N(CH3)2 . However.more polar organic solvent like acetonitrile. For e. small carbohydrate and t-RNA. EtOH. The number of plates increases as the available surface area per unit length of the column becomes greater – in other words as the matrix particle become smaller. However. The pressure used affects the resolution of the bands. Use of shorter hydrocarbon chains less densely packed is called Hydrophobic Interaction Chromatography HIGH PRESSURE LIQUID CHROMATOGRAPHY [HPLC] Conventional liquid chromatography uses plastic or glass columns that can range from a few centimeters to several meters. there may be insufficient time for molecules in the mobile phase equilibrate and the bands are also broadened. or mixtures of these solvents. with the longer columns finding use for preparative-scale separations. The stationary phase is a bonded siloxane with a polar functional group. being used in analytical columns. This decreases the resolution. fast analytical columns. ethylene glycol. or chloroform. and the larger particles being used in preparative-scale HPLC. At atmospheric pressure diffusion spreading causes the bands to overlap. if pressure (and hence flow rate is very high. Reverse-phase chromatography is the most common form of liquid chromatography. water. or mixtures of these with H2O. can be used. DMSO. The most common bonded phases are n-octyldecyl (C18) and n-decyl (C8) chains. The most common lengths are 10-100 cm. typically of 10-30 cm in length and 3-5 mm inner diameter. methylene chloride. primarily due to the wide range on analyte that can dissolve in the mobile phase. Diffusional spreading can be reduced if the transit time of the mobile phase in the column made small. HPLC is known for its rapid separation with extraordinary resolution of peaks. peptide. It is because of its small fraction of the void volume. The use of very fine particle and very high pressure to maintain adequate flow rate is called as HPLC or high performance or high pressure liquid chromatography. Highperformance liquid chromatography (HPLC) columns are stainless steel tubes.

. it utilizes the property of biological affinity of the substances to be separated. and it has become essential with the growing demand of the biotech in the 1980s and 1990s. With new materials and new demands. it is capable of giving absolute purification. but suitable support materials were not available until much more recently. in a single process. the ligand.Biotech research has used affinity chromatography because of its ability to separate one desired species from a host of other biological molecules. As the name suggests. Specificity based on three aspects of affinity—the matrix. even from complex mixtures. and the attachment of the ligand to the matrix—is the hallmark of this process and the reason for its success. the technique became extremely useful in the 1960s. As a consequence. The technique has been known for almost a century.Chromatographic Separation principle Commercial name Phase Type of support Adsorption Partisil C8 Corasil Pellumina Partisil Micro Pak Al Bondapak C18 ULTRA pak TSK ODS Octylsilane Silica Alumina Silica Alumina Porous Pellicular Pellicular Microporous Microporous Pellicular Porous Ion exchange Partisil-SAX MicroPak-NH2 Strong base Weak base Porous Porous Exclusion Styragel Bio-glass Nature of stationary Superpose Fractogel TSK Glass Polystyrene-divenyl benzene Agarose Polyvinylchloride Rigid solid Semi-rigid gel Soft-gel Semi-rigid gel Affinity chromatography Principle Affinity chromatography is almost exclusively used for the purification of biological molecules such as proteins and other macromolecules.

During this process. and most can be affixed to the matrix and used to isolate the desired molecule. but it has been extended to nucleotides. M + L ML For the success of the experiment following steps must be kept in mind. 4. Then. The most useful material is the Agarose. If the ligand chosen can bind to more than one molecule in the sample in question. exhibit minimum absorption. It should not absorb any chemical or substance to be purified itself. leaving the desired product in the column. nucleic acids. 2. A ligand must bind tightly. carbonyl. Biological systems have millions of ligand (also known as receptors). immunoglobulin. adding the third aspect of selection for affinity chromatography. During the elution the matrix should not be destroyed. The desired molecule is then removed from the column by using a wash (typically changing the pH) that lowers the dissociation constant and allows recovery of a nearly pure sample.Affinity chromatography operates on the principle that ligand (as stated in the Table). that ligand could be used in the size exclusion chromatography and affinity column. Matrix materials are often polysaccharides. and does not denature after application of extreme pH and ionic strength. hydroxyl. Decontamination is typically performed by rinsing the column with sodium hydroxide or urea. All other compounds in the solution will elude. or all will be for naught. Different matrix materials are stable in different pH ranges. but the only ligand that binds the molecule also trapped two additional. then a technique called negative affinity. attached to a matrix made up of an inert substance. if a ligand were found that attached to the two unwanted molecules. that have many hydroxyl groups that can be activated. are easily activated and can serve as the sites to which the ligand attach. Matrix materials simply hold the active ligand and provide a pore structure to increase the surface area to which the molecules can bind. such as amino. The ligand should be coupled without altering its binding properties. maintain good flow property after coupling. and thio groups. Matrix should have broad range of thermal and chemical stability. in addition to requiring activation. then an ML complex will be formed. such as agarose. allowing the desired molecule to elute while the other two were retained in the column. if you were looking for a molecule from a cell. bind to the desired molecule within a solution to be analyzed . and polyacrylamide. which uses ligand to remove everything but the target molecule from the solution. For example. The technique can be shown by the following equation: if we assume M is the macromolecule and L ligand is attached to the matrix. The technique was originally developed for the purification of enzymes. The matrix. The ligand must bind strongly with the molecule that is to be recovered. 3. since they have broad range of thermal tolerance. must also often stand up to decontamination when purifying pharmaceutical compounds. and membrane receptors and even to whole cells and cell fragments. you could run a normal affinity column and collect these three molecules. different molecules. Substituent groups within the matrix. Choosing the correct ligand is the first hurdle. 1. For example for . the ligand will interact only with the desired molecule and form a permanent bond. the ligand must also remain active toward the target molecule. The choice of ligand depends on the specificity of the substance. Ideally. Ligand attachment requires that the matrix be activated and then react with the ligand to fix them onto the matrix. may be used.

Con A Sepharose They selectively can binds to the N-acetyl glucosamine residue. Therefore they can be used for the lymphocyte separation. METHOD OF LIGAND IMMOBILIZATION Linking or coupling of the ligand to matrix material is called as immobilization. CNBR activated agarose CNBr is negatively charged so they can react strongly with the amino group. Co-enzyme. catecholamine Heparin-Agarose Blood protein. In addition to using small ligand to separate large molecules. It is extremely useful for coupling enzyme. sugar. d) Spacer can be attached by reacting functional group C00H (by using Agarose) and NH2 (group of 6AHA). nucleic acid and most protein to agarose.Can be Used or sulpher containing protein. Coupling of N-nucleophile to CNBr. indicating that the molecule is more tightly bound to the ligand. a) Used mainly for small ligand.This is useful for linkage of sugars and carbohydrate or any material containing OH gp. Carbomyl diimidazole activated agarose. affinity chromatography can be used to determine dissociation constants of ligand and molecules. Loukas and colleagues studied the existence of one type of suspected opiate receptor in the brain (1994). These studies may one day lead to a better understanding of how drugs such as opium affect our brains. it should be treated with the cysteine. This continues to be the primary and extremely important field for affinity chromatography. a reversible inhibitor. D-glucosamine Can be separated by clam NADP+ Can be separated by using 2. 5’ ADP. Early applications were in the separation of biomacromolecules from other biological compounds.6 DIAMINO HEXANE. Applications The applications of affinity chromatography have been numerous. and Papain. Maltose Can be separated by the jack bean. the broader the chromatographic peak. e) Epoxy –activated Agarose. 6-AMINO-HEXENOIC ACID & 1. For example. Cu +2 Can be separated Octyl –agarose Protein Can be separated Thio-propul Sepharose Can be separated by clam Protein. amino gp. b) They can solve the steric problem c) Only a Spacer can be attached between matrix and ligand. Ribosome. Before using. thiol gp. antibody. Urease.an enzyme ligand must be the substrate. This results in isourea linkage that carries potential charge and thus can act as an ion-exchanger. The longer a molecule stays on the column. pea nut can be used for separation of lactose. 1. inhibitors. such as molecules from an entire cell. DNA polymerase. Helix pomatia lectin Used for separation of the pure T-cell Lactose Caster bean. antigen. 3. researchers have immobilized large molecules on the matrix and used them to separate the small molecules that bind to them. In addition to biological separation. androgen receptor Imino-diacetic acid Zn+2. Protein A Can be used for separation of IgG (using Fc region) Poly A Can be separated of m-RNA Boronate polyacrylamide RNA. 2 Affinity chromatography Substance Use Lectin Polysaccharides and glycoprotein present on the RBC membrane. This quantitative information can be used in further studies of the ligand . Table9. or an allosteric activator. f) Thiopropyl-Agarose. 2. They separated the ( -opioid binding protein by using an opioid receptor antagonist that was specific to the ( -opioid binding protein. but they are predominantly in the field of biochemistry. S.

