1. Industrial Production of Enzyme

3. Biologically active enzymes may be extracted from any living organism: Of the hundred enzymes being used industrially, - over a half are from fungi - over a third are from bacteria with the remainder divided between animal (8%) and plant (4%) sources. Sources of enzymes

4. Sources f Enzyme Microbes are preferred to plants and animals as sources of enzymes because: -they are generally cheaper to produce. -their enzyme contents are more predictable and controllable. - plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants).

5. Enzyme EC SourcesApplication  -Amylase 3.2.1.1AspergillusEBaking Catalase1.11.1.6AspergillusIFood Cellulase3.2.1.4TrichodermaEWaste Dextranase3.2.1.11PenicilliumEFood Glucose oxidase1.1.3.4AspergillusIFood Lactase3.2.1.23AspergillusEDairy Lipase3.1.1.3RhizopusEFood Rennet3.4.23.6 Mucor miehei ECheese Pectinase3.2.1.15AspergillusEDrinks Protease3.4.23.6AspergillusEBaking E: extracellular enzyme; I: intracellular enzyme Fungal enzymes

6. Enzyme SourcesApplication  -Amylase 3.2.1.1BacillusEStarch  -Amylase 3.2.1.2BacillusEStarch Asparaginase3.5.1.1 Escherichia coli IHealth Glucose isomerase 5.3.1.5BacillusI Fructose syrup Penicillin amidase 3.5.1.11BacillusI Pharmace utical Protease3.4.21.14BacillusEDetergent Bacterial enzymes

7. The screening procedure for commercial enzymes is to screen ideas: - to determine the potential commercial need for a new enzyme. - to estimate the size of the market and to decide how much potential users of the enzyme will be able to afford to pay for it. E.g. entirely novel substance, or to improve a process agreement, discussions with potential users PROCEDURES

8. -use all available databases to search for mention of the enzyme in the academic and patents literature. -screen for new microbial strains Procedures Location of a sources of enzyme

9. - temperature for optimum productivity and stability - pH optimum and stability - kinetic constants (Km, Vmax) - whether there is substrate or product inhibition - the ability to withstand components of the expected feedstock other than substrate. - select a reactor Procedures Determination of the Properties of Enzyme

10. Various decisions must be made concerning the acceptability of the organism to the regulatory authorities: -the productivity of the organism. -the way in which the enzyme is to be isolated, utilised (free or immobilised) and, if necessary, purified. If the organism is unacceptable from a regulatory viewpoint two options exist; - to eliminate that organism & continue the screening operation. - to use recombinant DNA technology. Procedures Determination of the acceptability of Enzyme

11. The selected strain(s) of microbe will be grown in pilot plant conditions. - achieve accurate costing of processes. -reveal imperfections, or at least areas of ignorance which must be corrected at the laboratory scale. -produce samples of the enzyme preparation to be used by customers. -produces samples for safety and toxicological studies. Protect intellectual property generated by patenting the enzyme or its production method or the process. Procedures Scale up of Production

12. Determination of Enzyme Activity Specific activity: the number of units of enzyme activity per amount of total protein. Unit: the amount of enzyme that gives a predetermined amount of catalytic activity under specific conditions.

13. Determination of Enzyme Activity To measure the amount of glucoamylase in a crude enzyme preparation, 1 ml of the crude enzyme preparation containing 8 mg protein is added to 9 ml of a 4.44% Lintner starch solution. One unit of activity of glucoamylase is defined as the amount of enzyme which produces a µmol of glucose per min in a 4% solution of starch at pH 4.5 and at 60 o C. Initial rate experiments show that the reaction produces 0.6 µmol of glucose/ml-min. What is the specific activity of the crude enzyme preparation?

14. Determination of Enzyme Activity To determine the total amount of glucose produced: 10 ml X 0.6 µmol of glucose/ml-min = 6 µmol of glucose/min = 6 units of activity The specific activity is: 6 units of activity / total protein added = 6 units of activity / (1ml protein solution X8 mg protein/ml) = 0.75 units/mg protein

15. Cost of purification The effect of number of steps on the yield and costs in a typical enzyme purification process. Step Specific activity Total cost Cost per weight Cost per activity 11.001 131.1041.47 291.20192.13 3271.30833.08 4811.403584.92 52431.5015366.32

16. Enzyme Production at a Large Scale Hydrolase: proteases, pectinase, lipase, lactase Isomerases: glucose isomerase Oxidases: glucose oxidase Transferases: Rhodanase

17. Application of Industrial Enzyme Food industrial: Starch saccharification: amylase cheese: rennase cleaves the principal protein of milk and causes milk to curdle and aids digestion. Bread : amylase, protease, hemicellulases. Fruit juice: pectinases to degrade pectins in cell walls of fruits and vegetables Beer: amylase, acetolactate decarboxylase.

