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BATANGAS STATE UNIVERSITY

College of Engineering, Architecture and Fine Arts
Gov. Pablo Borbon Campus II,
Alangilan, Batangas City, Philippines 4200

ChE526
BIOCHEMICAL ENGINEERING

BIOCHEMICAL ENGINEERING
& ENZYME KINETICS

Valencia, Jenna Mariz
Victorio, Shenjin
Villamin, Princess
Yaba, Tricia Elena
Yurong, Jackie Meileen
ChE-5202

Dr. Sicily B. Tiu

January 23, 2017

BIOCHEMICAL ENGINEERING

biological products separated. . o Cell culture can be scaled up. or devices. food. such as bioreactors. Its applications are in the petrochemical industry. Biochemical engineering Biochemistry is the branch of science concerned with the chemical and physicochemical processes that occur within living organisms. implementation and operation of processes for handling biological materials and biocatalysts. such as bioreactors. and water treatment industries. o It is an important area in modern biotechnology. Biochemical engineering is more about rearranging these interactions to make life work better. and how those interactions are important in the processes that keep us alive. Biochemists figure out how different proteins interact with each other. Biochemical engineers often make modified proteins. pharmaceutical. biotechnology.Definition Biochemical engineering is a branch of chemical engineering that mainly deals with the design and construction of unit processes that involve biological organisms or molecules. Biochemistry is more about discovering how life works.processing. o It is usually divided into biochemical reaction engineering and bio separations.  Biochemical Engineering o It is the extension of chemical engineering principles to systems using a biological catalyst to bring about desired chemical transformations. development. purified and prepared on a large scale. Core concepts of biochemical engineering Introduction  BIOTECHNOLOGY Biotechnology is the art and science of converting reactants into useful products by the action of microorganisms or enzymes. The role of biochemical engineers has become more important in recent years due to the dramatic developments of biotechnology. Biochemical Engineering is concerned with design. o It mainly deals with the design and construction of unit processes that involve biological organisms or molecules. which help regulate human health when it's out of balance. It is concerned with conducting biological processes on an industrial scale.  BIO-PROCESSING Any process in which microorganisms play an essential role in getting transformation of feed into useful products is called as bio. Biochemistry vs. providing a link between biology with chemical engineering.

a biochemical engineer can utilize various separation techniques developed in chemical processes such as distillation. Hourly wages typically start from $25. How can the system be operated and controlled for the maximum yield? For the optimum operation and control. filtration. safety operation and quality of product output  Biochemical engineers aren’t limited to a lab alone. and leaching. How can the products be separated with maximum purity and minimum costs? For this step. adsorption. 4.830. Salaries typically start from $52. drying. they can also include the overall supervision of a plant. o It is the key for biotechnology development to intensify the researches into biological reactors and the separation.01 and go up to $72.52.11. it is important to know how fast the process can take place. 3. extraction. How fast will the process take place? If a certain process can produce a product. 2.010 and go up to $150. What change can be expected to occur? To answer this question. Roles of Biochemical Engineers  The main role of a biochemical engineer is to optimize the growth of microbes under aerobic conditions in a reaction mixture with volume of around a thousand liter  They design economic processes in which maximum biomass yield is obtained with lesser input of raw material and at cheap operating costs  They ensure the maximum efficiency. absorption. Food Industries 2. precipitation. Pharmaceutical Industries 3. the downstream processing (or bioseparation). BIOCHEMICAL INDUSTRIES 1. Brewery and Distilling Industries 4. one must have an understanding of the basic sciences for the process involved. Average Biochemical Engineer Yearly in the US: Biochemical Engineers earn a median salary of $95. and providing technical management services  Chemical engineer who has immense knowledge about the basics of life science and biotechnology TRIVIA: Average Biochemical Engineer Hourly Wage in the US: Biochemical Engineers earn a median hourly wage of $46. Waste Treatment The basic questions which need to be asked for the process development and design are as follows: 1. . purification technologies for biological products.900 per year. reliable on-line sensing devices need to be developed.