The layer is formed must be thin Mobile phase is passed through the stationary phase under capillary force. Two factor that affect the TLC has. ion-exchange. This is also helpful in the activation of the plate. For e. allowing for easy densitometry studies.5 cm from edge by means of micropipette or microsyringe. The movement of analyte is expressed by the retardation factor. fluorescent dye (this can be absorbed by the UV light. Detection is done by the several methods. H2SO4 and KMN03 for hydrocarbon. A wider range of sorbant can be used and also due to easy detection of spot and separation of chromatogram. HPTLC and HPPLC Thin-layer chromatography (TLC) gets the high-performance (HP) treatment through several optimization steps leading to improved efficiency and automation. . ADVANTAGE OF TLC OVER OTHER CHROMATOGRAPHY TECHNIQUE IT has great resolving power and greater speed of separation. Sample is applied to the plate 2—2. HPTLC shows better optical properties than standard TLC. Equally if not more important. Distribution process is based on the fact of adsorption. The distribution coefficient is represented by the Kd. The plate is dried at 100—1200C. and the analyte is distributed well in the layer. Br2 vapour is used for olefins detection.g. High surface to volume ratio and the weight ratio. partition.25 mm thick slurry is prepared in water is applied to glass with the help of plate spreader. The mobile liquid phase passed through the thin layer plate. If necessary 2D gels electrophoresis can be used in the TLC plate this can be used by placing the plate in the buffer at right angle to first separation.25 mm).02 mm instead of 0. Movement of compound can be observed by the specific Rf values. H2SO4 for organic acid. they have smaller mean grain sizes of the coating particles (down to 7 µm instead of 12–30 µm). Commonly used separating agent is the Ninhydrin for detection of the amino acid. Distance moved by the solute from origin / Distance moved by the solvent from K’ can be calculated by = 1—Rf/ Rf Process of preparation of the thin layer plate A thin layer of about . Thin layer chromatography Principle in this type of chromatography the stationary phase is generally the glass plastic or metal foil plate. and the grain size is more uniform. SbCl2 for steroid and tarpenoid.5 cm it is left for one hour. Rhodamine B is used for the detection of the lipid. For radiolabel led compound autoradiography can be used. CaSO4 is mixed to facilitate adhesion of the adsorbent to the plate.or molecule. Better resolution as well as a 10-fold improvement in detection limits are key to HPTLC’s increasing popularity. exclusion chromatography. For investigating the unsaturated compound I2 vapour can be used. The layers are made smaller (0. The TLC plate is dipped in the developing phase to depth of about 1. Rf= origin.

Traditional CE uses polar solvents. who built tube electrophoresis units as early as 1959. Water soluble but strong ionic compound---ion-exchange chromatography. These cations pull the rest of the fluid with them. It took two more decades before Jorgen son and Lukacs developed the first truly useful CE instrument and showed that it had exceptional resolution (1981). This area is then attracted by the cathode and starts to flow toward the product end of the column.By using a forced-flow mobile phase and a sealing chamber. It describes the migration of a charged particle under the influence of electric field. The substances separated at the low . they will elute last because of their struggle to reach the anode. including routine use in the pharmaceutical industry and clinical analysis. iv. Electrodes are placed in both reservoirs. Compound differ in molecular size—exclusion chromatography. especially to the speed of the run. HPTLC can also be used with centrifugation to force the sample outward in what is known as rotational planar chromatography (RPC). researchers can transform HPTLC into high-pressure planar liquid chromatography (HPPLC). in food analysis. Terabe and colleagues developed a variant of CE that allows determination of nonpolar molecules (1984. However. which ushered in the widespread use of gels in biological studies. But in the early 1980s. Capillary electrophoresis was pioneered by S. vi. 1985). but they will also exit in a fashion similar to liquid partition chromatography. gaining the added benefits that pressure provides. Arne Tiselius rediscovered this technique in 1937 and used it to separate human serum proteins. which interferes with charged species migration. ionisable group. CE instruments consist of a capillary column between two reservoirs of buffer solution (the solution used as the solvent). Applications of these modern forms are extensive. Hjerten. creating a dense +ve area. the separation of molecules is based on their polarity as polar molecules are slowed by entering the micelle. Low polar compound---liquid chromatography. Many important biomolecules can be separated by this technique like: amino acid. In the 1960s. cations migrate toward the column wall. was first discussed by L. development focused on new gel materials that minimized convection.However. The gel is prepared from the material like agarose or polyacrylamide and the separation of compound is done in the buffer. A reversed-phase HPLC packing material is used to retain the analytes at different rates on the liquid stationary phase. Michaelis (1909). CE uses the phenomenon of electroosmotic flow to separate analytes. natural products analysis (including a variety of botanicals and herbals). the technology on which this technique depends. In subsequent years. The column itself becomes negative if the cathode is on the product side and the anode is on the sample side. migration due to an electric field. These adaptations are collectively responsible for what is known as modern TLC. precluding the identification and analysis of polar compounds. Water soluble but weakly ionic---reverse phase liquid chromatography. protein. nucleotide. ii. Micellar electrokinetic capillary chromatography (MEKC) uses surfactants or polymers to form micelles—small spheres with hydrophobic centers and ionic shields around the outside—that travel against the liquid flow toward the anode (the charges could be switched with different surfactants) but are swept with the electroosmotic flow toward the cathode. had yet to mature. Volatile compound ----gas liquid chromatography. such as high-quality capillary silica tubing and extremely sensitive detectors. polyacrylamide gels were developed. iii. Hydrophobic molecules will enter the micelle. Electrophoretic technique Electrophoresis. Selection of chromatographic system i. Ligand having specific binding property—Affinity chromatography. Therefore. v. which relies heavily on instrumental analysis. When the column is negative. vii. peptide. which places a potential across the column. Theory. and in environmental analysis for determining such things as pesticides in drinking water. For compound having different functional group—adsorption chromatography.

