Nanyang Technological University Biochemical Engineering Lecture Notes PDF

CH3104 Biochemical Engineering PART2 Lecture 1: Course outline & Enzyme Asst. Prof. CHEN Lin 1 Asst. Prof. CHEN Lin [email protected] Office: N1.2-B1-15 Office Hours: Wednesday 2-3 pm (or via prior schedule)...

CH3104 Biochemical Engineering PART2 Lecture 1: Course outline & Enzyme Asst. Prof. CHEN Lin 1 Asst. Prof. CHEN Lin [email protected] Office: N1.2-B1-15 Office Hours: Wednesday 2-3 pm (or via prior schedule) 2  Class schedule Lectures: Friday 9.30am-11.30am (CBE-LT) Tutorials: Monday 3.30pm-4.30pm (CBE-SR2) Monday 4.30pm-5.30pm (CBE-SR2) Friday 12.30pm-1.30pm (CBE-SR2) Friday 1.30pm-2.30pm (CBE-SR2) Consultation: Wednesday 2-3 pm (or via prior schedule) Email: [email protected] Room: N1.2-B1-15 Discussion Forum 3  Tentative Assessment One quiz (announced): 20% Final examination (closed Book): 60% 4  Course Policy There will be no make-up quizzes. Zero points for no show up. Exceptions for absent due to medical reasons (with valid proof). In this case, points will be awarded based on your performance in the final examination. Active note taking in the class is encouraged. Please provide feedback on teaching (such as too fast or too slow), and feedback on your expectation from the course during the semester. 5 How to increase the rate of a reaction: 1. Increase the concentration of a reactant. 2. Increase the temperature of the reactants. 3. Increase the surface area of a reactant. 4. Add a catalyst to the reaction. 6 Learning Objectives Introduction to enzymes How enzymes work Enzymes applications 7 Introduction Enzymes are biological catalysts synthesized by living cells that accelerate biochemical reactions The orderly course of metabolic processes is only possible because each cell is equipped with its own genetically determined set of enzymes It is only this that allows coordinated sequences of reactions - metabolic pathways Involved in many regulatory mechanisms Almost all enzymes are proteins except catalytically active ribonucleic acids, the ribozymes 8 Enzymes in pathways 9 Introduction Enzymes are characterized by three distinctive features: Catalytic Power Ability to catalyses biochemical reaction Accelerating reaction rates as much as 1016 over uncatalyzed levels - far greater than any synthetic catalysts Specificity A given enzyme is very selective Both in the substances with which it interacts and in the reaction that it catalyzes Regulation Metabolic inhibitors and activators 10 Introduction Traditionally, enzymes often named by adding the suffix –ase to the substrate which they acted (e.g., urease) or the reaction catalyzed (e.g., alcohol dehydrogenase) Confusion arose from these trivial naming. A new system of nomenclature of enzyme was developed based on nature of reaction it helps Six classes of reactions are recognized (Within each class are subclasses, and under each subclass are sub-subclasses within which individual enzymes are listed) 11 IUB Classification of Enzyme Enzyme are classified on the basis of action it performs 12 IUB Classification of Enzyme Enzyme are classified on the basis of action it performs 13 IUB Classification of Enzyme Enzyme are classified on the basis of action it performs 14 Enzyme Commission Number (E.C.) The assignment of E.C. number is described in guidelines set out by the International Union of Biochemistry and Molecular Biology, and follows the format E.C. w.x.y.z, where numerical values are substituted for w, x, y, and z. The value of w is always between 1 and 6 and indicates one of six main divisions. The values of x, y, z indicate the sub classification and for father identification 15  Intracellular and extracellular enzymes Intracellular o enzymes are synthesized and retained in the cell for the use of cell itself. o They are found in the cytoplasm, nucleus, mitochondria and chloroplast. Example: Oxydoreductase catalyses biological oxidation, enzymes involved in reduction in the mitochondria. Extracellular o enzymes are synthesized in the cell but secreted from the cell to work externally. Example : Digestive enzyme produced by the pancreas, are transported to the duodenum. 