an activator or inhibitor's binding is reversible. . This is called competitive inhibition. is a reversible inhibitor. by attaching to the active site. are chemicals that aren’t consumed in a reaction but can speed up a reaction. Therefore. It blocks activity of a viral enzyme that helps the virus make more copies of itself. but we will look at two important groups: competitive and noncompetitive inhibitors. plant. ENZYMES . This is part of the molecule that has just the right shape and functional groups to bind to one of the reacting molecules. only the inhibitor or the substrate can be bound at a given moment. For example. A major function of enzymes in a living system is to catalyze the making and breaking of chemical bonds. Some important types of drugs act as reversible inhibitors. Molecules that increase the activity of an enzymes are called activators. noncompetitive In many well-studied cases. especially eukaryotic cells. and that affect enzyme function by different routes. . There are many kinds of molecules that block or promote enzyme function. That is. because the inhibitor “competes” with the substrate for the enzyme. like any other catalysts. This is called the activation energy. Enzymes can be regulated by other molecules that either increase or reduce their activity. We won't discuss all of the types here. they increase the rate of reaction without themselves undergoing permanent chemical changes. Breaks down hydrogen peroxide Hydrogen Peroxide – produced naturally in chemical reactions How enzymes work For two molecules to react they must collide with one another. The reacting molecule that binds to the enzyme is called the substrate. Competitive vs. . Catalase – one of the most common enzymes that is found in almost all living cells. for example. are biological catalysts that are protein molecules in nature. Enzymes have an active site. They must collide in the right direction (orientation) and with sufficient energy. the drug tipranivir.  An inhibitor may bind to an enzyme and block binding of the substrate. meaning that the molecule doesn't permanently attach to the enzyme. Produced by living cells (animal. . while molecule that decrease activity of an enzyme are called inhibitors. and microorganism) and are absolutely essential as catalysts in biochemical reactions. Sufficient energy means that between them they have enough energy to overcome the energy barrier to reaction. which is used to treat HIV. Reversible inhibitors are divided into groups based on their binding behavior.

but it causes other changes in the enzyme so that it can no longer catalyze the reaction efficiently. This inhibition is said to be "noncompetitive" because the inhibitor and substrate can both be bound at the same time. is just any form of regulation where the regulatory molecule (an activator or inhibitor) binds to an enzyme someplace other than the active site. In noncompetitive inhibition. The competitive inhibitor binds to the active site and prevents the substrate from binding there. Instead. The noncompetitive inhibitor binds to a different site on the enzyme. Diagram illustrating competitive and noncompetitive inhibition. Allosteric regulation Allosteric regulation. the inhibitor doesn't block the substrate from binding to the active site. . it attaches at another site and blocks the enzyme from doing its job. broadly speaking. it doesn't block substrate binding. The place where the regulator binds is called the allosteric site.

The allosteric inhibitor binds to an enzyme at a site other than the active site. The left part of this diagram shows allosteric inhibition. Allosteric enzymes typically have multiple active sites located on different protein subunits.^{3}3start superscript. causing an increase in the function of the active site. unless bound to other non- protein helper molecules called cofactors. allowing substrate to bind at a higher affinity. Also. the activity of the other active sites goes up. or even at all. 3. The right part of this diagram shows allosteric activation. the ones where the inhibitor binds elsewhere than the active site) are forms of allosteric regulation. or permanently through stronger covalent bonds. in a process called cooperativity. When an allosteric inhibitor binds to an enzyme. There are also allosteric activators. Common cofactors include inorganic ions such as iron \text {(Fe}^{2+})(Fe2+)left . Cofactors and coenzymes Many enzymes don’t work optimally. some enzymes that are allosterically regulated have a set of unique properties that set them apart. are often given the name of allosteric enzymes. However. Some allosteric activators bind to locations on an enzyme other than the active site. all active sites on the protein subunits are changed slightly so that they work less well. These may be attached temporarily to the enzyme through ionic or hydrogen bonds. the substrate itself can serve as an allosteric activator: when it binds to one active site. The allosteric activator binds to an enzyme at a site other than the active site. end superscript This is considered allosteric regulation because the substrate affects active sites far from its binding site. Pretty much all cases of noncompetitive inhibition (along with some cases of competitive inhibition. which include some of our key metabolic regulators. The shape of the active site is altered so that the enzyme can no longer bind to its substrate. These enzymes. The shape of the active site is changed.