18. Constituent Composition (%) Sodium tripolyphosphate (water softener, loosens dirt) a a 38.0 Sodium alkane sulphonate (surfactant)25.0 Sodium perborate tetrahydrate (oxidising agent)25.0 Soap (sodium alkane carboxylates)3.0 Sodium sulphate (filler, water softener)2.5 Sodium carboxymethyl cellulose (dirt-suspending agent) 1.6 Sodium metasilicate (binder, loosens dirt)1.0 Bacillus protease (3% active)0.8 Fluorescent brighteners0.3 Foam-controlling agentsTrace PerfumeTrace Waterto 100% Detergent

19. EnzymeEC numberUse Asparaginase3.5.1.1Leukemia Collagenase3.4.24.3Skin ulcers Glutaminase3.5.1.2Leukemia Lysozyme3.2.1.17Antibiotic Ribonuclease3.1.26.4Antiviral  -Lactamase 3.5.2.6Penicillin allergy Urokinase3.4.21.31Blood clots Rhodanase2.8.1.1Cyanide poisoning Uricase1.7.3.3Gout Medical Application

20. Summary of Enzyme Enzyme classification Enzyme have common catalytical features - decrease the reaction activation energy - does not affect equilibrium Enzyme special catalytic features - Efficient - Specific - regulated - versatile

21. Summary of Simple Saturation Kinetics Michaelis-Menten Approach Briggs-Haldane Approach Use these two approaches to derive enzyme catalytic reaction. Use experimental data to obtain parameters of Michaelis-Menten kinetics.

26. V= K 5 [ES] 2 V= K5K5

27. Estimation of inhibited enzyme kinetics Determine the type of inhibition. Determine the parameters for Michaelis- Menten equation without inhibition. Determine the parameter of KI for inhibited kinetics.

29. Substrate inhibition

36. Summary of Inhibited Kinetics For reversible enzyme inhibition, there are - competitive - noncompetitive - uncompetitive - substrate inhibition Determine parameters for all these types of inhibition kinetics.

37. Estimation of inhibited enzyme kinetics

38. Substrate inhibition

42. Summary of Immobilization Methods Methods of Enzyme immobilization: - Entrapment - matrix - membrane (microencapsulation) - Surface immobilization - physical adsorption - ionic binding - covalent binding - Cross-linking

43. Summary of Diffusion Effects in Immobilized Enzyme System - Determine the support to be non-porous or porous. - Identify the substrate determining the reaction rate. - Conduct mass balance of the substrate of interest. Accumulation of substrate of interest = rate of substrate gain - substrate consumption rate (production formation rate, or reaction rate) At steady state, Rate of substrate gain = substrate consumption rate

44. Summary of Diffusion Effects At steady state, the reaction rate per unit surface area is equal to the rate of net substrate gain in regard to the external diffusion. In surface-bound enzymes on nonporous support materials. Consider external diffusion rate (liquid film mass transfer rate) E+S

45. Diffusion effects in surface-bound enzymes on nonporous support materials. At steady state, the reaction rate is equal to the external diffusion rate: With the equation and known S b, K L, V m ’ or K m, to determine numerically or graphically: - The substrate concentration at the surface. - The reaction rate.

48. Graphical solution for reaction rate per unit of surface area for enzyme immobilized on a non-porous support

49. Diffusion effects in surface-bound enzymes on nonporous support materials. To increase the overall reaction rate with external diffusion limitation -Increase the bulk concentration of substrate. -Increase the liquid film mass transfer coefficient k L.

50. Summary of Diffusion Effects At steady state, the reaction rate per unit volume is equal to the rate of net substrate gain in regard to the intraparticle diffusion. In surface-bound enzymes on porous support materials. Consider intraparticle diffusion rate.

51. is the effectiveness factor. the rate is diffusion limited. the rate is reaction limited. β η Ф η

52. At specific conditions (T, P) for a fixed system, To increase the intra-particle mass transfer rate: - Decrease the size of immobilized enzyme particle - Increase the substrate concentration - Increase the porosity or specific surface area of the particle

53. Electrostatic and Steric Effects in Immobilized Enzyme Systems - The optimum pH for immobilized enzyme system will shift from that of soluble free enzyme Electrostatic effect - The activity of enzyme toward a high-molecule- weight substrate may be reduced. Steric hindrance