Module 1: Introduction Lecture1: Introduction

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 Biocatalysts and biocatalysis Biocatalysts are either proteins (enzymes) or nucleic acids (RNA or ribozymes and DNAs) capable of catalyzing myriad of reactions in living systems for survival and reproduction. Isolated proteins or nucleic acids can also catalyze these reactions (in vitro) outside living cells and these processes are known as biocatalytic methods. These excellent properties of some biomolecules are utilized in enzyme technology/biocatalysis. They can be used as biocatalysts to catalyze chemical reactions on an industrial scale in a sustainable manner. Application of biocatalysts covers the production of desired materials for all human needs (food, animal feed, detergents, pharmaceuticals, fine and bulk chemicals). Biocatalytic processes are especially suited to the production of compounds from biomass as the raw material (figure-1).

Biomass

Biological systems in technical apparatus

Products (API, bioactive materials, food ingredients)

Byproduct(s) and waste

Recycling

Schematic view of an ideal sustainable biotechnological production process

Enzymes the catalysts of choice: 1. 2. 3. 4. Highly selective operation in complex mixtures, Stereo- and regiospecificity of conversions, Absence of side reactions leading to simpler separation processes and higher yields. Savings in energy and waste treatment cost owing to mild reaction conditions

Specificity

Enzymes are usually very specific

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Complementary shape, charge (electrostatic/ion-dipole interactions) and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity. Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes. Promiscuous enzymes: Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.
Biological functions of enzymes in living systems Enzymes serve a wide variety of the following functions inside living organisms. Signal transduction and cell regulation Muscle contraction for movement generation Active transport through ion-pump Exotic functions (luciferase in fireflies for light generation) Regulation of digestive systems in animals through metabolic pathway Metabolic pathway: In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel, this can allow more complex regulation. Historical background of biocatalysis Early progress: Manufacture and preservation of food and alcoholic drinks in ancient times had successfully done by enzymes. Cheese making has always involved the use of enzymes. Homers Iliad mentions the uses of a kids stomach for making cheese (400 BC). In 1833 Payen and Persoz investigated the hydrolysis of starch by diastase which was later acknowledged by Berzelius as a biocatalytic reaction in 1835. In 1874 the first company (Christian Hansens Labpratory) was set up for the marketing of enzyme preparations for cheese making in Copenhagen. Scientific advances since 1890: In 1894 Emil Fischer described the elegant aspects of enzyme catalysis. Enzyme specificity the term is coined by Fischer. Later on he described his famous Lock and Key hypothesis,

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 which he e considered d a precondition for the potential p of an enzyme to have a ch hemical effe ect on the subst trate.

In 1897, Eduard Buc chner submit tted his first paper on the e ability of yeast y extracts that lacked d any living yea ast cells to ferment sug gar. In a se eries of experiments at the Univers sity of Berlin n, he found tha at the sugar r was fermen nted even when w there were w no livin ng yeast cells in the mix xture. He name ed the enzy yme that bro ought about the ferment tation of suc crose "zyma ase". In 1907, he received the Nobel Prize P in Che emistry "for his biochem mical researc ch and his discovery d of f cellfree ferm mentation". Following F Bu uchner's exa ample, enzym mes are usu ually named according to t the reaction they carry out. o Typically y, to genera ate the name e of an enzy yme, the suf ffix -ase is added a to the na ame of its substrate s (e. .g., lactase is the enzyme that clea aves lactose e) or the type of reaction (e.g., DNA polymerase p f forms DNA polymers). a proteins had been justified j by crystallizatio on of Ureas se by The final proof that enzymes are a the know wn cases the pure enzym me crystals tu urned out to be proteins. Summer in 1926. In all ested a mod dification to the t lock and d key model l: since enzy ymes In 1958, Daniel Koshland sugge er flexible structures, s th he active sit te is continually reshap ped by inter ractions with h the are rathe substrate e as the sub bstrate intera acts with the enzyme. As A a result, th he substrate e does not simply bind to a rigid active site; the am mino acid sid de chains which make up u the active e site are mo olded into the precise pos sitions that enable the enzyme to perform its s catalytic fu unction. In some s cases, such as glycosidases, th he substrate e molecule also a change es shape slig ghtly as it enters e e site. The active site continues c to o change un ntil the subst trate is completely boun nd, at the active which po oint the final shape and charge is determined. d Induced fit t may enhan nce the fidel lity of molecula ar recognitio on in the presence of o competitio on and no oise via the e conformat tional proofread ding mechan nism. It is of ften termed as Koshland ds induced fit hypothe esis. 1. A At the early of 20th cent tury, plant li ipases (lipid d breaking enzymes) e w were isolated d and utilized for pro oduction of fatty f acids. P en nzymes are used for chill-proofing of f beer. 2. Proteolytic 3. In n 1898 the use u of isolate ed enzymes s for the production of le eather playe ed a major ro ole in th he area of ap pplied biocat talysis. 4. In n 1920, othe er enzymatic sources (ex xcept plant/a animals) wer re needed to o provide existing enzymatic pro ocesses with h the necess sary biocata alysts. And the t attention n was focuse ed on m microorganis ms such as bacteria, fungi and yeasts as a potential source of new enzymes.

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Development since 1950 1950-1970: With advancement of scientific and technical knowledge, market demands for enzymes for use in Washing process Starch processing Cheaper raw materials for sweeteners Optically pure amino acids Stimulated further development in applied biocatalysis. Novel enzymatic processes are identified such as, 1. Penicilin amidase (acylase) used for the hydrolysis of penicillin. 2. Glucose isomerase for converting glucose to fructose. Enzyme immobilization was introduced to enable the reuse of costly enzymes. In 1970s with the help of gene technology improved and cheaper biocatalyst production became possible. Today most of the enzymes used as biocatalysts except for food processing are recombinant. Consequently, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. Industrial applications of enzymes in tabular forms Application Enzymes used Uses Food processing Amylases from fungi and Production of sugars. plants Baby foods Trypsin Predigest baby foods Brewing industry Amylases, glucanases, Split polysaccharides and proteases proteins into malt Fruit juices Cellulase, pectinases Clarify fruit juices Dairy industry Renin, Lipases, lactases Manufacturing of cheese. Meat tenderizers Papain To soften meat for cooking Starch industry Glucose isomerase Converts glucose into fructose Paper industry Amylase, Xylanases, Degrades starch to lower Cellulases viscosity. Biofuel industry Cellulases Used to break down cellulose into sugars that can be fermented. Biological detergents Proteases Helping with removal of protein stains from clothes Contact lens cleaners Proteases Remove proteins on contact lens to prevent infections Rubber industry Catalase To generate oxygen from peroxide to convert latex into foam rubber Molecular biology Restriction enzymes, DNA Manipulate DNA in gene ligases and polymerases engineering

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Advantages and disadvantages of cells and enzymes as biocatalysts in comparison with chemical catalysts Advantages * Stereo, regio and chemoselective * Low temperatures (0-1100C) required * Low energy consumption * Active at pH 2-12 * Less byproducts * Non-toxic when correctly used * Can be reused (immobilized) * Can be degraded biologically * Can be produced in unlimited quantities * Cells and enzymes are - unstable at high temperature - unstable at extreme pH-values - unstable in aggressive solvents - inhibited by some metal ions - hydrolyzed by peptidases * Some enzymes - are still very expensive - require expensive cofactors * When inhaled or ingested enzymes are, as all foreign proteins, potential allergens

Disadvantages

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