2.Introduction to Enzyme biotechnology.pptx

To give knowledge about enzyme biotechnology.

1. Enzyme biotechnology Prepared by: Ms. Harshada R. Bafna. M. Pharm (Quality Assurances) 1

2. Enzymes are soluble, amorphous, colloidal, proteinases, bioactive organic catalyst products by living cell. Enzymes are mainly classified as extracellular enzymes (exoenzyme) and intracellular enzymes (Endoenzyme). Exoenzymes are secreted outside the cell such as cellulose, polyglucturonase, pectinmethylesterase etc. Endoenzymes are isolated by breaking the cells by means of a homogenizer or a bead mill and extracting them through the biochemical processes. 2

3. Enzymes- Biological catalysts- promote chemical reactions in living organisms Have the ability catalyze reactions under very mild conditions with high degree of substrate specificity thus decreasing the formation of by products. “ Immobilized enzyme ” 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” Immobilization refers to restricting the mobility of an enzyme or protein and fixing it into a state without disturbing its functional ability . An immobilized enzyme is fixed to an inert , carrier that not only allows the exchange of substrate and product outflow but also restricts the changes in enzyme conformation due to change in pH or temperature. 3

4. Advantages of immobilized enzymes: Increase functional efficiency of enzyme Enhanced reproducibility of the process Continuous use of enzyme Minimum reaction time More stability of product Less chance of contamination in product Less labor input in process improved process control 4

5. The use of enzymes in industrial applications is limited because most of enzymes are relatively unstable after immobilization. High cost of isolation, purification and recovery of active enzymes from the reaction mixtures after the completion of catalytic process. Enzymes are inactivated by the heat generated in the system Disadvantages 5

6. Carrier matrix: The materials used for immobilization of enzymes are inert matrices and are polymer in nature. they should have following properties : Cost effective Inert Good physical strength Reduced product inhibition Reduction in non specific adsorption Reduction in microbial contamination Carrier matrix: Enhancement in specificity of an enzyme Renewability of an immobilized enzyme Adequate stability 6

7. Methods of enzyme immobilization Immobilization methods Surface Immobilization Within Surface Immobilization Adsorption Covalent Bonding Complexation Encapsulation Entrapment 7

8. Adsorption: It is the simplest and oldest method. It was first used by Nelson and Griffin in 1916 when they immobilized Invertase on an activated charcoal . Enzyme is adsorbed on the physical outer surface of the support. Being a nonchemical method it can affect the functional ability of enzyme by blocking its active / modulating site. Weak bond or interactions (Hydrogen bond, Vander wall forces) play role in stabilizing the enzyme to carrier. Carriers used in adsorption can be Mineral based support : Aluminum oxide or clay, alginate beads Organic Bimolecular based support : Starch , Cellulose Modified ion exchange resin : Sepharose 1. Adsorption 8

9. Enzymes: Invertase , Catalase, glucose oxidase, Alpha- amylase Methods for adsorbing enzymes on carrier surface: Static Method: In this method the carrier is just dipped and left in the enzyme solution without any stirring. Dynamic Method: In this method carrier is placed in the enzyme solution and stirring is maintained to load the enzyme on carrier surface. Reactor loading Method: In this method the carrier is placed in reactor and enzyme solution is loaded with continuous stirring. Electrode position Method: In this method carrier is put in vicinity of an electrode. Now the enzymes migrate to carrier in presence of electric current. 9

10. ADVANTAGES Simple and economical method Limited loss of activity. No reagent required Can be Recycled, Regenerated & Reused. DISADVANTAGES Relatively low surface area for binding. Exposure of enzyme to microbial attack. Disturbance of the enzyme resulting from changes in temperature, pH, and ionic strength. 1. Adsorption 10

11. Carriers used in Covalent Bonding are: Biomolecules : Carbohydrates are commonly used as a carrier like Cellulose, DEAE Cellulose etc. Synthetic Molecules : Synthetic molecules like polyacrylamide are used having good chemical reactivity to form bonds. Protein carriers: Protein carriers like Collagen, gelatin are also used. 2. Covalent Bonding: Covalent Bonding: As the name suggests this method utilizes chemical groups present on both enzyme and carrier for immobilization. A Covalent bond is formed in between chemical groups of enzyme and carrier . This is one of the most widely used methods for enzyme immobilization. Hydroxyl groups and amino group of support or enzyme from covalent bonds more easily. 11