And thus protein is analyzed in qualitative way. Generally pore size of the 1% Agar has pore size larger than proteins and nucleic acid. The pore size is decided by the initial concentration of the agarose. Electrophoretic mobility (μ) is defined as ratio of velocity to field strength.N. b. For protein separation SDS –PAGE can be used the usual percentage lies between the 3 to 30 % of acrylamide. protein can be denatured into the simple protein.temperature to save it from heating effect. Agar can be used more for analysis of DNA.anhydro galactose.6. Note that bis acrylamide is essentially two acrylamide linked by the methylene group. It allows the free movement of the protein so that one type of protein either same in the molecular weight or charge can stalk at one position. c. This cross linking attaches new anti conventional properties. Free radical generation can be done by the photo polymerization. Force can be represented by the following equation: F= qv/ velocity q-= charge and V is the potential difference. small concentration of agarose gives larger pore size. The main problems occurs during electrophoresis is the phenomenon of electro endosmosis. Polyacrylamide Gel Electrophoresis After the polymerization of acrylamide it forms a polymer compound called as polyacrylamide. After this process the protein is separated in the gel having smaller pore size. Generally the agarose concentration is used in the 1% to 3% . Oxygen can be used for the removal of the free radicals. And so electrophoresis is called as polyacrylamide gel electrophoresis or PAGE. Agarose has property to dissolve in the hot water making liquid solution while it solidifies in the cooled medium and forms a rigid gel. Low percentage gel can be used for the separation of the DNA. Stalking gel generally uses lower percentage of the gel that provides the larger pore size. The basic repeat unit is the galactose and 3. The function of buffer is to maintain the constant ionization pattern of the molecule. and size exclusion chromatography on it is resolved by the resolving gel. While. On average it has seen that one SDS can binds to the two amino acid. S2O8 + eSO4 2+ SO4 – IF this free radical is represented as R’. This can be avoided by the substituting some other functional group like sulphate. For this purpose vertical slab gel can be used. and the TEMED or N. SDS-PAGE SDS is an anionic detergent (CH3—(CH2)10 –CH2OSO3-Na+). With β mercaptoethanol and SDS. THERE is some necessary component for the polymerizations of the acrylamide like the N. The gel is formed due to inter and intra-H bonding. Problem arises during the electrophoresis is the settling of sample and exact tracking of the . It helps in the better separation of the protein. a. 1% Agar is used for the immuno-elctrophoresis or flat bed electrophoresis. Since protein separation done in the two steps: first it is separated in the stalking gel. N’ bis acrylamide.N’N’’ tetramethylenediamine. Initiation of the polyacrylamide is difficult so some more compound is added like ammonium persulphate. Agarose Gel Electrophoresis Agarose is prepared from natural compound isolated from Gracilaria and Geladium. All these process can be done by the vertical slabs. High concentration of agarose gives usually smaller pore size. Actually addition of TEMED IS necessary as it acts like the catalyst.

Just beyond the isoelectric point the protein is bind to the positive charge of the ionexchange column. which have lower mobility than chloride ion of the loading buffer and the stacking gel Cl. Here riboflavin is used for initiation of the polymerization of the gel. Staining of gel is done for 2-3 hrs. But note that glycinate ion have a lower electrophoretic mobility than protein –SDS complex.7) and according to the different electrophoretic nobilities and the sieving effect of the gel. By plotting a graph of distance moved against log Mr for each of standard proteins. This can allow separation of the protein in the range of 100. The gel is then placed in the destain solution. Generally 15 % polyacrylamide gel is used in the separating gel. Native (buffer) gel In this method SDS is not used for the separation of the protein subunit prior to loading. And destaining requires overnight. and will initially migrate towards the cathode.8. it can be separated easily. 000 7.000 to 10. Here protein is separated according to the native charge of protein at pH of the gel (normally pH 8. g. f. a standard IP of known protein can be determined and with the help of this calibration curve. The negatively charged protein is attracted toward the anode. Protein having pH lower than iso-electric point will be positively charged. The ion-exchanger is fixed in the column and set to a particular temperature and pH. a calibration curve can be constructed. 000. Chromatofocussing Is suitable for the protein separation. For protein of molecular weight more than 150. A protein generally have single band in the protein unless it has two same subunit. Molecular weight of protein Mr can be determined by the comparing its mobility with those of a number of protein used as standard. IEF is highly sensitive technique and used for studying heterogeneity of the protein. As we know that protein is the ampholytes. Iso-electric focusing: IEF gel: It utilizes the horizontal gels and separates the protein according to the isoelectric point of the protein. Gradient gel The protein is separated according to the gradient formed by the different concentration of the gel.and glycinate ion. Chromatofocussing gives a good resolution of quit complex mixture of protein provided that there is close difference in their isoelectric point.> PROTEIN-SDS > GLYCINATE So protein SDS band lies in between the Cl. For determining the IP of the protein. This is done to sharpen the protein band. glycine is added. After the protein is reaches the bottom. Generally 5% at the top.protein position in the gel. .8 and resolving gel has pH about 8. For settling problem sucrose solution is used. the charge on the protein decreases slowly and at one point protein stops where IP is equal to the charge. it gives poor resolution of compound having same isoelectric point. while 15% gel is used at the bottom. The method is generally used for the enzyme –substrate reaction and total protein content. As they proceed further. d. On adding protein at top of column the protein moves at its isoelectric point. The main advantage is that – very similar molecular weight can be resolved. The protein first of all loaded in the stalking gel. e. the gel is removed and placed in stain more generally the Coomassie blue. and for tracking the protein bromo-phenol blue is used. The distance moved by the protein of unknown Mr is then measured and its log Mr and hence Mr can be determined from calibration curve. The difference of pH lies between 3-4 unit from upper and bottom. In this master mix. IP of unknown protein can be determined.5% gel is used. PH of the stacking gel is generally 6. This is based on forming a pH gradient.

In this well. It is difficult to observe in the gel. i.1 μg of protein. In the mean time. Electrophoresis tank is filled with the buffer. means it can detects the 0. Quantitative analysis of the protein can be done by the method called as scanning densitometry. In the first dimension –isoelectric focusing is done in polyacrylamide gel in narrow tubes in the presence of ampholytes. Protein can be further analyzed and protein can be purified.N.TETRAMETHYLENE DIAMINE (TEMED). Also open DNA moves slower in compare to the closed circular DNA. This prevents the protein from being washed out while it is stained. Now a days to reduce so much of the labor. whereas 2% gels can be used for the separation of the sample between 0. Coomassie blue is highly sensitive. N’ –methylene bis acrylamide (bis-acrylamide). Protein band can be cut out of protein and sequence by the gas phase sequencer.N’. It also helps in isolating the extra protein in the expression. Note that – single stranded DNA is always expressed in nt (nucleotide) while double stranded DNA is expressed always in the base pair or kilo base pair. The volume is calculated by measuring the length and breadth of the gel tank.3% will separated double stranded DNA molecules of about 5 and 60 kb size. This acid –methanol mixture acts as a denaturant to precipitate or fix the protein in the gel. DETECTION.2D-PAGE This technique utilizes the technique of both IEF and the SDS-PAGE.N’. Denatured protein is separated in the gel according to the isoelectric point. while separation of protein is called as southern blotting. It is 100 time more sensitive than CBB. ESTIMATION AND RECOVERY OF PROTEIN GELS. Ethidium bromide is introduced in between the DNA double stranded DNA. Staining is done in the 0.8% gels which are suitable for separation for the DNA molecule in the range of 0. It gives pink red bands. Most of the lab uses 0.5 kb ---10 kb. so that well can form in the gel. This requires the presence of smaller amount of the N. Actually the bis –acrylamide is two acrylamide joined by the methylene. Agarose gel of 0. Polyacrylamide gel Polymerization of the acrylamide results in the formation of the polyacrylamide. the comb is introduced in the gel. Ag+ ion is reduced to metallic silver on the protein. The molecular weight of the DNA can be known from the calibration curve prepared from the known standard of the DNA. Silver stain is more sensitive. Preparation of the gel Gel is prepared by dissolving agarose in the water according to the need. Glycoprotein can be detected by the stain called as PAS (periodic acid stiff stain). The gel is boiled and poured in the tank and left for the solidifying. and is initiated by the addition of ammonium persulphate and the base N.1% (w/v) CBB in methanol: water: glacial acetic acid. Generally the technique is used in the translational product of the gene or mRNA. Along with the sample some dye and glycerol is also loaded so that they can’t float in the buffer. AGAROSE GEL ELECTROPHORESIS OF DNA As we know that DNA is larger than proteins and therefore they are unable to enter the polyacrylamide gel. Note: separation of protein is called as western blotting. where the silver is deposited to give black band. DNA material is loaded. It can be used immediately after the electrophoresis. CBB or Coomassie blue R -250 is most commonly used for the detection of the protein in the gel. The smaller fragment of the DNA moves faster in the gel in compare to the larger fragments. So the convenient way is to use agarose to analyze the DNA. In another step the protein is run in presence of SDS. TEMED catalyses the decomposition of the persulphate ion to give a free radical (a molecule with . So they are also called as PAGE.1 and 3 kb. Polyacrylamide polymerization is the free –radical catalysis. 8M urea and non-ionic detergent. computerized 2D is done. Bromophenol blue is generally used and also ethidium bromide. The gel is sealed surrounding the gel tank which is later on opened before placing them in the electrophoresis tank.