16 Enzyme constituents Most of enzymes carry out their functions relying solely on their protein structure Many others require non-protein components that participate directly in substrate binding / catalysis, termed prosthetic group, cofactor, coenzyme Prosthetic Non-protein part group Coenzyme Metal ion Protein part Apoenzyme Holoenzyme 17 Co-factor types Co-factors are required to prepare the active site for proper substrate binding and/or participate in catalysis Co-factors Coenzymes Prosthetic groups Metal ions (loosely bound) (tightly bound) Activator metal Metal ions of ions metalloenzymes (loosely bound) (tightly bound) 18 Co-factors: Heat stable, low molecular weight, non-protein, organic/non-organic compound required for enzyme activity Vitamin Coenzyme Metal B1-Thiamine Thiamine pyrophosphate (TPP) Magnesium (Mg²⁺) B2-Riboflavin Flavin adenine dinucleotide (FAD) Zinc (Zn²⁺) Flavin adenine mononucleotide (FMN) B3-Niacin Nicotinamide adenine dinucleotide Iron (Fe²⁺/Fe³⁺) (NAD) NADP Biotin Enzyme bound Biotin Copper (Cu²⁺) B5-Pantothenic Coenzyme A Manganese (Mn²⁺) acid B6-Piridoxine Pyridoxal phosphate (PP) Calcium (Ca²⁺) B12-Cobalamin Methylcobalamin Cobalt (Co²⁺) Deoxyadenosylcobalamin Folic acid THF (Tetrahydrofolic acid) Molybdenum (Mo) 19 Enzymes vs. Non-biological catalysts Feature Enzyme Non-biological catalyst Structure Proteins Varies from metal ions to complex molecules Mode of action Catalysis occur via active site Catalysis takes part as a whole Specificity Highly specific Less specific/catalyze different reactions Saturation Can be saturated with substrate Most do not show saturation Temperature & pH stability Have optimum condition Not sensitive Nature Generally produced by living cells Reacts outside the living cells & acts inside living cells https://www.youtube.com/watch?v=yk14dOOvwMk 20 Mechanism of Enzyme Action: Active Sites Enzymes are proteins which are chains of amino acids They are folded into a complex 3-D structure: globular shape Their shape determine the enzyme’s function Human pancreatic amylase 21 Mechanism of Enzyme Action: Active Sites The active site of an enzyme is the region that binds substrates, co-factors (metal ion, coenzymes and prosthetic groups) contain residue that helps to hold the substrate. Active site has a specific shape due to tertiary structure of protein. 22 Mechanism of Enzyme Action: Active Sites A change in the shape of protein affects the shape of active site and function of the enzyme. Active sites generally occupy less than 5% of the total surface area of enzyme. 23 Mechanism of Enzyme Action: Active Sites Active site can be further divided into: 24 Action model: Lock and key Proposed by EMIL FISCHER in 1894. Lock and key hypothesis assumes the active site of an enzymes are rigid in its shape. There is no change in the active site before and after a chemical reaction. Enzyme 25 Action model: Lock and key The lock and key model of enzyme action, proposed earlier this century, proposed that the substrate was simply drawn into a closely matching cleft on the enzyme molecule. Substrate Products Enzyme Symbolic representation of the lock and key model of enzyme action. 1. A substrate is drawn into the active sites of the enzyme. 2. The substrate shape must be compatible with the enzymes active site in order to fit and be reacted upon. 3. The enzyme modifies the substrate. In this instance the substrate is broken down, releasing two products. 26 Action model: Induced fit model More recent studies have revealed that the process is much more likely to involve an induced fit model(proposed by DANIAL KOSH LAND in 1958). According to this, exposure of an enzyme to substrate cause a change in enzyme, which causes the active site to change its shape to allow enzyme and substrate to bind. 27 Action model: Induced fit model More recent studies have revealed that the process is much more likely to involve an induced fit. The enzyme or the reactants (substrate) change their shape slightly. The reactants become bound to enzymes by weak chemical bonds. This binding can weaken bonds within the reactants themselves, allowing the reaction to proceed more readily. The enzyme changes shape, forcing the The resulting end substrate molecules to product is released by Two substrate combine. the enzyme which molecules are returns to its normal drawn into the cleft shape, ready to of the enzyme. undergo more reactions. 