BT8403/ Enzyme Technology And Biotransformation 2018-2019 DEPARTMENT OF BIOTECHNOLOGY Faculty Name : Ms. K.Archana Faculty Code : HTS 1407 Subject Name : Enzyme Technology and Biotransformation Subject Code : BT8403 Year & Semester : II& IV BT8403/ Enzyme Technology And Biotransformation 2018-2019 DEPARTMENT OF BIOTECHNOLOGY COURSE DETAILS Faculty Name : Ms. K. Archana Faculty Code: HTS 1407 Subject Name: Enzyme Technology and Biotransformation Subject Code: BT8403 Department: Biotechnology Year & Semester: II & IV COURSE OUTCOMES On completion of this course, the students will be able to Knowledge CO No Course Outcomes Level Describe about the enzyme and its classification, reaction in order to C213.1 proceed towards various concepts in biotechnology K1 C213.2 Understand about the enzyme kinetics which will provide the K2 importance and utility of enzyme towards research C213.3 Discuss about the enzyme immobilization techniques and its application in food, pharmaceutical and chemical industries K2 C213.4 Elaborate about production and purification of enzyme at industrial K2 scale C213.5 Explain about the biotransformation application of enzyme K2 Mapping of Course Outcomes with Program Outcomes and Program Specific Outcomes BT6404 PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3 PSO4 C213.1 3 1 - - - 1 - - - 1 1 1 2 1 - - C213.2 3 - - - - 1 1 - - 1 1 1 2 1 - - C213.3 2 1 - - - 1 2 - - 1 1 - 2 - - - C213.4 2 1 - - - 1 1 - - - 1 - 2 - - - C213.5 1 - - - - 1 2 - - - 1 - 2 - - - BT6404 PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3 PSO4 C213 3 1 - - - 1 1 - - 1 1 1 2 1 - - K1 – Remember; K2 – Understand; K3 – Apply; K4 – Analyse; K5 – Evaluate; K6 - Create Mapping Relevancy 1: Slight (Low) 2: Moderate (Medium) 3 Substantial (High) - : No correlation BT8403/ Enzyme Technology And Biotransformation 2018-2019 Unit – 1 INTRODUCTION TO ENZYMES Part-A 1. What are enzymes? (May 2017) Enzymes are biomolecules that catalyze chemical/biochemical reactions and the rate is increased. 2. What are the differences between enzymes and chemical catalysts? Enzymes are very efficient catalysts, often far superior to conventional chemical catalysts, Foremost amongst these are their specificity and selectivity not only for particular reactions but also in their discrimination between similar parts of molecules 3. List the six classes of enzymes. (May 2015) Oxidoreductases, Transferases, hydrolases, Lyases, Isomerases and ligases. 4. What are the general properties of enzymes? (i) They lower the activation energy of reaction, (ii) They do not participate in the reaction, and return to their original form at the end of reaction; (iii) they only increase the reaction rat 5. What are the objectives of Enzyme Engineering? (i) Improved kinetic properties, (ii) Elimination of allosteric regulation, (iii) Enhanced substrate and reaction specificity, (iv) Increasedthermostability, (v) Alteration in optimal pH, (vi) Suitability for use inorganic solvents, (vii) increased/decreased optimal temperature, etc. 6. What are Isozymes? Give example. Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters. An example of an isozyme is glucokinase, a variant of hexokinase 7. What are Oxidoreductases? Give example. Oxidoreductasesare involved in redox reactions, i.e., transfer of hydrogen or oxygen atoms between molecules. This class includes: dehydrogenases (hydride transfer), oxidases (e- transfer to O2), oxygenase (oxygen atom transfer from O2), and peroxidases (e- transfer to peroxides). Example, glucose oxidase (EC 1.1.3.4). 8. What is activation energy? The free energy needed to elevate a molecule from its stable ground state to the unstable transition state is known as activation energy (denoted by ∆G*). 9. What are coenzymes? Give example. An organic cofactor is commonly known as coenzyme. Some of these chemicals such as riboflavin, thiamine and folic acid. 10. What are Transferases? Give example. Transferasescatalyse the transfer of an atom or group of atoms (like acyl-, alkyl- and glycosylgroups) between two molecules. The transferred groups are different from those transferred by the other classes of enzymes like Oxidoreductases, etc. Example, aspartate aminotransferase (EC 2.6.1.1) 11. Define metallo –enzymes. Enzymes that use a metal in the active site are called metallo-enzymes. 12. Describe the enzyme specificity. One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze. A few enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. 