end superscript Coenzymes are a subset of cofactors that are organic (carbon-based) molecules. For example: Reaction which is catalyzed + ase alcohol dehydrogenase ethanol + NAD+ acetaldehyde + NADH2 glucose isomerase glucose fructose glucose oxidase D-glucose + O2 + H20 gluconic acid lactic acid dehydrogenase lactic acid pyruvic acid Commercial Applications of Enzymes Due to on-going research by biotechnologists. the enzyme that builds DNA molecules. F.curding of milk to start cheese-making processcr pepsin . For example. end superscript. 4. Some vitamins are precursors to coenzymes and others act directly as coenzymes. Nomenclature of Enzymes Originally enzymes were given nondescriptive names such as: rennin .^44start superscript. enzymes now have a large number of commercial applications. a very common . plus. plus. For example. In this industry. right parenthesis and magnesium (\text {Mg}^{2+})(Mg2+)left parenthesis.parenthesis. 2.hydrolyzes proteins at mild alkaline pH Name of substrate + ase a-amylase starch glucose + maltose + oligosaccharides lactase lactose glucose + galactose lipase fat fatty acids + glycerol maltase maltose glucose urease urea + H20 2NH3 + CO2 cellobiase cellobiose glucose The nomenclature was later improved by adding the suffix -ase to the name of the substrate with which the enzyme functions. start superscript. end superscript. requires magnesium ions to function. 2. M. They are used for improving production methods and for fabric finishing. g. e. The most common sources of coenzymes are dietary vitamins. vitamin C is a coenzyme for several enzymes that take part in building the protein collagen. They carry many advantages. Commercial applications of enzymes:  Enzymes are widely used in the textile industry. with one important one being that enzymes are specific to only one catalytic reaction and so they therefore do not produce a range of unwanted by-products. DNA polymerase. or to the reaction that is catalyzed. start superscript. a key part of connective tissue.hydrolyzes proteins at acidic pH trypsin . right parenthesis.

polishing of cotton o Catalase – removing hydrogen peroxide o Pectinase – for bioscouring (a way to scour fabrics) o Alpha amylase – for desizing at low temperatures  The food and drink industry has to be one of the largest markets for enzymes. and printing properties. enzymes are added to the dough when baking bread to ensure that the bread is high in quality and has a better volume (that there is more of it). Compared with the uncoated paper. improved gloss. This industry. They are used in many household and industrial detergents. Enzymes also have the ability to preserve bread.’[2] In the manufacturing of coated papers. Examples of enzymes that may be used in the textile industry: o Cellulase – for stonewashing denim. they reduce water consumption through more effective release of soil. Amylase is used for modification of starch coating and xylanases to reduce the consumption of bleach chemicals are very well known applications. esterases is used for stickies removal and amylases and cellulases are used for improved deinking and cellulases for fiber modification have become an integral part of the chemical solutions used in the pulp and paper mills. a smoother texture. they are biodegradable so they do not really effect the environment that much. In the baking industry. keeping it fresh for a longer period of time and therefore increasing its shelf life. but nowadays ‘lipases for is used for pitch control. a starch-based coating formulation is used in order to coat the surface of the paper. the coating provides a number of benefits. They contribute to a: better overall cleaning performance. o Fungal alpha amylase – for dough improvement in the bread making industry o Glucoamylase – used in fermentation o Papain enzymes – for fermentation in the brewing industry o Beta glucanse – for filtration o Protease – used in biscuit production  Enzymes are also used in the pulp and paper industry. . including. This is because the enzymes are very effective at relatively low temperatures and pH values. o Cellulase – can be used for pulp deinking and pulp refining o Xylanase – for pulp bleaching o Alpha amylase – starch modification Enzymes are used in detergents and in personal care and hygiene. application is the use of the enzyme amylase in order to remove starch size. in addition to the food processing industry is currently one of the largest application areas for enzymes.

. 2. Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in a reactor as If you measure the concentrations of substrate and product with respect to time. the product concentration will increase and reach a mmaximum value. it is important to know how the reaction rate is influenced by reaction conditions such as substrate. and optimum economic condition. which are important in the design of an effective bioreactor. The rate equations developed from the kinetic studies can be applied in calculating reaction time. whereas the substrate concentration will decrease as shown in Figure 2. product.SIMPLE ENZYME KINETICS Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various chemical and physical conditions.2. yields. and enzyme concentrations.1 The rate of reaction can be expressed in terms of either the change of the substrate Cs or the product concentrations C as follows: In order to understand the effectiveness and characteristics of an enzyme reaction. since the reaction rate changes gradually from first order to zero order as the substrate concentration is increased. 3. first-order reaction) when the substrate concentration is in the low range. The reaction rate is proportional to the substrate concentration (that is. The maximum reaction rate rmax is proportional to the enzyme concentration within the range of the enzyme tested. Kinetic studies of enzymatic reactions provide information about the basic mechanism of the enzyme reaction and other parameters that characterize the properties of the enzyme. From these curves we can conclude the following: 1. we obtain a series of curves like the one shown in Figure 2. The reaction rate does not depend on the substrate concentration when the substrate concentration is high. If we measure the initial reaction rate at different levels of substrate and enzyme concentrations.