etc. special equipment. 1997). Some of these include alginate. reagents and technical skills is required. Alginate is not compatible with phosphate buffer. health and safety for process workers and end product users. and enzymes such as proteases. magnetite. inertness to enzyme(s). organic solvents. [b] Chemical properties chemical property like hydrophilicity. Immobilization requires carriers to bind and to support the enzymes. fibers). In SDS gel. such as carrier-binding. medical or pharmaceutical applications. Beads. pH. available functional groups for modification. residual enzyme activity on storage. kappa-carrageenan. [d] Resistance Immobilized enzyme must be resistance against bacterial or fungal attack. and (c) the properties and limitations of the support system (Bickerstaff. substrate(s) or cofactor(s). polyurethane. [e] Safety Immobilized enzyme must be safe from toxicity of component reagents. Aim of Enzyme Immobilization Enzyme immobilization is aimed to restrict the freedom of movement of an enzyme. shape or form (eg. Carriers must be "safe" if the end product is to be used for food. A variety of carriers have been tested with different enzymes. It is generally believed that the best support for one enzyme may not work as well for another enzyme. available surface area. [f] Economic Availability and cost of carrier materials. Interestingly. (b) requirements of the specific application.unpaired electron). Carriers or support may be selected on the basis of the following considerations: [a] Physical properties Strength. or entrapment (Katchalski-Katzir. It must be noted that all oxygen should be removed prior to use of the gel by degassing it in the vacuum. Chapter -13 Enzyme Immobilization Introduction The word "immobilized enzyme" was coined by Katchalski-Katzir in 1971 (KatchalskiKatzir. For example. but it needs riboflavin which generated the free radical. pore volume. There is sufficient variety to accommodate almost all available industrial enzymes. porosity. Generally. permeability. acrylamide is toxic. the operational conditions. Induction of the polymerization can be done by a method called as photo-polymerization. flow rate. pressure drop are the main requirements of the immobilization. ie. density. 1993).g. temperature. mechanical stability of support material when subject to pressure or water flow. matrix) and decide on the method of immobilization. disruption by chemicals. compressibility of carriers. chemicals. 10% to 20 % acrylamide are used in techniques such as SDS gel electrophoresis. [c] Stability Immobilized enzyme must have stability on storage. one must select the carrier (support. Beside this industrial scale chemical . Compatibility with certain buffers e. 1993). and that each enzyme ought to be evaluated to determine the optimal carrier matrix system and conditions. there are very few detailed studies to compare the efficacy of various immobilization methods or immobilization supports. ability to be regenerated or reused. cross-linking. the decision will depend on (a) the various characteristics of the enzymes. Acrylamide gel can be made with a content between 3 to 30 %. Enzymes may be immobilized on solid carriers by various techniques. When one wants to do enzyme immobilization. sheets.

effective working life. due to steric difficulties. or within. one phase containing the enzyme and the other phase containing the product. The enzyme is imprisoned within its phase allowing its re-use or continuous use but preventing it from contaminating the product. some other material. also known as substrates (not to be confused with the enzymes' reactants). and re-usability must not be very costly. This activity residue remains to contaminate the product and its removal may involve extra purification costs. they retain some activity after the reaction which cannot be economically recovered for re-use and is generally wasted. The productivity of an enzyme. are able to move freely between the two phases. Several hundred enzymes are commercially available that are very costly. covalent binding III. they are not directly used up by the processes in which they are used. its use may be undesirable for cleanup. membrane confinement Figure 6. An important factor determining the use of enzymes in a technological process is their expense. A wide variety of insoluble materials. 3. continuous processing. It also allows continuous processes to be practicable. where any of the reactants are also insoluble. with a considerable saving in enzyme. they should be stabilised against denaturation and utilised in an efficient manner. rather than free (soluble). Immobilized enzymes must not be biodegradability for example in waste treatment polyurethane is not naturally occurring and is not easily degraded. When they are used in a soluble form. however. including the reactants. These are usually inert polymeric or inorganic matrices. therefore. they do lose activity with time. 2. other molecules. adsorption II. As enzymes are catalytic molecules. Their high initial cost. but conditions are usually available where these properties are little changed or even enhanced. minimising downstream processing costs and possible effluent handling problems. although some are much cheaper and many are much more expensive. Immobilised enzyme systems. 2. labour and overhead costs. Reaction: Immobilized enzymes must not show any diffusion limitations on mass transfer of cofactors. simple and economic methods must be used which enable the separation of the enzyme from the reaction product.1): I. However due to denaturation. Methods of immobilizations There are four principal methods available for immobilising enzymes (Figure 3. Immobilisation often affects the stability and activity of the enzyme.1. (a) enzyme non-covalently adsorbed to an . feasibility for scale-up. although this is often the case.preparation. Advantages of Immobilizations 1. and give an improved productivity. substrates or products. may be used to immobilise the enzymes by making them insoluble. The most important benefit derived from immobilisation is the easy separation of the enzyme from the products of the catalysed reaction. Insoluble immobilised enzymes are of little use. is greatly increased as it may be more fully used at higher substrate concentrations for longer periods than the free enzyme. enzymes. 3. The easiest way of achieving this is by separating the enzyme and product during the reaction using a two-phase system. If possible. so immobilised. Immobilisation of enzymes often incurs an additional expense and is only undertaken if there is a sound economic or process advantage in the use of the immobilised. Limitations of Immobilized Enzyme 1. The term 'immobilisation' does not necessarily mean that the enzyme cannot move freely within its particular phase. should only be incidental to their use. This prevents the enzyme contaminating the product. entrapment IV. Therefore. Side reactions. This is known as immobilisation and may be achieved by fixing the enzyme to. particularly if the enzyme is noticeably toxic or antigenic. In order to eliminate this wastage.

The surface density of binding sites together with the volumetric surface area sterically available to the enzyme. The forces involved are electrostatic. etc. shape. ________________________________________ Carrier matrices Carrier matrices for enzyme immobilisation by adsorption and covalent binding must be chosen with care. but there are sufficiently large to allow reasonable binding. Easily reversible. Some physical factors such as flow rate. ideally they should be cheap enough to discard. cheap and immobilization can be done quickly. A substantial saving in costs occurs where the carrier may be regenerated after the useful lifetime of the immobilised enzyme. and does not require chemical activation or modification. The form. . The nature of the support will also have a considerable affect on an enzyme's expressed activity and apparent kinetics. agitation by stirring. which catalytically removes the inactivating hydrogen peroxide produced by most oxidases). one does not see any damage to the enzyme by the carriers. It will increase the enzyme specificity (kcat/Km) whilst reducing product inhibition. The manufacture of high-valued products on a small scale may allow the use of relatively expensive supports and immobilisation techniques whereas these would not be economical in the large-scale production of low added-value materials. No chemical changes to carriers or enzyme. 4.g. Figure 6.e. contaminant binding.g. The actual capacity will be affected by the number of potential coupling sites in the enzyme molecules and the electrostatic charge distribution and surface polarity (i. density. Simple. and discourage microbial growth and non-specific adsorption. such as van der Waals forces. the enzyme is bound to the carrier material via reversible surface interactions. (c) enzyme entrapped within an insoluble particle by a cross-linked polymer.insoluble particle. shift the pH optimum to the desired value for the process. Little or no damage to the enzyme. Clearly most supports possess only some of these features. and possibly hydrophobic forces. 2. temperature. ionic and H-bonding interaction. magnetic iron oxide. Adsorption of enzymes In adsorption. manganese dioxide. (b) enzyme covalently attached to an insoluble particle. The forces are generally weak. or a reductive surface environment (e. Normally. Some disadvantages of adsorption include Desorption or leakage of enzyme from support. a catalytic surface (e. The ideal support is cheap. Desorption may occur on changing environmental conditions (such as pH. The carriers with adsorption properties are selected on the basis of knowledge of their compatibility with the enzyme. 3. enabling transfer of the biocatalyst by means of magnetic fields). for enzymes inactivated by oxidation). (d) enzyme confined within a semipermeable membrane. pore size distribution. Some matrices possess other properties which are useful for particular purposes such as ferromagnetism (e. physically strong and stable. inert. the hydrophobic-hydrophilic balance) on both the enzyme and support. titania.g. ionic strength) or on conformational changes arising from substrate/cofactor binding. but a thorough understanding of the properties of immobilised enzymes does allow suitable engineering of the system to approach these optimal qualities. Adsorption utilizes existing surface interactions between enzyme and carrier.2 showing the carrier that adsorbs the Enzyme Some advantages of adsorption include 1. determine the maximum binding capacity. porosity. operational stability and particle size distribution of the supporting matrix will influence the reactor configuration in which the immobilised biocatalyst may be used. Of particular relevance to their use in industrial processes is their cost relative to the overall process costs.

