1. Pharmaceutical Biotechnology PHR 403 Chapter 3: Enzyme Technology

2. Learning objectives: In this chapter, students will learn about-  Enzymes  commercial uses of enzyme  immobilized enzyme technology and reactors  application of immobilized enzymes in manufacturing and preparation of different biopharmaceuticals.  Other uses of enzyme technology In this chapter, students will learn about-  Enzymes  commercial uses of enzyme  immobilized enzyme technology and reactors  application of immobilized enzymes in manufacturing and preparation of different biopharmaceuticals.  Other uses of enzyme technology

3. Sources Animal Lipases Tripsin Rennets Plant Papain Proteases Amylase Soyabean lipoxygenase Microbial Extracellular enzyme Cellulase Plymethylgalacturonase Polygalacturonase Pectimethylesterase Intracellular enzyme Invertase Uric oxidase Asparaginase

4. Comparsion Animal/ Plant Vs. Microbial enzyme Microbial enzymes have 2 advantages over animal and plant enzymes- Firstly:  Economical, can be produced on large scale, within the limited space and time.  Easy to extract and purify Secondly:  They are capable of producing large variety of enzyme  Can grow in wide range of environment.  Show genetic flexibility  Short generation times Microbial enzymes have 2 advantages over animal and plant enzymes- Firstly:  Economical, can be produced on large scale, within the limited space and time.  Easy to extract and purify Secondly:  They are capable of producing large variety of enzyme  Can grow in wide range of environment.  Show genetic flexibility  Short generation times

5. Historical perspective 1913 Enzyme containing detergent was developed But it was less stable 1965 New stable enzyme protease was introduced 1970 First commercial process was used to produce fructose from glucose by isomerization Present 2000 enzymes have been isolated 1000 enzymes are recommended for various applications. 50 microbial enzymes has industrial applications.

6. Properties of enzyme  Presence of species specificity  Macromolecule differ in different species  Phylogenetic variations gave rise to variation in different macromolecules  Example: Protease of 2 closely related microorganisms may be related in some properties but differs in many other properties.  Variations in activity and ability  Most microbial enzymes are extracellular  Extracellular enzyme are influenced by temperature, pH etc.  Optimum environment for extracellular enzyme correlates with the optimum growth environment for the bacteria. Bacillus coagulans (Thermophillic) has different amylase activity than Bacillus licheniformis (Mesophillic)  Intracellular enzymes are not that much influenced by environment  Optimum temperature and pH  Presence of species specificity  Macromolecule differ in different species  Phylogenetic variations gave rise to variation in different macromolecules  Example: Protease of 2 closely related microorganisms may be related in some properties but differs in many other properties.  Variations in activity and ability  Most microbial enzymes are extracellular  Extracellular enzyme are influenced by temperature, pH etc.  Optimum environment for extracellular enzyme correlates with the optimum growth environment for the bacteria. Bacillus coagulans (Thermophillic) has different amylase activity than Bacillus licheniformis (Mesophillic)  Intracellular enzymes are not that much influenced by environment  Optimum temperature and pH

7. Properties of enzyme  The stability of the enzyme is also increase by many factors, such as: a)High concentration of respective enzyme b)Presence of their substrate c)Presence of ion. eg.Ca 2+ d)Reduced amount of water content  Substrate specificity  Activation and inhibition  Same enzymes isolated from diferent sources show different responses. i.e., β- galactosidase from fungal origin does not require Cobalt, where the same enzyme from bacterial origin needs that.  The stability of the enzyme is also increase by many factors, such as: a)High concentration of respective enzyme b)Presence of their substrate c)Presence of ion. eg.Ca 2+ d)Reduced amount of water content  Substrate specificity  Activation and inhibition  Same enzymes isolated from diferent sources show different responses. i.e., β- galactosidase from fungal origin does not require Cobalt, where the same enzyme from bacterial origin needs that.

8. Methods of enzyme production Isolation of microorganism, strain development and preparation of inoculums Medium formulation and preparation Sterilization of medium, maintenance of culture and fluid filtrations Purification of enzyme

9. Isolation of microorganism, strain development, and preparation of inoculum Aim: 1. Production of enzyme in high amount 2. Completion of fermentation process in short time 3. Maximum utilization of culture medium in low cost. Aim: 1. Production of enzyme in high amount 2. Completion of fermentation process in short time 3. Maximum utilization of culture medium in low cost.