13. What are Hydrolases? Give example. Hydrolasesare those enzymes, which catalyze hydrolytic reactions (and their reversals); this class includes esterases, glycosidases, proteases and lipases. Example, chymosin or rennin (EC 3.4.23.4). 14. What do you mean by the active site of an enzyme? One particular portion of the enzyme surface has a strong affinity for the substrate 15. What are Lyases? Give example. Lyasesare involved in elimination reactions resulting in the removal of a group of atoms from the substrate molecule. This class includes aldolases, decarboxylase, dehydratases and some pectinases. Example, histidine ammonia lyase (EC 4.3.1.3). BT8403/ Enzyme Technology And Biotransformation 2018-2019 16. What are the assumptions in induced fit model? (May 2017) The active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. 17. What are Isomerases? Give example. Isomerasescatalyse the formation of isomers of molecules; they include epimerases, racemases and intramoleculartransferases. Example, xylose isomerase (EC 5.3.1.5). 18. What is the role of entropy in catalysis.(Nov 2015) Entropy is composed of translational, rotational, and internal entropies. When two molecules react without a catalyst there is a loss of rotational and translational entropies. 19. What are Ligases? Give example. Ligases or synthetases catalyze the formation of covalent bonds between two molecules utilizing the energy obtained from hydrolysis of a nucleoside triphosphate like A TP or GTP. Example, glutathione synthase (EC 6.3.2.3). 20. What is meant by active site?(May 2015, Nov 2016) An active site is the part of an enzyme that directly binds to a substrate and carries a reaction. It contains catalytic groups which are amino acids that promote formation and degradation of bonds. By forming and breaking these bonds, enzyme and substrate interaction promotes the formation of the transition state structure. 21. List the six classes of enzymes and brief on the role of each class. (May 2016) Class Chemical Reaction Catalyzed Sample Enzymes Oxidoreductase Oxidation-reduction in which oxygen and Cytochrome oxidase, lactate hydrogen are gained or lost dehydrogenase Transferase Transfer of functional groups, such as an Acetate kinase, alanine amino group, acetyl group, or phosphate deaminase group Hydrolase Hydrolysis (addition of water) Lipase, sucrase Lyase Removal of groups of atoms without Oxalate decarboxylase, hydrolysis isocitratelyase Isomerase Rearrangement of atoms within a molecule Glucose-phosphate isomerase, alanine racemase Ligase Joining of two molecules (using energy Acetyl-CoA synthetase, usually derived from the breakdown of ATP) DNA ligase 22. Outline the concept of active site and energetics of ES complex formation (May 2016) The enzyme's active site binds to the substrate. Increasing the temperature generally increases the rate of a reaction, but dramatic changes in temperature and pH can denature an enzyme, thereby abolishing its action as a catalyst. The induced fit model states an enzyme binds to an active site and both change shape slightly, creating an ideal fit for catalysis. When an enzyme binds its substrate it forms an enzyme-substrate complex. Enzymes promote chemical reactions by bringing substrates together in an optimal orientation, thus creating an ideal chemical environment for the reaction to occur. The enzyme will always return to its original state at the completion of the reaction. 23. Explain the Koshland Induced fit Hypothesis (Nov 2015) Daniel E Koshland formulated this hypothesis in 1959.According to this hypothesis the active site does not have a rigid lock and key conformation. The binding of the substrate moleculeto the enzyme molecule induces to modify the shape of the active site so that it becomes complementary to the BT8403/ Enzyme Technology And Biotransformation 2018-2019 substrate molecule. This is called induced fit. Induced fit is possible because of the flexibility of the protein molecules. 24. What are monomeric and oligomeric enzymes? Give examples (Nov 2016) Enzymes having only one polypeptide chain are called monomeric enzymes. eg. DNA Polymerase Enzymes formed by non covalent bonding of a few monomers are called oligomeric enzymes. eg. pyruvate kimase. PART B 1. How the enzyme commission developed a system of classification and its recommendations on nomenclature. (Nov 2015, Nov 2016 ) The first general principle of these 'Recommendations' is that names purporting to be names of enzymes, especially those ending in -ase, should be used only for single enzymes, i.e. single catalytic entities. They should not be applied to systems containing