________________________________________ Figure 6.1 Preparation of immobilised invertase by adsorption (Woodward 1985) Support type % bound at DEAE-Sephadex anion exchanger CM-Sephadex cation exchanger pH 2. Glu. Ion-exchange matrices. The driving force causing this binding is usually due to a combination of hydrophobic effects and the formation of several salt links per enzyme molecule. The bond is normally formed between functional groups on the carrier and the enzyme. Overloading of carrier possible. surface area. clays.cellulose (carboxymethyl). a process which may simply involve washing off the used enzyme with concentrated salt solutions and re-suspending the ion exchanger in a solution of active enzyme. carboxyl (COOH) group from Asp. Care must be taken that the binding forces are not weakened during use by inappropriate changes in pH or ionic strength. Those on the enzymes are usually amino acid residues such as amino (NH2) group from Lys or Arg.1). Although the physical links between the enzyme molecules and the support are often very strong. functional groups on the carriers can be altered to different forms to accommodate different kinds of covalent bonds to be formed with the enzyme. Tests must be run to ensure the formation of covalent bonds will not inactivate the enzyme. This increases the range of immobilization methods that can be used for a given carrier. Through chemical modifications. pH. chemical modification of -OH group can give rise to AEcellulose (aminoethyl). although more expensive than these other supports. Schematic diagram showing the effect of soluble enzyme concentration on the activity of enzyme immobilised by adsorption to a suitable matrix.7 100 75 pH 7. The particular choice of adsorbent depends principally upon minimising leakage of the enzyme during use. CM. ionic strength. under appropriate conditions of pH and ionic strength. they may be reduced by many factors including the introduction of the substrate. porosity. For example. ________________________________________ Table 6. and DEAE-cellulose (diethylaminoethyl). porous carbon. The amount adsorbed depends on the incubation time. followed. cofactor or contaminants to the carrier may result in diffusion limitations and mass transfer problems.2. While many supports exist. and sulfhydryl (SH) group from Cys. after a sufficient incubation period. by washing off loosely bound and unbound enzyme will produce the immobilised enzyme in a directly usable form (Figure 3. Non-specific binding by substrate. glasses and polymeric aromatic resins.5 0 100 pH 4. and the physical characteristics of both the enzyme and the support. Possible steric hindrance by the carrier material. an important factor for enzyme immobilization appears to be hydrophilicity. This may in turn change the kinetic properties of the reactions.collision or abrasion can cause desorption. hydroxyl (OH) group from Ser. This may result in reduced catalytic activity. Examples of suitable adsorbents are ion-exchange matrices (Table 3. which helps to maintain enzyme activity in a hydrophilic .2). Adsorption of enzymes on to insoluble supports is a very simple method of wide applicability and capable of high enzyme loading (about one gram per gram of matrix). may be used economically due to the ease with which they may be regenerated when their bound enzyme has come to the end of its active life.0 100 34 ________________________________________ Covalent coupling This involves the formation of a covalent bond between the enzyme and the carrier material. Binding of protons to the support may change the pH of the microenvironment and change the reaction rate. hydrous metal oxides. Thr. Simply mixing the enzyme with a suitable adsorbent.

Glutaraldehyde is another bifunctional reagent which may be used to cross-link enzymes or link them to supports (Figure 13. Chemically it is an agarose (poly{β1.3b). charged status) and roughly follows the relationship -S.2). It is particularly useful for .11. This reacts with primary amino groups (i. The reactivity of the protein side-chain nucleophiles is determined by their state of protonation (i.3d). if rather expensive.e. Carbodiimides (Figure 13.2 Relative usefulness of enzyme residues for covalent coupling Residue Content Exposure Reactivity Stability of couple Use Aspartate + ++ + + + Arginine + ++ ± Cysteine ± ++ Cystine + ± ± Glutamate + ++ + + + Histidine ± ++ + + + Lysine ++ ++ ++ ++ ++ Methionine ± Serine ++ + ± + ± Threonine ++ ± ± + ± Tryptophan ± Tyrosine + + _+ + C terminus ++ + + + N terminus ++ ++ ++ + Carbohydrate . ________________________________________ Table 6.> -NH2 > -COO.> -SH > -O. once formed (Table 3. especially in slightly alkaline solutions. for covalent link formation depends upon their availability and reactivity (nucleophilicity). often involving chloroformates. The strength of binding is very strong.5. They also appear to be only very rarely involved in the active sites of enzymes. mild and often successful method of wide applicability. Lysine residues are found to be the most generally useful groups for covalent bonding of enzymes to insoluble supports due to their widespread surface exposure and high reactivity.3a). The hydroxyl groups of this polysaccharide combine with cyanogen bromide to give the reactive cyclic imido-carbonate. using less than a gram of enzyme) involves Sepharose. in addition to the stability of the covalent link. activated by cyanogen bromide. found in enzymes. and very little leakage of enzyme from the support occurs.> -OH >> -NH3+where the charges may be estimated from a knowledge of the pKa values of the ionising groups (Table 1. Figure 3.e.~ ++ The most commonly used method for immobilising enzymes on the research scale (i.3-D-galactose-α-1. Only small amounts of enzymes may be immobilised by this method (about 0.3c) are very useful bifunctional reagents as they allow the coupling of amines to carboxylic acids.milieu.1) and the pH of the solution. Sepharose is a commercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack.3 gram per gram of matrix has been reported.4-(3.e. however. The high toxicity of cyanogen bromide has led to the commercial.~ ++ .02 gram per gram of matrix) although in exceptional cases as much as 0. Careful control of the reaction conditions and choice of carbodiimide allow a great degree of selectivity in this reaction. production of ready-activated Sepharose and the investigation of alternative methods. to produce similar intermediates (Figure 3. Immobilisation of enzymes by their covalent coupling to insoluble matrices is an extensively researched technique.6-anhydro)-L-galactose}) gel. This is a simple. mainly lysine residues) on the enzyme under mildly basic conditions (pH 9 .~ ++ ++ + + ± Others . Functional groups that affects the covalent coupling The relative usefulness of various groups.