12. Enzyme Carrier Matrix Cellulase , Pectinase, Glucose Isomerase Polyurethane Amino Group Alpha Carboxyl At C Terminal Methods of Covalent Bonding are: Diazoation: this reaction occurs between amino group of the carrier and Tyrosil and Histidyl group of the enzyme. Peptide Bond: this is the most common bond formed between amino acids and carri er. It occurs in amino and carboxyl groups of enzyme and carrier . Poly functional Agent: In this method a multifunctional agent like Gluteraldehyde is used to form bond between amino group of enzyme and carrier . 12

13. Advantages: The strength of binding is very strong, so leakage of enzymes from the support is absent or very little. This is a simple, mild and often successful method of wide applicability Enzymes are chemically modified and so many are denatured during immobilization. Only small amounts of enzymes may be immobilized (about 20 mg per gram of matrix). Disadvantages: 13

14. Entrapment: In this method an enzyme is entrapped within a porous matrix . Bonds involved can be covalent or non-covalent in nature . The matrix used is hydrophilic in nature . Type of polymer varies with the type of enzyme and its application. Pore size is determined on the basis of the size of enzyme so that it does not leak out of the matrix. Matrices used in entrapment method are: Polyacrylamide gels, Agar, Gelatin, Alginate. 1. Inclusion in gels: In this method enzyme is trapped inside the gel . Gel is formed by the polymer. Pore size of the gel is smaller than that of the enzyme so that there is no leakage. Pore size depends on the concentration of polymer used. Eg : Poly acrylamide gel, Poly vinyl alcohol gels. 3. Entrapment: 14

15. 2. In fibers: In this method the enzymes are supported on the fibers of the supporting material forming the matrix. The fibers are skeleton of the matrix in which enzyme is trapped. Eg : Cellulose and Poly -acryl amide gels. If the gel pore size is not proper then it may lead to leak of enzyme Low substrate accessibility of respective enzyme Disadvantages: 15

16. Encapsulation: In this method an enzyme is encapsulated within a capsule made up of semi permeable membranes like nitro celluloses, nylon, hemi cellulosic structures etc. In this method the effectiveness depends on the stability of the enzyme inside the capsule...3 D conformation is maintained inside the capsule. Advantages: Faster and cheaper method Large amount of enzymes can be encapsulated at a time. Disadvantages: Pore size limitation Size of substrate and product a limitation in exchange through membrane. 4. Encapsulation: 16

17. This entrapment involves the formation of spherical particle called as “microcapsule” in which a liquid or suspension of biocatalyst is enclosed within a semi permeable polymeric membrane. Microcapsules: In this microcapsules are formed in which enzymes are entrapped. Most commonly used polymers in microcapsules are polyamines and sodium alginate. Microencapsulation: 17

18. Advantages: Faster and cheaper method Limited or no conformational change of enzyme Mild conditions are required Easy at small scale application Disadvantages: Leakage of enzyme Pore diffusion is a limitation both for substrate and product Microbial contamination Limited to small scale operations 18

19. Cross linking: As the name suggests enzymes are immobilized in the cross-linked matrix formed by the polymer. There is no any carrier or surface molecule ligand for attachment present in this method. This method is also called as copolymerization. Covalent bonds are formed in between poly functional agents and enzyme . Active site should not be covered in cross linking otherwise the activity will be reduced. 5. Cross linking: 19

20. Various polymers used for cross linking enzymes are: Gluteraldehye Diazonium salts Advantages: Faster and cheaper method Simpler process Disadvantages: Structural modification Risk of denaturation by poly functional agents 20

21. Industrial production : Widely used in the commercial production of antibiotics, beverages, amino acids and secondary metabolites etc. of industrial grade. Biomedical applications: Immobilized enzymes are most commonly used in the fast diagnostic kits like ELISA and treatment of many pathogenic diseases. Food industry: Enzymes like Pectinases and Celluloses, amylases are immobilized on suitable carriers or matrices and are successfully used in the commercial production of jams, jellies and syrups from fruits and vegetables. E.g. Lactase is immobilized on cellulose fibers and produce lactose free milk from milk and whey. Large Scale Production of bio-diesel from vegetable oils using bioreactors. Applications of Enzyme Immobilization: 21