28 Mechanism of Enzyme Action The catalytic efficiency of enzymes is explained by two perspectives: Thermodynamic Processes at the changes active site Acid base Metal ion Catalysis by Catalysis by Covalent catalysis catalysis proximity strain catalysis 29 Thermodynamic changes All chemical reactions have energy barriers between reactants and products The difference in transitional state and substrate is called activation barrier Enzymes lower a reaction’s activation energy 30 Common types of catalysis Covalent catalysis A group on the enzyme becomes covalently modified during reaction, e.g., by forming a covalent bond to the substrate during the reaction. Enzyme is released unaltered after completion of reaction. 31 Common types of catalysis Acid-base catalysis A group on the enzyme acts as an acid or base: it removes a proton from or donates a proton to the substrate during the reaction. Mostly undertaken by oxido-reductases enzyme. His 12 acts as a general base and His 119 as a general acid to Promote nucleophilic attack and bond cleavage. RNase A 32 Common types of catalysis Metal ion catalysis A metal ion (e.g., Zn²⁺, Mg²⁺, Fe²⁺/Fe³⁺) is used by the enzyme to facilitate a chemical rearrangement or binding step by stabilizing charged intermediates. Metalloenzymes vs. Metal-activated enzymes Carbonic anhydrase 33 Common types of catalysis Catalysis by proximity In this catalysis, molecules must come in bond forming distance. The enzyme holds two substrates near in space and in precisely the correct spatial orientation to optimize their reaction. 34 Common types of catalysis Catalysis by bond strain Mostly undertaken by lyases. The enzyme-substrate binding causes reorientation of the structure of site due to in a strain condition. (Most enzymes use a combination of several of these strategies) 35 Changing the active activity: denature Changes to the 3D shape of the active site/protein enzyme will result in a loss of function. Enzymes are sensitive to various factors such as temperature, pH & exposure to chemicals. During denaturation, the weak bonds (hydrogen bonds, ionic bonds, hydrophobic interactions, and disulfide bridges) that hold the enzyme's structure together are disrupted. 36 Changing the active activity: denature Often irreversible (e.g., cooking an egg, where heat causes permanent denaturation of proteins). In some cases, if the denaturing agent is removed, the enzyme may recover. Denaturation usually leads to a complete loss of enzyme activity because the enzyme's structure is critical to its function. 37 Changing the active activity: inhibition The decrease or complete loss of enzyme activity due to the binding of an inhibitor molecule to the enzyme. Inhibition does not necessarily involve a loss of the enzyme’s structure, but rather a reaction interference. Inhibitor blocks active site Inhibition occurs when an inhibitor molecule binds to the enzyme, either at the active site or at another site. This binding prevents the enzyme from interacting with its substrate or slows down its catalytic activity. 38 Types of inhibition Inhibition Reversible Irreversible Non- Competitive Uncompetitive Mixed competitive 39 Irreversible inhibition This type of inhibition involves the covalent attachment of the inhibitor to the enzyme. The catalytic activity of enzyme is completely lost. Examples: Aspirin which targets and covalently modifies a key enzyme cyclooxygenase (COX) involved in inflammation is an irreversible inhibitor. 