(E) with its active site unchanged and ready to accept the substrate molecule (S). the attachment of tyrosine groups through diazo-linkages.3e). (d) Distortion of the active site produces an inactive immobilised enzyme. Distortion can be prevented by use of molecules which can sit in the active site during the coupling process. (e) The use of trialkoxysilane to derivatise glass. by cross-linking the enzyme plus a non-catalytic diluent protein within a porous sheet (e. (e) 3-aminopropyltriethoxysilane Figure 6.3] (b) Chloroformates may be used to produce similar intermediates to those produced by cyanogen bromide but without its inherent toxicity. (a) Immobilised enzyme. ________________________________________ Entrapment and Encapsulation In both of these methods. This can. (c) Enzyme bound in a non-productive mode due to the inaccessibility of the active site. Conditions are chosen to minimise the formation of the inert carbamate.3] (c) Carbodiimides may be used to attach amino groups on the enzyme to carboxylate groups on the support or carboxylate groups on the enzyme to amino groups on the support.4). ________________________________________ Figure 6. Both (c) and (d) may be reduced by use of 'spacer' groups between the enzyme and support.e.4. The effect of covalent coupling. for use in biosensors. (b) Ethyl chloroformate [6. The activity of the immobilised enzyme is then simply restored by washing the immobilised enzyme to remove these molecules.producing immobilised enzyme membranes.g.3] (d) Glutaraldehyde is used to cross-link enzymes or link them to supports. the enzyme remains free in solution.3. (c) Carbodiimide [6. as shown in (b). The product of the condensation of enzyme and glutaraldehyde may be stabilised against dissociation by reduction with sodium borohydride. There are numerous other methods available for the covalent attachment of enzymes (e. ________________________________________ It is clearly important that the immobilised enzyme retains as much catalytic activity as possible after reaction.3] . product or a competitive inhibitor ensures that the active site remains unreacted during the covalent coupling and reduces the occurrence of binding in unproductive conformations. cyanogen bromide [6. on the expressed activity of an immobilised enzyme. but restricted in . be ensured by reducing the amount of enzyme bound in non-catalytic conformations (Figure 3. lens tissue paper or nylon net fabric). (d) Glutaraldehyde [6. Immobilisation of the enzyme in the presence of saturating concentrations of substrate.g. Conditions are chosen to minimise the formation of the inert substituted urea. (a) Activation of Sepharose by cyanogen bromide. as shown. or by the use of a freely reversible method for the coupling which encourages binding to the most energetically stable (i. The use of trialkoxysilanes allows even such apparently inert materials as glass to be coupled to enzymes (Figure 13. and lysine groups through amide formation with acyl chlorides or anhydrides). in part. effectively displacing the enzyme away from the steric influence of the surface. The reactive glass may be linked to enzymes by a number of methods including the use thiophosgene.. ________________________________________ A. It usually consists of an equilibrium mixture of monomer and oligomers. native) form of the enzyme. Nonproductive modes are best prevented by the use of large molecules reversibly bound in or near the active site.

for example. all of which depend for their utility on the semipermeable nature of the membrane. where calcium alginate is widely used.6) shell around the aqueous droplets which traps the enzyme. in most configurations. This must confine the enzyme whilst allowing free passage for the reaction products and. This is then dispersed in a solution of hexanedioic acid in the immiscible solvent. The resultant reaction forms a thin polymeric (Nylon-6. The micro-capsules and liposomes are washed free of non-confined enzyme and transferred back to aqueous solution before use. Entrapment of enzymes Entrapment of enzymes within gels or fibers is a convenient method for use in processes involving low molecular weight substrates and products. animal and plant cells. while allowing free movement of substrates. This product may then be copolymerised and cross-linked with acrylamide (CH2=CH-CO-NH2) and bisacrylamide (H2N-CO-CH=CHCH=CH-CO-NH2) to form a gel. The entrapment process may be a purely physical caging or involve covalent binding.3 presents a comparison of the more important general characteristics of these methods. Encapsulation of Enzymes Enzymes. Liposomes are concentric spheres of lipid membranes. encapsulated within small membrane-bound droplets or liposomes. They are formed by the addition of phospholipids to enzyme solutions. red blood cells can be used as encapsulation capsules. cofactors and products. surrounding the soluble enzyme. Although costly. chloroform. As an example of this latter method. The simplest of these methods is achieved by placing the enzyme on one side of the semipermeable membrane whilst the reactant and product stream is present on the other side. the enzymes' surface lysine residues may be derivitized by reaction with acryloyl chloride (CH2=CH-CO-Cl) to give the acryloyl amides. Enzymes may be entrapped in cellulose acetate fibers by. these are very easy to use for a wide variety of enzymes (including regenerating coenzyme systems. Notably. see Chapter 8) without the additional research and development costs associated with other immobilisation methods.6-diaminohexane. Hollow fiber membrane units are available commercially with large surface areas relative to their contained volumes (> 20 m2 l-1) and permeable only to substances of molecular weight substantially less than the enzymes. Entrapment is the method of choice for the immobilisation of microbial. ________________________________________ Table 6. the enzyme is dissolved in an aqueous solution of 1. the difficulty which large molecules have in approaching the catalytic sites of entrapped enzymes precludes the use of entrapped enzymes with high molecular weight substrates. However. The porosity of the gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzyme.movement by the lattice structure of a gel.3 Generalised comparison of different enzyme immobilisation techniques. Encapsulation generally refers to larger capsules that allow for co-immobilization of different combinations of enzymes for selected applications. may also be used within such reactors. Amounts in excess of 1 g of enzyme per gram of gel or fiber may be entrapped. Characteristics Adsorption Covalent binding Entrapment Membrane confinement Preparation Simple Difficult Difficult Simple Cost Low High Moderate High Binding force Variable Strong Weak Strong Enzyme leakage Yes No Yes No Applicability Wide Selective Wide Very wide Running Problems High Low High High Matrix effects Yes Yes Yes No . As an example of the former. Membrane confinement Membrane confinement of enzymes may be achieved by a number of quite different methods. the substrates. followed by extrusion through a spinneret into a solution of an aqueous precipitant. Table 6. making up an emulsion of the enzyme plus cellulose acetate in methylene chloride.

This can be done through chemical (covalent bonding) or physical means (flocculation). Just as biotechnology is transforming the pharmaceutical industry. Tests must be done to ensure the active site remains free and available for catalytic activity. signal cells to turn on and off and perform other complex functions. the desired enzyme can then be manufactured in commercial quantities. They are also easier to scale up. textiles. but in each protein molecule . In humans. can break down several types of protein. Other animals use enzymes to break cellulose into sugar or break up proteins. 1997). paper and pulp and specialty chemicals. Enzymes are proteins produced by all living organisms. for commercial applications result from the exploitation of rDNA methods in the manufacturing process or for the improvement of the catalysts themselves. Scientists can locate enzymes in the natural environment with special functional characteristics that have significant commercial value. e. In some cases. Using biotechnology. bases. 3-D structure without any support. each enzyme can break down or synthesize one particular compound. and also allows their easy separation from the medium.g. Crosslinking This method joins the enzyme to each other to form a large. for instance. such as enzymes. Contrary to inorganic catalysts such as acids. Most proteases. Today up to 90% of the enzymes used in large scale. In other words. Most enzymes are manufactured in fermentation systems like human therapeutic proteins. Both improved economics as well as the manufacture of novel products not possible or practical by traditional chemical approaches have been achieved. The improved stability and ease of separation of immobilized enzymes make them suitable to be used in a variety of bioreactors. The progress made in applying the techniques of genetic engineering in the development of industrial-scale processes that produce or utilize enzymes has been simply amazing. Encapsulation often improves the operational stability and sometimes efficiency of enzyme catalysis under industrial conditions. In addition. The manufacturing process uses renewable resources as a raw material feedstock. metals and metal oxides. Even today. Chapter-14 Introduction to Industrial biotechnology Industrial biotechnology applies the techniques of modern molecular biology to improve the efficiency and reduce the environmental impacts of processes in industries like food production. encapsulation allows for ease of handling and storage of enzymes. substrate or cofactor should be selected. grain cleaning. enzymes help digest food. enzymes are very specific. as listed below. For example. Industrial biotechnology companies develop biocatalysts. This allows for easier recovery of the enzymes for repeat use. to be used in chemical synthesis. the entrapment method may not work well with cellulose substrate because the substrate itself is large and will not readily go into the entrapment matrix to reach the enzyme. Methods compatible with the enzyme.Large diffusional barriers No No Yes Yes Microbial protection No No Yes Yes ________________________________________ ________________________________________ There are several methods of immobilization (Bickerstaff. much of the science is focused on the development of new technological approaches that will allow the future solution of problems of fundamental understanding as well as practical application. Sometimes other properties of the enzyme may be altered by immobilization. altered pH-activity profile. some observers predict the same impact in the industrial sector. they limit their action to specific bonds in the compounds with which they react.

