Enzyme immobilization

This slide was prepared using the book 'Introduction to Biotechnology' by Giriraj Kulkarni.

1. Amrita Sutradhar B. Pharm, M. Pharm (JU) Lecturer Department of Pharmacy Primeasia University Enzyme Immobilization

2. What is enzyme? Enzymes are biological catalysts which enhance the rate of biochemical reactions from 10 6 - 10 12 times when compared to those of uncatalyzed reactions. All enzymes are proteins and contain an active site where substrates are converted to products. They possess both speed of action and precision.

3. Mechanism of enzyme action In biochemical reaction, the molecules possessing high internal energy can go to a reactive form instantly. This reactive form is called transition state. To reach transition state, all the molecules must possess activation energy. The enzyme can accelerate the reaction by combining with substrate. The substrate-enzyme complex is then converted to products and enzyme is liberated. The transition state of this enzyme substrate complex has much lower activation energy than transition state of substrate alone.

4. Mechanism of enzyme action Due to this reduction in activation energy, more substrate molecules participate in the reaction and hence reaction rate will be increased. The enzyme recognizes substrate which fits into its active site perfectly. Once enzyme-substrate complex is formed, making and breaking of chemical bond occurs which converts the substrates into products and the enzyme is set free. Thus the enzyme and substrate complex can be compared with a lock and key model.

5. Sources of enzymes Enzymes are usually obtained from three major sources- Plants Animals Micro-organisms The advent of fermentation technology led to the preparation of purer form of enzymes from microbes. Today, most of the industrially used enzymes are from microbial source.

6. Advantaged of microbes over other sources of enzymes: Microbes have a short generation time and hence the times required for enzyme production in large quantities is less compared to plants and animals. Enzymes can be easily extracted from microbes. If the enzymes are extracellular, they will be secreted into the medium and can be purified easily. Micro-organisms produce enzymes throughout the year and no seasonal variation is found which is common in case of plants and animals.

7. Advantaged of microbes over other sources of enzymes : 4. The enzymes obtained from microbes are stable under extreme environmental conditions. 5. Micro-organisms can produce a large varieties of enzymes which is not possible with plants and animals. They can be manipulated easily by mutation or by genetic engineering to give high yield of enzymes.

8. Enzyme immobilization Immobilization is defined as the imprisonment of an enzyme in a distinct phase that allows exchange with, but is separated from the bulk phase in which the substrate and product molecules are dispersed. Imprisonment refers to the physical or chemical trapping of the enzyme either onto or into a polymer matrix.

9. Why is enzyme immobilization necessary? Most of the enzymes are expensive and hence they should be used as efficiently as possible. An important function of enzyme is their capacity to perform same function again and again. But if the enzymes are used in pure or crude form, it is not possible to separate them from the products after completion of catalysis since both of them are in dissolved state. If they are separated, the process becomes very costly and difficult. Some enzymes are too costly and the process of enzymatic catalysis becomes cost effective only if they are reused. Hence, immobilization techniques have been developed. The immobilization involves the conversion of water soluble enzymes into a solid form of catalyst which is insoluble.

10. Advantages of immobilized enzymes: Reuse: The same enzyme can be used repeatedly. Continuous use: Continuous production system can be designed easily. Cost effectiveness: If the enzymes are immobilized, they can be used continuously or repeatedly and hence cost effectiveness is achieved. Less contamination: Since the immobilized enzymes remain within the polymer matrix, it will not contaminate the final product.

11. Advantages of immobilized enzymes: 5. Stability: Immobilization increases the thermal stability of the enzymes. For example, immobilized glucose isomerase is stable at 65 C for almost one year whereas the pure enzyme is denatured within few hours at a temperature of 45 C. 6. Process control: Since the immobilized enzymes have a standardized activity, the process control becomes very easy. 7. Enzyme Substrate ratio: It is very high in immobilized enzymes and this increases cost effectiveness.