40 Reversible inhibition It is an inhibition of enzyme activity in which the inhibiting molecular entity can associate and dissociate from the protein‘s binding site. 41 Competitive inhibition In this type of inhibition, the inhibitors compete with the substrate for the active site. Formation of E.S complex is reduced while a new E.I complex is formed. The relative concentrations of substrate and inhibitor and their affinities determine the inhibition degree. At high substrate concentrations, the inhibitory effects can be reversed: substrate outcompete the inhibitor from binding 42 Examples of competitive inhibition Statin drug as example of competitive inhibition: Statin drugs such as lipitor compete with HMG-CoA(substrate) and inhibit the active site of HMG CoA-reductase (that bring about the catalysis of cholesterol synthesis). Lipitor (atorvastatin) 43 Non-competitive inhibition The inhibitor binds to a site on the enzyme other than the active site (allosteric site), and this can happen whether the substrate is bound or not. It alters the enzyme's activity but not the substrate binding. Enzyme is inactivated when the inhibitor binds, regardless of the presence of substrate. This form of inhibition is particularly effective because it results in impaired function of the enzyme, even if the substrate is present in large amounts. 44 Uncompetitive inhibition The inhibitor binds only to the enzyme-substrate complex, preventing the complex from releasing the product. This form of substrate inhibition is particularly effective in situations where control of the reaction is critical after binding. 45 Examples of uncompetitive inhibition Methotrexate and Dihydrofolate Reductase (DHFR): Methotrexate is a drug used to treat certain cancers (such as leukemia) and autoimmune diseases (such as rheumatoid arthritis). It targets the enzyme dihydrofolate reductase (DHFR), which is crucial for DNA synthesis and cell division. 46 Enzyme specificity Enzymes are highly specific in nature, interacting with one or few substrates and catalyzing only one type of chemical reaction. Example: Oxydoreductase do not catalyze hydrolase reactions and hydrolase do not catalyze reaction involving oxidation and reduction. 47 Enzyme specificity Enzymes show different degrees of specificity: Bond specificity. Group specificity. Substrate specificity. Optical or stereo-specificity. Dual specificity. 48 Bond specificity In this type, enzyme acts on substrates that are similar in structure and contain the same type of bond. Example : Amylase which acts on α-1-4 glycosidic bond in starch dextrin and glycogen, shows bond specificity. 49 Group specificity In this type of specificity, the enzyme is specific not only to the type of bond but also to the structure surrounding it. Example : Pepsin is an endopeptidase enzyme, that hydrolyzes central peptide bonds in which the amino group belongs to aromatic amino acids e. g phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp). 50 Substrate specificity In this type of specificity, the enzymes acts only on one substrate. Example : Uricase, which acts only on uric acid, shows substrate specificity. Maltase, which acts only on maltose, shows substrate specificity. 51 Optical/stereo-specificity In this type of specificity , the enzyme is not only specific to substrate but also to its optical configuration. Example : D-amino acid oxidase acts only on D amino acids. L-amino acid oxidase acts only on L amino acids. 52 Dual specificity There are two types of dual specificity. The enzyme may act on one substrate by two different reaction types. Example : Isocitrate dehydrogenase enzyme acts on isocitrate (one substrate) by oxidation followed by decarboxylation(two different reaction types). 53 Dual specificity The enzyme may act on two substrates by one reaction type. Example : Xanthine oxidase enzyme acts on xanthine and hypoxanthine (two substrates) by oxidation (one reaction type) 54 Why we use enzymes for industries? Enzymes speed up chemical reactions in a natural way. Enzymes target specific substrates, ensuring precise reactions and fewer by-products. Enzymes work by weakening bonds which lowers activation energy, thus under mild conditions. 55 Advantages and disadvantages of different catalyst groups 56 Merits of using biocatalysts for industries 57 Production of enzymes Commercial sources of enzymes are obtained from three primary sources, i.e. animal tissue, plants and microbes. These naturally occurring enzymes are quite often not readily available in sufficient quantities for food applications or industrial use. However, by isolating microbial strains that produce the desired enzyme and optimizing the conditions for growth, commercial quantities can be obtained. 