2.3 Cocoa butter substitutes Nitrile hydratase 4.1.x Formic acid (from waste cyanide) Glucoamylase 3. therefore. Due to their efficiency.3.2.1. Enzymes are very efficient catalysts. is so efficient that in one minute one enzyme molecule can catalyze the breakdown of five million molecules of hydrogen peroxide into water and oxygen.4 Aspartame a Dihydropyrimidinase 1-High-fructose corn syrups (HFCS) With the development of glucoamylase in the 1940s and 1950s it became a . sometimes need to run at very high or very low temperatures or high or low pHs.Being formed to work in living cells.1.1. the specific action of enzymes allows high yields to be obtained with a minimum of unwanted byproducts. the mild conditions in which they work and their high biodegradability.1. pressure or corrosion. Application of Immobilised-Enzyme Processes Introduction Immobilise -enzyme systems are used where they offer cost advantages to users on the basis of total manufacturing costs.1.1.2 D.23 Lactose-free milk and whey Lipase 3.only certain bonds will be cleaved depending on which enzyme is used. Most enzymes function optimally at a temperature of 30-70°C and at pH values which are near pH 7. For certain technical applications. The plant size needed for continuous processes is two orders of magnitude smaller than that required for batch processes using free enzymes.1.12 L-Alanine Cyanidase 3.2.1. including paper manufacturing.3. The capital costs are.5. However. which is found abundantly in the liver and red blood cells.11 Penicillins Raffinase 3.I.3 Urocanic acid Hydantoinasea 3. The opportunity to identify unique bioactive molecules is vast. Chemical processes.26 Invert sugar Lactase 3.1.14 L-Amino acids Aspartate ammonia-lyase 4.5 High -fructose corn syrup Histidine ammonia-lyase 4. Less than 1 percent of the microorganisms in the world have been cultured and characterized. Companies involved in industrial biotechnology are constantly striving to discover and develop high-value enzymes and bioactive compounds that will enhance current industrial processes. Enzyme processes are therefore potentially energysaving and save investing in special equipment resistant to heat. The challenge is to find organisms that can survive and even thrive in these environments.3.x Acrylamide Penicillin amidases 3.Nature provides rich resources for innovative solutions for many industries. the enzyme catalase.2. special enzymes have been developed that work at higher temperatures.1.and L-amino acids Invertase 3. enzymes are very well suited to a wide range of industrial applications.5.2.22 Raffinose-free solutions Thermolysin 3. textile processing. ________________________________________ Table 7.24.2.1 Some of the more important industrial uses of immobilised enzymes Enzyme EC number Product Aminoacylase 3.1 L-Aspartic acid Aspartate 4-decarboxylase 4.3 D-Glucose Glucose isomerase 5. considerably smaller and the plant may be prefabricated cheaply off-site Immobilised enzymes offer greatly increased productivity on an enzyme weight basis and also often provide process advantages (see Chapter 6) Currently used immobilised-enzyme processes are given in Table 7.1.1. specific action.5. For example.2.5. oil field chemicals and specialty chemical synthesis reactions.1. enzymes can work at atmospheric pressure and in mild conditions in terms of temperature and acidity. In industrial processes. no enzyme can withstand temperatures above 100°C for long.5.

2). Fructose is 30% sweeter than sucrose. the majority being converted to various hydroxy acids). on a weight basis.0 using sodium carbonate and magnesium sulphate is added to maintain enzyme activity (Mg2+ and Co2+ are cofactors). The Ca2+ concentration of the glucose feedstock is usually about 25 m. immobilised glucose isomerase was used in a batch process. Although differerent immobilisation methods have been used for enzymes from differerent organisms. Nowadays most isomerisation is performed in PBRs (Table 14. Immobilisation is generally by cross-linking with glutaraldehyde. left from previous processing. All glucose isomerases are used in immobilised forms. as they have specificities for glucose and fructose which are not much different from that for xylose and ways are being found to avoid the necessity of xylose as inducer. these have shortcomings as objects of commerce: D-glucose has only about 70% of the sweetness of sucrose. these should perhaps now no longer be considered as xylose isomerases.3). but the immobilisation methods now used fix the cobalt ions so that none needs to be added to the substrate streams. and is comparatively insoluble. 0. Also. Originally. causing inhibition. after cell lysis or homogenisation. the principles of use are very similar. They are used with high substrate concentration (35-45% dry solids. on a weight basis. which have the additional benefit of substantially stabilising the enzymes at higher operational temperatures. Batches of 97 DE glucose syrup at the final commercial concentration (71% (w/w)) must be kept warm to prevent crystallisation or diluted to concentrations that are microbiologically insecure. enzymes were found that catalyse the conversion of D-xylose to an equilibrium mixture of D-xylulose and D-xylose in bacteria. the existence of such an enzyme was not suspected. Excess Mg2+ is uneconomic as it adds to the purification as well as the isomerisation costs. 2-GLUCOSE ISOMERASE Glucose is normally isomerised to fructose during glycolysis but both sugars are phosphorylated. This proved to be costly as the relative reactivity of fructose during the long residence times gave rise to significant by -product production. can produce such glucose isomerases: The commercial enzymes are produced by Actinoplanes missouriensis. and this presents a problem.22 M KOH at 5°C under nitrogen for 3. The vast majority of glucose isomerases are retained within the cells that produce them but need not be separated and purified before use. The pH is adjusted to 7. these xylose isomerases were found to isomerise α-D-glucopyranose to α-D –fructofuranose. At higher concentrations of calcium a Mg2+ : Ca2+ ratio of 12 is recommended. and twice as soluble as glucose at low temperatures so a 50% conversion of glucose to fructose overcomes both problems giving a stable syrup that is as sweet as a sucrose solution of the same concentration (see Table 14. They are remarkably friendly enzymes in that they are resistant to thermal denaturation and will act at very high substrate concentrations. However. difficulties were encountered in the removal of the added Mg2+ and Co2+ and the recovery of the catalyst. ________________________________________ . Bacillus coagulans and various Streptomyces species. until the late 1950s. plus in some cases a protein diluent. When supplied with cobalt ions. The isomerisation is possible by chemical means but not economical. Ca2+ competes successfully for the Mg2+ binding site on the enzyme.straightforward matter to produce high DE glucose syrups.g.1 M glucose 'isomerised' with 1. giving tiny yields and many by-products (e. At about this time. The need for Co2+ has not been eliminated altogether. The use of this phosphohexose isomerase may be ruled out as a commercial enzyme because of the cost of the ATP needed to activate the glucose and because two other enzymes (hexokinase and fructose-6-phosphatase) would be needed to complete the conversion.5-8.5 months gives a 5% yield of fructose but only 7% of the glucose remains unchanged. mainly bacteria. At this level the substrate stream is normally made 3 mM with respect to Mg2+. Now it is known that several genera of microbes. Only an isomerase that would use underivatised glucose as its substrate would be commercially useful but. 93-97% glucose) at 5560°C.

sometimes after treatment with flocculants.2. The half-life of most enzyme preparations is between 50 and 100 days at 55°C. there still remains the possibility of inhibition due to oxidised by-products caused by molecular oxygen. half-lives 0.2 < 0. Insoluble material is removed by filtration.8 6. an enzyme bed volume of about 4 m3 is needed.e. £ tonne-1 500 30 5 All processes start with 45% (w/w) glucose syrup DE 97 and produce 10000 tonnes per month of 42% fructose dry syrup. £ tonne-1 5 5 1 Conversion cost.1 Product refining Filtration C-treatmenta Cation exchange Anion exchange C-treatment Cation exchange Anion exchange C -treatment Capital. labour and energy costs. after three half -lives). Comparison of glucose isomerisation methods Parameter Batch (soluble GI) Batch (immobilised Gl) Continuous (PBR) Reactor volume (m3) 1100 1100 15 Enzyme consumption (tonnes) 180 11 2 Activity. At equilibrium at 60°C about 51 % of the glucose in the reaction mixture is converted to fructose. because of the excessive time taken for equilibrium to be attained and the presence of oligosaccharides in the substrate stream. In its lifetime 1 kg of . ________________________________________ Precautions It is essential for efficient use of immobilised glucose isomerase that the substrate solution is adequately purified so that it is free of insoluble material and other impurities that might inactivate the enzyme by chemical (inhibitory) or physical (pore-blocking) means. Typically a batch of enzyme is discarded when the activity has fallen to an eighth of the initial value (i. and soluble materials are removed by ion exchange resins and activated carbon beads. the feed flow rate is adjusted according to the enzyme activity. This may be removed by vacuum de-aeration of the substrate at the isomerisation temperature or by the addition of low concentrations (< 50 ppm) of sulphite. Activity decreases.6 Colour formation (A420) 0. half-life (h) 30 300 1500 Active life. However. this means that glucose produced by acid hydrolysis cannot be used.8 7. most manufacturers adjust flow rates so as to produce 42-46% (w/w) fructose (leaving 47-51 % (w/w) glucose). a Treatment with activated carbon. Several reactors containing enzyme preparations of different ages are needed to maintain overall uniform production by the plant (Figure 14. In effect. Some of the improvement that may be seen for PBR productivity is due to the substantial development of this process.7 2 3 Residence time (h) 20 20 0. To produce 100 tonnes (dry substance) of 42% HFCS per day. following a first-order decay equation.Table 7.5 Co2+ (tonnes) 2 1 0 Mg2+ (tonnes) 40 40 7 Temperature (°C) 65 65 60 pH 6. This done.7 0. To maintain a constant fructose content in the product.10). as its low quality necessitates extensive and costly purification.