12. Immobilization ‘in’ a support This technique involves- Entrapment Microencapsulation

13. Entrapment In this technique, the enzyme is entrapped within a cross linked polymer matrix. Enzyme is dissolved in a solution of precursors of the polymer and then polymerization is initiated. The enzyme is physically entrapped within the matrix and it can’t escape by permeation. However, substrate molecules can diffuse in, can be acted upon by the enzyme, and product molecules can diffuse out of the matrix. The common polymers used include- Polyacrylamide gel, Cellulose triacetate, Gelatin etc.

14. Drawbacks of entrapment Immobilization of enzymes by entrapment technique has the following problems- Due to wide pore size distribution in the gel, there will be continuous leakage of enzyme. Since enzyme is bound within the matrix, substrate accessibility is reduced. Some of the enzyme activity is lost or reduced due to free radicals produced during polymerization.

15. Microencapsulation In this method, the enzyme is immobilized by enclosing a droplet of enzyme in a semipermeable membrane capsule. Once encapsulation is done, the enzyme can not escape whereas the low molecular weight substrate and products can diffuse through the membrane. The capsule can be made up of either permanent materials like nylons or biodegradable materials like poly-lactic acid.

16. There are two general methods of encapsulating an enzyme – The membrane are prepared by phase separation, just like emulsification. Both phases are immiscible and form coacervate when mixed. The enzyme is entrapped in this coacervate. Water insoluble membrane can be prepared by chemical polymerization and the enzyme can be entrapped during polymerization.

17. Immobilization ‘On’ a support Three techniques- Adsorption Covalent binding Cross-linking

18. Adsorption The adsorption occurs due to nonspecific bonding like electrostatic, hydrophobic or affinity bonding to a specific ligand. Some adsorbents used in this technique are Carboxy methyl Cellulose (CMC), Silica gel etc. During immobilization the enzyme concentration and P H should be controlled carefully because a change in P H may lead to desorption.

19. Adsorption In the process of immobilization by adsorption, the adsorbent is packed in a water jacketed column and washed with a preconditioning solution. The enzyme solution is prepared and buffered fairly. Such solution is then circulated through the column for several hours. Then the solution is drained and column is washed with water, then with 0.5 M NaCl and again with water. The immobilized enzyme column is now ready for use.

20. Covalent binding In this process the enzyme forms a covalent bond with the active groups on a polymer support. This can be done by two ways- Through the reactive groups on the side chains of its amino acids such as lysine, arginine and tyrosine. Through the terminal amino and carboxyl groups of polypeptide chains.

21. Cross linking Three techniques are used for immobilization of enzymes by cross linking- Cross linking of the enzyme with gluteraldehyde to form an insoluble aggregate. Adsorption of the enzyme onto a surface followed by cross linking. For example, the enzyme can be cross linked and adsorbed onto the surface of particles like colloidal silica. Impregnation of porous matrix materials with the enzyme and cross linking of the enzyme within the pores. The common porous matrix used is collodion membrane.

22. The enzyme applications are broadly classified into four categories- Medical use Analytical use Manipulative use Industrial use

23. Medical use: Treatment of leukemia can be done by administering asparaginase from bacteria. Leukemic cell require exogenous asparagine for their growth and this will be destroyed by the enzyme. Lysozyme, a protective body fluid e.g. nasal mucosal fluid, lacrimal fluid etc. is used as antibacterial agent. Hyaluronidase, an enzyme is used in dental work to facilitate drug penetration. Penicillinase is used to stop Penicillin hypersensitivity. Glucose oxidase is used to diagnose the presence of glucose in urine.

24. Analytical use: Enzymes can be used to analyze the blood concentration of D-glucose, D-alanine, D- galactose , Urea etc.

25. Manipulative use Restriction enzymes are used in recombinant DNA technology to cut DNA. DNA ligase is used to join foreign genes to plasmid DNA. Alkaline phosphatase is used to prevent the recirculation of cut plasmid. Reverse transcriptase is used to produce DNA from RNA. DNA Polymerase is used in the formation of DNA

26. Industrial use Invertase is used to convert glucose to fructose. Penicillin acylase is used to produce semisynthetic Penicillin. Proteolytic enzymes are used to soften skin in leather industry.

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