the rate becomes constant (zero order) and equal to rmax. The mechanism of one substrate enzyme reaction can be expressed as One of the original theories to account for the formation of the enzyme-substrate complex is the "lock and key" theory. 1986) and proposed the rate equation where rmax and KM are kinetic parameters which need to be experimentally determined. d(CES)/dt = O.5). 1913): It is assumed that the product-releasing step. (2. Eq. This is also known as the pseudo-steady-state (or quasi-steady-state) . p. and the slow step determihes the rate. (2. we need to find the kinetic mechanisms which support this equation. Eq. 1925): The change of the intermediate concentration with respect to time is assumed to be negligible.Therefore. The amount of an enzyme is very small compared to the amount of substrate. 100. 3. (2.3. while the other is at equilibrium. The rate is proportional to Cs (first order) for low values of Cs.6).4) expresses the three preceding observations fairly well.4) describes the experimental results well. that is. structural compatibility between an enzyme and a substrate which optimally favors the recognition of the substrate as shown in Figure 2. In addition to the preceding assumptions. Eq. there are three different approaches to derive the rate equation: 1. the enzyme molecules have large and complicated three-dimensional structures. is much slower than the reversible reaction. 2. Briggs-Haldane approach (Briggs and Haldane. Michaelis-Menten approach (Michaelis and Menten. the formation of the enzyme substrate complex does not significantly deplete the substrate. (2. Even though the enzyme is soluble in water. This is an assumption which is often employed in heterogeneous catalytic reactions in chemical kinetics. The total enzyme concentration stays constant during the reaction. The reaction rate equation can be derived from the preceding mechanism based on the following assumptions: 1. The complex then breaks down to the products and regenerates the free enzyme. Since Eq. that is. but with higher values of Cs.Henri observed this behavior in 1902 (Bailey and Ollis. The product concentration is so low that product inhibition may be considered negligible. Brown (1902) proposed that an enzyme forms a complex with its substrate. CEO= CES + C 2. The main concept of this hypothesis is that there is a topographical.

10) into Eq. (2. (2. the free-enzyme concentration CE can be related to the initial enzyme concentration CEO So. MICHAELIS-MENTEN APPROACH Assumption:  Product releasing step is slower than Reversible reaction. determines the overall rate of reaction. the rate of reaction can be expressed as a function of Cs and CE.  Reversible reaction is in Equilibrium.7). KM is equal to the dissociation constant K1 or the reciprocal of equilibrium constant Keq as . (2.7). (2. Numerical solution: Solution of the simultaneous differential equations developed from Eqs.11) is known as the Michaelis constant.8) into Eq.4).8) into Eq. now we have three equation"s from which we can eliminate C E and CES to express the rate expression as the function of substrate concentration and the initial enzyme concentration.5) and (2. By substituting Eq. If we assume that the total enzyme contents are conserved. the rate of product formation and substrate consumption is proportional to the concentration of the enzyme-substrate complex as: The concentration of the enzyme-substrate complex CES in Eq. (2. KM in Eq. (2. In the Michaelis-Menten approach.6) without simplification.assumption in chemical kinetics and is often used in developing rate expressions in homogeneous catalytic reactions. can be related to the substrate concentration C S and the free-enzyme concentration C from the assumption that the first reversible reaction Eq. (2. Then. 3. of which CE cannot be easily determined.9) for CE and rearranging for CES' we obtain Substitution of Eq. (2. Product-releasing step. If the slower reaction. (2. the forward reaction is equal to the reverse reaction so that By substituting Eq. (2.5) is in equilibrium.7) results in the final rate equation which is known as Michaelis-Menten equation and is identical to the empirical expression Eq. (2.