Diagram showing the production rate of a seven-column PBR facility on start -up.1] . assuming exponential decay of reactor activity.10. The columns are brought into use one at a time. A shorter PBR operating time also results in a briefer start-up period and a more uniform productivity. This is produced by using vast chromatographic columns of zeolites or the calcium salts of cation exchange resins to adsorb and separate the fructose from the other components. Then. although basically very simple.1). but the thermodynamics do not favour the conversion so means must be found of removing sucrose from the system as soon as it is formed..4. The glucose-rich 'raffinate' stream may be recycled but if this is done undesirable oligosaccharides build up in the system. the pH of the syrup is lowered to 4 . The thermodynamics of the system favour fructose production at higher temperatures and 55% fructose syrups could be produced directly if the enzyme reactors were operated at around 95°C. Another ambition of the corn syrup industry is to produce sucrose from starch. It may be seen that the final average production rate is higher when the PBRs are individually operated for shorter periods but this 29% increase in productivity is achieved at a cost of 50% more enzyme.-.4.51 times the initial productivity of one column. Immobilised glucoamylase is used in some plants to hydrolyse oligosaccharides in the raffinate.7). ________________________________________ After isomerisation. .immobilised glucose isomerase (exemplified by Novo's Sweetzyme T) will produce 10 -11 tonnes of 42% fructose syrup (dry substance). 9% glucose) is blended with 42% fructose syrups to give the 55% fructose (42% glucose) product required. money available) is sufficient: phosphorylase starch (Gn) + orthophosphate glucose isomerase starch (Gn-1) + α-glucose-1-phosphate [7.5% initial activity) before replacement. The use of miscible organic co -solvents may also produce the desired effect. due to the more rapid replacement of the biocatalyst in the PBRs.e.The high-fructose syrups can be used to replace sucrose where sucrose is used in solution but they are inadequate to replace crystalline sucrose. ——— PBR activities allowed to decay through three half-lives (to 12.1. glucose isomerase and sucrose phosphorylase (EC 2. is only economic if run continuously. The final average productivity is 3. This can be done using a combination of the enzymes phosphorylase (EC 2.PBR activities allowed to decay through two half-lives (to 25% initial activity) before replacement. here the substrate concentration is comparatively low (around 20% dry solids) so the formation of isomaltose by the enzyme is insignificant. The fructose stream (90% (w/w) fructose. reactor is being refilled with fresh biocatalyst. whilst the seventh.. The fractionation process. For use in the better colas. This market is still expanding and ensures that HFCS production is the major application for immobilised-enzyme technology.Clearly the need for a second large fructose enrichment plant in addition to the glucose isomerase plant is undesirable and attention is being paid to means of producing 55% fructose syrups using only the enzyme.1.23 times the initial productivity of one column. This will not be easy but is achievable if the commercial pull (i. At any time a maximum of six PBRs are operating in parallel..For many purposes a 42% fructose syrup is perfectly satisfactory for use but it does not match the exacting criteria of the quality soft drink manufacturers as a replacement for sucrose in acidic soft drinks..5 and it is purified by ion-exchange chromatography and treatment with activated carbon. ________________________________________ Figure 7. 55% fructose is required. The final average productivity is 2. exhausted. it is normally concentrated by evaporation to about 70% dry solids. Both these alternatives present a more than considerable challenge to enzyme technology! The present world market for HFCS is over 5 million tonnes of which about 60% is for 55% fructose syrup with most of the remainder for 42% fructose syrup.

the bed of invertase-char being 2 ft (60 cm) deep in a bed of char 20 ft (610 cm) deep. The preparation was very stable. Figure [7. the product did not have the subtlety of flavour of the acid-hydrolysed material and the immobilised enzyme process was abandoned when the acid became available once again. Plainly. 50°C).glucose fructose [7. The galactose released is destroyed in the alkaline conditions of the first stages of juice purification and does not cause any further problems while the sucrose is recovered. where the invertase -char mix is stabilised by cross-linking and has a half-life of 90 days in use (pH 5. the limiting factors being microbial contamination or loss of decolourising power rather than loss of enzymic activity. Provided they are able to release sucrose without hydrolysis when the stress is released. The scale used was large.5. was unavailable. raffinoseutilizer. Immobilised raffinase may also be used to remove the raffinose and stachyose from soybean milk. This is grown in a particulate form and the particles harvested. Although free invertase may be used (with residence times of about a day). The revival is due. it would be totally unacceptable to use an enzyme preparation containing invertase to remove this material during sucrose production. This process results in a 3% increase in productivity and a significant reduction in the costs of the disposal of waste molasses.3] A further possible approach to producing sucrose from glucose is to supply glucose at high concentrations to microbes whose response to osmotic stress is to accumulate sucrose intracellularly. 5-Production of amino acids Another early application of an immobilised enzyme was the use of the aminoacylase from Aspergillus oryzae to resolve racemic mixtures of amino acids. The process was cost-effective but. A layer of the bone char containing invertase was included in the bed of bone char already used for decolourising the syrup.4] Chemically synthesised racemic N-acyl-DL-amino acids are hydrolysed . previously used in the manufacture of Golden Syrup. not surprisingly. Recently. Yeast cells were autolysed and the autolysate clarified by adjustment to pH 4. followed by filtration through a bed of calcium sulphate and adsorption into bone char. it has been relaunched using BrimacTM.7. A productivity of 16 tonnes of inverted syrup (dry weight) may be achieved using one litre of the granular enzyme. 4-Use of immobilised Invertase Invertase was probably the first enzyme to be used on a large scale in an immobilised form (by Tate & Lyle). It is impossible to produce inverted syrups of equivalent quality by acid hydrolysis. stirring is stopped and the juice pumped off the settled bed of enzyme. These sugars are responsible for the flatulence that may be caused when soybean milk is used as a milk substitute in special diets. such microbes may be the basis of totally novel processes. 3-Use of immobilised raffinase The development of a raffinase (α-D-galactosidase) suitable for commercial use is another triumph of enzyme technology. relatively low conversion and batch variability problems of acid hydrolysis. dried and used directly as the immobilised-enzyme preparation. In the period 1941 -1946 the acid. Mortierella vinacea var. however. It has been necessary to find an organism capable of producing an -galactosidase but not an invertase. lost by physical attrition. fills the requirements. to the success of HFCS as a high-quality low-colour sweetener. so yeast invertase was used instead. high salt-ash. Enzyme. the cost of 95% inversion (at 50% (w/w)) being no more than the final evaporation costs (to 75% (w/w)).2] sucrose phosphorylase -glucose-1 -phosphate + fructose sucrose + orthophosphate [7. the use of immobilised enzymes in a PBR (with residence time of about 15 min) makes the process competitive. Enzymic inversion avoids the high-colour. It is stirred with the sugar beet juice in batch stirred tank reactors. is replaced by new enzyme added wi