10. Medium formulation and preparation Carbohydrate sources: Molases, Barley, Corn, wheat and starch hydrolase Protein: Meals of Soybean, Cotton seed, peanut and whey, corn steep liquor and yeast hydrolysate. Carbohydrate sources: Molases, Barley, Corn, wheat and starch hydrolase Protein: Meals of Soybean, Cotton seed, peanut and whey, corn steep liquor and yeast hydrolysate.

11. Sterilization Continuous sterilization Surface culture Vs Submerged culture Anti-foaming agents. Eg: Oils Continuous sterilization Surface culture Vs Submerged culture Anti-foaming agents. Eg: Oils

12. Purification Preparation of concentrated solution Clarification of concentrated enzyme Addition of preservatives Precipitation of enzyme Drying Packaging Preparation of concentrated solution Clarification of concentrated enzyme Addition of preservatives Precipitation of enzyme Drying Packaging

13. Enzyme immobilization technology Enzymes can increase the rate of reaction many folds. But, majority of the enzyme are fairly unstable Industrial application is often hampered by a lack of long-term operational stability and the technically challenging recovery process and reuse of the enzyme. In order to make enzyme utilization in biotechnological processes more favourable, different methods for cost reduction have been put into practice and, immobilization is one of them Enzymes can increase the rate of reaction many folds. But, majority of the enzyme are fairly unstable Industrial application is often hampered by a lack of long-term operational stability and the technically challenging recovery process and reuse of the enzyme. In order to make enzyme utilization in biotechnological processes more favourable, different methods for cost reduction have been put into practice and, immobilization is one of them

14. Immobilized enzyme The term ‘immobilized enzymes’ refers to ‘enzymes physically confined or localized in a certain defined region of space.  with retention of their catalytic activities  which can be used repeatedly and continuously substantially simplifies the manipulation with the biocatalyst and the control of the reaction process while enhancing the stability of the enzyme under both storage and operational conditions. The term ‘immobilized enzymes’ refers to ‘enzymes physically confined or localized in a certain defined region of space.  with retention of their catalytic activities  which can be used repeatedly and continuously substantially simplifies the manipulation with the biocatalyst and the control of the reaction process while enhancing the stability of the enzyme under both storage and operational conditions.

15. What is enzyme immobilization?  The term “immobilized enzymes” refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously.”  It is a process of confining the enzyme molecules to a solid support over which a substrate is passed and converted to products.  The major components of an immobilized enzyme system are- enzyme, matrix, and the mode of attachment.

16. Continued…  The enzymes can be attached to the support by interactions ranging from- reversible physical adsorption and ionic linkages to stable covalent bonds.  However, as a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered.

17.  Besides the application in industrial processes, the immobilization techniques are the basis for making a number of biotechnological products with applications in- diagnostics, bioaffinity chromatography, and biosensors.Continued…

18. Choice of Support  The characteristics of the matrix are of paramount importance in determining the performance of the immobilized enzyme system.  Ideal support properties include- physical resistance to compression, hydrophilicity, inertness toward enzymes, ease of derivatization, biocompatibility, resistance to microbial attack, and availability at low cost

19. Classification of Support  According to their chemical composition, supports can be classified as- inorganic and organic.  The organic supports can be subdivided into- natural and synthetic polymers

20. Classification of Support Organic Natural polymers: Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate Proteins: collagen, albumin Carbon Synthetic polymers: Polystyrene Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides, vinyl, and allyl-polymers

21. Continued… Inorganic Natural minerals: bentonite, silica Processed materials: glass (nonporous and controlled pore), metals, controlled pore metal oxides.

22. Immobilization criteria  There are a number of requirements to achieve a successful immobilization: The biological component must retain substantial biological activity after attachment It must have a long-term stability The sensitivity of the enzyme must be preserved after attachment Overloading can block or inactivate the active site of the immobilized biomaterial, therefore it must be avoided

23. Methods of enzyme immobilization  Irreversible enzyme Immobilization 1. Covalent bond formation 2. Entrapment  Reversible enzyme Immobilization 1. Adsorption a) Non-specific adsorption b) Ionic binding c) Hydrophobic adsorption d) Affinity binding 2. Chelation or metal binding 3. Formation of disulfide bond

24. Methods of enzyme immobilization Alternatively it can be classified as- 1.Immobilization ‘inside’ a support 2.Immobilization ‘on’ a support 1.Immobilization ‘inside’ a support a. Entrapment b. Microencapsulation 2. Immobilization ‘on’ a support a. Adsorption b. Covalent bonding c. Cross linking

25. Advantages of immobilized enzyme  Reuse  Continuous use  Less labour intensive  Saving in capital cost  Minimum reaction time  Less chance of contamination of products  More stability  Improved process control and  High enzyme : substrate ratio.  Reuse  Continuous use  Less labour intensive  Saving in capital cost  Minimum reaction time  Less chance of contamination of products  More stability  Improved process control and  High enzyme : substrate ratio.