58 Production of enzymes They can be made to produce abundant quantities of enzymes under suitable growth conditions. Microorganisms can be cultivated by using inexpensive media and production can take place in a short period. In addition, it is easy to manipulate microorganisms in genetic engineering techniques to increase the production of desired enzymes. Recovery, isolation and purification processes are easy with microbial enzymes than that with animal or plant sources. In fact, most enzymes of industrial applications have been successfully produced by microorganisms. Various fungi, bacteria and yeasts are employed for this purpose.. 59 Production of enzymes Different organisms' contribution in the production of enzymes Fungi – 60% Bacteria – 24% Yeast – 4% Streptomyces – 2% Higher animals – 6% Higher plants – 4% Ex. Aspergillus niger - A unique organism for production of bulk enzymes There are well over 40 commercial enzymes that are conveniently produced by A. niger. These include a-amylase, cellulase, protease, lipase, pectinase, phytase, catalase and insulinase. 60 Production of enzymes Steps Involved: 1. Selection of organisms 2. Formulation of medium 3. Production process 4. Recovery & Purification of enzymes 61 Industrial enzymes market global forecast to 2028 North America EU APEC South America RoW The global industrial enzymes market is expected to be worth USD 10.2 billion by 2028, growing at a CAGR of 6.6% during the forecast period 62  Carbohydrases used in industries Industry Enzymes (example) Baking α- and β-Amylase Beverage Celluloses Sweeteners Glucosyltransferase Prebiotics β-D-Fructosyltransferase Biofuels Cellulase Agriculture Invertases Dairy Lactase Animal feed Xylanase Pharmaceuticals β-Glucocerebrosidase Detergents Cellulases Wastewater treatment Cellulase Paper Xylanase Textile Pectinase 63 Industrial applications of enzymes 64 Industrial applications of enzymes 65 Enzyme used in food industry Dairy Wine and fruit Baking Brewing Meat industry Production juice industry Rennet Maltogenic amylase β-Glucanase Pectinase Protease Lactase Glucose oxidase α- Amylase β- Glucanase Papain Protease Pentosenase Protease Catalase Amyloglucosidase 66  Enzyme used in dairy industry S. Enzyme Purpose / Function No. 1. Rennet Coagulant In cheese product 2. Lactase Hydrolysis of lactose to give lactose-free milk product. 3. Protease Hydrolysis of whey protein. 4. Catalase Removal of Hydrogen peroxide. 67 Enzyme used in washing powders Lipases break down fatty, insoluble lipid stains like grease and oil into smaller, soluble fatty acids and glycerol, making them easier to remove during washing Proteases breaks down the colored, insoluble proteins that cause stains to smaller, colorless soluble polypeptide Can wash at lower temperature 68 Diagnostic importance of enzyme Estimation of enzyme activities in biological fluid is of great clinical importance. The enzyme can be divided in 2 groups Plasma Specific or plasma functional enzyme Non-plasma specific or plasma non-functional enzyme 69 Plasma specific Present in the plasma normally and have specific functions Their values are higher in plasma than tissue They are mainly synthesized in liver and enter the circulation Ex: Lipoprotein lipase, plasmin, thrombin, choline esterase, ceruloplasmin Impairment of liver function or genetic disorder – leads to enzyme deficiency Thrombin Thrombosis 70 Non-plasma specific These enzymes are present in the low level in plasma compared to the tissue Estimation of activities of these enzymes serves for the diagnosis and prognosis of several disease - markers of disease The raised enzyme level may indicate Cellular damage Increased rate of cell turnover Proliferation of cells Increased synthesis of enzymes 71 Important diagnostic enzymes Amylase – Acute pancreatitis Serum glutamate pyruvate transferase (SGPT) – liver disease (hepatitis) Serum glutamate oxaloacetate transaminase (SGOT) – Heart attacks (myocardial infarction) Alkaline phosphatase – Rickets, obstructive jaundice Acid phophatase – cancer of prostate gland Lactate dehydrogenase (LDH) – heart attacks, liver disease Creatinine phosphokinase (CPK) – myocardial infarction Aldolase – Muscular dystrophy 72 Key Takeaways Enzyme: definition, classification, and constituent Catalysis type, inhibition, and specificity 73