2). Competitive Inhibition In competitive inhibition. ENZYME INHIBITION Inhibitors are chemicals that reduce the rate of enzymatic reactions. The inhibitor binds only to the free enzyme. KM is an important kinetic parameter because it characterizes the interaction of an enzyme with a given substrate. (2. the value of KM is equal to the substrate concentration when the reaction rate is half of the maximum rate rmax (see Figure 2.11). Reaction Mechanism: Example: . not to the ES complex. They block the enzyme but they do not usually control it. Types of Inhibition 1. the inhibitor competes with the substrate for the same binding site. When KM is equal to Cs. The Michaelis-Menten equation is analogous to the Langmuir isotherm equation: where: • θ is the fraction of the solid surface covered by gas molecules • K is the reciprocal of the adsorption equilibrium constant. Therefore. r is equal to one half of rmax according to Eq.The unit of KM is the same as Cs.

It does not bind to the free enzyme so there is noel complex in the reaction mechanism. Reaction Mechanism: Example:  Fructose 1. Noncompetitive Inhibition In noncompetitive inhibition.6-biphosphatase is a key regulatory enzyme in the gluconeogenesis pathway. Regulation of fructose 1. High amounts of AMP signal that ATP levels are low and gluconeogenesis should be shut down while glycolysis is turned on. the inhibitor binds only to the ES complex.6-biphosphatase and phosphofructokinase by AMP prevents a futile cycle in which glucose is simultaneously synthesized and broken down. 2. Uncompetitive Inhibition In competitive inhibition. The inhibitor binds equally well to free enzyme and the ES complex. the inhibitor does not interfere with substrate binding and vice versa. discovered in the 1930s) were the first effective systemic anti-bacterial agents. Sulfanilamide is a competitive inhibitor of p- aminobenzoic acid.6-biphosphatase inhibition by AMP Fructose 1. The inhibitor binds enzyme regardless of whether the substrate is bound. sulfanilamides do not affect human cells. Reaction Mechanism: . Sulfanilamides (also known as sulfa drugs. Because we do not make folic acid.6-biphosphatase (shutting down gluconeogenesis) and activate phosphofructokinase (turning on glycolysis). High AMP levels inhibit fructose 1. 3.

alkaline phospatase catalyzes the release of inorganic phosphate from phosphate esters. Irreversible Inhibition In irreversible inhibition.Prototype for the nerve gas sarin . kidney. placenta. and leukocytes. Reaction Mechanism: Examples:  Diisopropylphosphofluoridate . Reaction Mechanism: . The function of alkaline phosphatase in other tissues is not known. bile ducts. 4. intestine. the inhibitor binds to the enzyme irreversibly through formation of covalent bond with the enzyme. including liver. It is found in a number of tissues.Permanently inactivates serine proteases by forming a covalent bond with the active site serin. Serum alkaline phosphatase levels are important diagnostics markers for bone and liver disease. Example:  Alkaline phosphatase inhibition by phenylalanine At alkaline pH. Enzyme is inactivated until all of the irreversible inhibitor is used up. bone. Alkaline phosphatase plays a role in the deposition of hydroxyapetite in osteoid cells during bone formation. permanently inactivating the enzyme.

especially the enzymes isolated from thermophilic organisms found in certain hot environments. EFFECT of pH The rate of an enzyme reaction is strongly influenced by the pH of the reaction solution both in vivo and in vitro The typical relationship between the reaction velocity and pH shows a bell-shaped curve Figure 2.3  Amylase from saliva pH 6.13.EFFECT OF TEMPERATURE The rate of a chemical reaction depends on the temperature according to Arrhenius equation as The temperature dependence of many enzyme-catalyzed reactions can be described by the Arrhenius equation. denaturation begins to occur at 45 to 50°C. denaturation processes progressively destroy the activity of enzyme molecules. However. the temperature is limited to the usual biological range. so that the overall reaction velocity drops. since the atoms in the enzyme molecule have' greater energies and a greater tendency to move. As the temperature rises. For many proteins.8  Chymotrypsin from the pancreas pH 7-8 . hydrogen) bonds. An increase in the temperature increases the rate of reaction. The optimum pH is different for each enzyme:  Pepsin from the stomach pH 2-3. This is due to the unfolding of the protein chain after' the breakage of weak (for example. Some enzymes are very resistant to denaturation by high temperature.