Enzyme Biotechnology Methods of enzyme immobilization and applications
“Confining the enzyme molecules or cells to a distinct phase from the one wherein the substrates and the products are present.” i.e. the enzymes are immobilized to convert the substrate (starting material) into the final product and separate both.
- Enzymes produced by micro-organisms can be isolated and used for the synthesis of different products.
- Cells and enzymes are used for carrying out various biochemical reactions like esterase enzyme are required for esterification, amylase for a breakdown of starch, protease for digestion of protein, etc.
- Various reactions are carried out using specific enzymes for specific reactions. E.g. Lipase enzymes are used for synthesizing esters from acid and alcohol as reactants. Esters which are obtained using lipase enzymes are in very pure form.
- The pure isomer is obtained using specific enzymes.
- Enzymes and cells are used for synthesizing drugs and drug intermediates.
- In chemical synthesis, racemic mixtures are formed and in some drugs the inactive form is toxic.
- When chemical methods are used, the racemic mixture is separated i.e. the active drug is separated from an inactive drug.
- The reaction is very stereo-specific i.e. the enzyme will attach the functional group at the proper location.
- The final product obtained is in a pure form, and the one formed by the synthetic process is racemic.
- The yield obtained is more compared to chemical synthesis.
- Enzymatic reactions are carried out in mild conditions such as low temperature, and less toxic chemicals.
- Enzymes can be re-used whereas chemicals can’t be re-used. Eco-friendly reactions if the enzymes are immobilized.
- Time-consuming process.
- It makes the drug costlier.
- Sometimes there are errors in the purification/isolation and the inactive toxic form remains in the final product and causes serious side effects.
DIFFERENCE BETWEEN ENZYMATIC SYNTHESIS AND CHEMICAL SYNTHESIS
METHODS USED FOR ENZYME IMMOBILIZATION
- Covalent binding
Adsorption is an easy technique, in this technique, the enzymes are adsorbed.
Enzymes are adsorbed onto a polymeric matrix.
Adsorption occurs due to electrostatic, hydrophobic or affinity binding.
During adsorption, pH should be controlled carefully because the change in pH may lead to desorption.
If the enzyme is released from the substrate, it gets mixed with the product and cannot be reused. Also, a purification step is then required.
Adsorbents used for enzyme immobilization are:
- CMC ( carboxy methyl cellulose )
- Silica gel
- DEAE cellulose (diethyl amino ethyl)
- Ion exchange resins
The amino groups on the enzyme form a covalent bond with the active groups on polymer support which is used to immobilize the enzymes
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.
Carrier matrix used:
- CM- Sephadex
- Porous glass, etc.
In this method, enzymes are entrapped within the matrix (gelatin) and cannot escape permeation.
Enzymes are entrapped in such a way that only product and substrate can diffuse through the pores of the matrix. The substrate enters the matrix, reacts with the enzyme and then the final product diffuses out of the matrix. So pure products can be obtained.
The enzymes and the polymeric substance remain immobilized.
Commonly used polymers are:
- Starch, etc.
The enzymes are confined into the capsule.
The capsules are made of permanent materials like nylon, collodion, etc.
Biodegradable materials like polylactic acid or phospholipid liposomes are also used.
There are pores in the capsule, the pore size is adjusted by changing the concentration of ingredients used for manufacturing of capsules.
Through these pores, the substrate can enter inside the capsule and interact with the enzyme and the product will be formed.
The product will come out through the pores of the capsule.
The final product obtained is pure and enzyme-free.
COMPARISON OF DIFFERENT METHODS OF IMMOBILIZATION
|2. Enzyme leakage||yes||no||yes||no|
|3. Binding force||variable||strong||weak||strong|
|4. Matrix effects||yes||yes||yes||no|
|5. Method of preparation||simple||difficult||difficult||simple|
|6. Applicability||wide||selective||wide||very wide|
|7. Large diffusional barriers||no||no||yes||yes|
|8. Running problem||high||low||high||high|
|9. Microbial protection||no||no||yes||yes|
APPLICATIONS OF ENZYME IMMOBILIZATION
Biosensors – Electrochemical sensors, glucose sensors, etc
Enzyme reactors – Batch reactors, continuous flow reactors, etc
Application of Immuno-adsorption technique – Immobilized enzyme-based immunoassay, immobilization based novel drug delivery system.
Analytical – Immobilization in affinity purification, affinity chromatography and purification.
Medical – Detoxification following a drug overdose.
Pharmaceutical – Selective hydrolysis of penicillin G, steroid modification, production of monoclonal antibodies, animal vaccines, etc.
SIMPLIFIED CONCEPTS OF BIOTECHNOLOGY By, Dr. Pramod Kadu & Ms. Suchita Vishwakarma
Third Year B Pharm Notes, Syllabus, Books, PDF Subjectwise/Topicwise