22. Waste water management: In Treatment of sewage and industrial effluents using packed bed reactors . Research: A research activity extensively uses many enzymes. Eg : Horse radish peroxidase in blotting experiments and different proteases for cell and organelle. Textile industry: S ouring, polishing and desizing of fabric Detergent industry: immobilization of lipase enzyme for effective dirt removal from cloths. 22

23. Enzyme kinetics specifically refers to the in-depth study of ‘enzymes in action’. The abnormally high inherent rate of the induced enzyme-catalyzed reactions enormously obviates and facilitates this study Factors affecting on enzyme activity: Concentration of enzyme: Concentration of substrate: Effect of temperature: Effect of pH: Effect of product concentration: Effect of activators: Factors affecting on enzyme activity: 23

24. Factors affecting Immobilized enzyme kinetics: Concentration of enzyme: Velocity of enzymatic reactions is enzyme conc. Concentration of substrate: The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached. The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present. Most enzymes show Michaelis-Menten kinetics, in which the plot of initial reaction velocity ( vo ) against substrate concentration ([S]), is hyperbolic. 24

25. ↓ of velocity with higher T. However, heat energy can also increase the kinetic energy of the enzyme to a point that exceeds the energy barrier for disrupting the noncovalent interactions that maintain its three dimensional structure. Enzymes from humans generally exhibit stability at temperatures up to 35-40°C. By contrast, enzymes from the thermophilic microorganisms that reside in volcanic hot springs or undersea hydrothermal vents may be stable up to or even above 100°C Effect of temperature: Raising the temperature increases the rate of both uncatalyzed and enzyme-catalyzed reactions by increasing the kinetic energy and the collision frequency of the reacting molecules. 25

26. Effect of pH: The rate of almost all enzyme-catalyzed reactions exhibits a significant dependence on hydrogen ion concentration. Most intracellular enzymes exhibit optimal activity at pH values between 5-9. For enzymes whose mechanism involves acid-base catalysis, the residues involved must be in the appropriate state of protonation for the reaction to proceed. The binding and recognition of substrate molecules with dissociable groups also typically involves the formation of salt bridges with the enzyme. Effect of product concentration: Product formed as result of enzymatic reaction may accumulate & may lower the enzymatic reaction by occupying active site of E. Under certain conditions of high conc. of P a reverse reaction may be favored from P→S. 26

27. Effect of activator: some enzyme need inorganic metallic catons like Mg 2+, Mn2+, Zn2+,Ca2+, Co2+, ect. For their optimum activity. Anions are also needed for enzyme activity rarely Eg.: Cloride ion, for amylase. Plant cell immobilization: Immobilization of cells refers to the technique of confining the cells in or on an inert support for their stability and functional reuse . By employing this technique, cells are made more efficient and cost-effective for their industrial use. Immobilization of plant cells would be one method of increasing productivity and hence reducing the costs . Immobilization is the newest culture technology of plant cell, and considered as to be the most “natural 27

28. NEED FOR IMMOBILIZATION: Protection from degradation and deactivation. Retention of cell. Cost efficiency. Enhanced stability. Allows development of multi-enzyme reaction Immobilization of plant cell would be one method of increasing productivity and hence reducing the cost. 28

29. Advantages of Plant Cell Immobilization: Retention of biomass enables its continuous reutilization as a production system. Separation of cells from medium: Immobilization allows a continuous process, which increase volumetric productivity and allows the removal of metabolic inhibitors. The immobilization reduces some of the physical problems associated with the cultivation of plant cells 29

30. Disadvantage of Plant Cell Immobilization: Secretion of secondary metabolites requires cellular transport or artificially altered membrane permeability. The immobilization process may reduce biosynthetic capacity. Products must be released from the cell into medium. Release of single cells from cell aggregate may make processing of the product more difficult.  Immobilization is an expensive affair often requiring sophisticated equipment. The possibility of loss of biological activity of an enzyme during immobilization or while it is in use. 30