28. 1. Adsorption Schematics of the three most common enzyme immobilization techniques: (A) physical adsorption, (B) entrapment and (C) covalent attachment/cross-linking

29. 1. Adsorption Enzyme is immbolized by bonding to either the external or internal surface of a career or support such as  Mineral support (Aluminium oxide, Clay)  Organic support ( Starch)  Modified sapharose and ion exchange resins. Bonds with low energy is involved (ionic, Hydrogen, van der waals forces) For external immobilization the carrier particle must be very small (500 Å- 1mm) Enzyme is immbolized by bonding to either the external or internal surface of a career or support such as  Mineral support (Aluminium oxide, Clay)  Organic support ( Starch)  Modified sapharose and ion exchange resins. Bonds with low energy is involved (ionic, Hydrogen, van der waals forces) For external immobilization the carrier particle must be very small (500 Å- 1mm)

30. 1. Adsorption Processes of immobilization using adsorption- 1.Static process: Enzyme is immobilized on the carrier simply by allowing the solution containing the enzyme to contact the carrier without stirring 2.The dynamic batch process: Carrier is placed into the enzyme solution and mixed by stirring or agitation 3.The reactor loading process: Carrier is placed into the reactor that will be subsequently employed for processing. The enzyme solution is transferred to the reactor and adsorption occurs in a dynamic environment. 4.The electrodeposition process: Carrier is placed proximal to one of the electrodes in enzyme bath, the current put on, enzyme migrates to the carrier and deposited on the surface. Processes of immobilization using adsorption- 1.Static process: Enzyme is immobilized on the carrier simply by allowing the solution containing the enzyme to contact the carrier without stirring 2.The dynamic batch process: Carrier is placed into the enzyme solution and mixed by stirring or agitation 3.The reactor loading process: Carrier is placed into the reactor that will be subsequently employed for processing. The enzyme solution is transferred to the reactor and adsorption occurs in a dynamic environment. 4.The electrodeposition process: Carrier is placed proximal to one of the electrodes in enzyme bath, the current put on, enzyme migrates to the carrier and deposited on the surface.

31. 1. Adsorption (Non-covalent Interactions) (a) Nonspecific Adsorption  The simplest immobilization method is nonspecific adsorption, which is mainly based on physical adsorption or ionic binding.  In physical adsorption the enzymes are attached to the matrix through- hydrogen bonding, van der Waals forces, or hydrophobic interactions Electrostatic interaction  Whereas in ionic bonding the enzymes are bound through salt linkages.

32. 1. Adsorption (Non-covalent Interactions)  Immobilization by adsorption is – mild, easy to perform process, and usually preserves the catalytic activity of the enzyme. Therefore, economically attractive,  But may suffer from problems such as enzyme leakage from matrix when the interactions are relatively weak.

33. 1. Adsorption (Non-covalent Interactions) Advantages: i. Simple and economical. ii. The weak interactions involved, hardly cause any distortion of the enzyme retaining maximum enzyme activity iii. Can be recycled, regenerated and reused. Disadvantages: i. Relatively low surface area for binding. ii. Exposure of enzyme to microbial attack iii. Yield are often low due to the inactivation and desorption. Use: i. Vinegar production: Adsorption of Acetobacter sp. onto the Wood chips ii. Manufacture of malted beverage

34. 1. Adsorption (Non-covalent Interactions) (b) Ionic Binding  Based on principles used in chromatography- the protein– ligand interactions.  For example, one of the first applications of chromatographic principles in the reversible immobilization of enzymes was the use of ion-exchangers.  The method is simple and reversible.  But it is difficult to find conditions under which the enzyme remains both strongly bound and fully active.

35. 1. Adsorption (Non-covalent Interactions) (c) Hydrophobic Adsorption  Another approach is the use of hydrophobic interactions.  In this method, it is not the formation of chemical bonds but rather an entropically driven interaction that takes place.  The strength of interaction relies on both the hydrophobicity of the adsorbent and the protein. (d) Affinity Binding  The principle of affinity between complementary biomolecules has been applied to enzyme immobilization.  The remarkable selectivity of the interaction is a major benefit of the method.  But it is cost-ineffective.