Enzyme biotechnology
Enzymes (biocatalyst) are the functional proteins which facilitated the execution of biochemical reactions. Enzymes provide suitable environment for the normal functioning, growth and proliferation of any living system. The enzymes work in mild temperature, pressure, pH, substrates specificity under suitable reaction conditions and for the production of desired products without any contaminations. Enzymes are used in variety of industries such as cosmetics, paper industry, textile industry, food industry, pharmaceutical industry, detergents, etc. Enzyme biotechnology refers to the application of enzymes in various processes for practical and industrial processes. In this article we will see about enzyme biotechnology, methods of enzyme immobilisations and its applications.
Enzyme immobilisation
Enzyme immobilisation is a process in which enzyme makes use of carrier phase for safe homing. Immobilisation of enzyme is defined as imprisonment of cell or enzyme in a support or matrix. The support or matrix on which enzymes are immobilised allows the exchange of medium containing substrate, effector or inhibitor molecules. Enzyme immobilisation technique overcomes the problem of low stability and high sensitivity. The first immobilised enzyme was amino acylase of aspergillus oryzae for the production of L-amino acids in Japan.
Advantages of immobilised enzymes
- Increased functional efficiency of enzymes
- Enhanced reproducibility of the biochemical reaction
- Reuse of enzyme
- Minimum reaction time
- Less chance of contamination in the final products
- More stability of products
- Improved process control with high quality enzyme
- Saving in cost and investment of process
Disadvantages of immobilised enzymes
- High cost for the isolation, purification and recovery of active enzyme
- Limited industrial application
- Catalytic properties of some enzymes are reduced or completely lost after their immobilisation on support carrier
- Some enzymes become unstable after immobilisation
- Enzymes become inactive due to heat generation
Matrix used in enzyme immobilisation
The matrix or support medium immobilises the enzyme by holding it permanently or temporally for a brief period of time. There are wide variety of matrixes or carriers or support medium available for immobilisation. The matrix used should be cheap and easily available. Their reaction with the components of medium or with enzyme should be minimum. The matrix can be classified into three major categories,
- Natural polymers: Alginate, chitosan, chitin, collagen, cellulose, gelatin, starch, pectin, etc.
- Synthetic polymers: Diethylaminoethyl (DEAE) cellulose, polyvinyl chloride (PVC), UV activated polyethylene glycol (PEG).
- Inorganic materials: Zeolites, ceramics, silica, glass, charcoal, activate carbon, etc.
Methods of enzyme immobilisation
Based upon the matrix type of bonds, there are five different method of enzyme immobilisation or whole cells.
- Adsorption
- Covalent bonding
- Entrapment
- Copolymerization or cross linking
- Encapsulation
Adsorption
It is the easiest and oldest techniques of enzyme immobilisation. The interaction between enzyme and surface of matrix is through weak forces by salt linkage, hydrogen bonds, hydrophobic bonds, ionic bonds and Vander wall forces. Materials used for adsorption are activated charcoal, alumina and ion exchange resins. The advantage of enzyme adsorption is minimum activation hence no reagents are required. It is cheap and easy way of enzyme immobilisation.
Methods of adsorption
- Static process: In the method enzyme immobilisation is done by allowing the solution containing enzyme to contact the carrier without stirring.
- Dynamic batch process: Carrier is placed in the enzyme solution and mixed by agitation or stirring.
- Reactor loading process: Carrier is placed in the reactor and then enzyme solution is added with continuous agitation.
- Electrode position process: Carrier is placed near end electrode in the enzyme bath and current is put on. Under the electric field, the enzyme migrates to the carrier and deposits on surface.
Covalent bonding
It involves the formation of covalent bonds between functional group of enzymes with the matrix. It is the widely use method of enzyme immobilisation. Hydroxyl group and amino groups of enzyme forms covalent bonds with matrix easily. Other functional groups are imino group, carboxyl group, thiol group, imidazole group and phenol rings.
Important functional groups of enzymes
- Alpha carboxyl group (C-terminal)
- Alpha amino group (N-terminal)
- Epsilon amino group of Lysine and Arginine
- Beta and gamma carboxyl group of Aspartate and Glutamate
- Phenol group of Tyrosine
- Thiol group of Cysteine
Carrier support commonly used for covalent bonding
| Carrier or matrix | Example |
| Carbohydrates | Cellulose, DEAE cellulose, agarose (C-terminal) |
| Synthetic agents | Polyacrylamide |
| Protein carriers | Collagen, gelatin |
| Amino group | Amino benzyl cellulose |
| Inorganic carriers | Porous glass, silica |
| Cyanide group | Cyanogen bromide agarose, cyanogen bromide, Sepharose |
Methods of covalent bonding
- Diazotization: In this method amino group of the matrix forms covalent bonding with tyrosine or histidine of enzyme.
- Peptide bonds: It is the bonding between amino or carboxyl group of the enzyme with the matrix.
- Polyfunctional reagents: In this method bi-functional or multi-functional reagent (glutaraldehyde) which forms covalent bonds between amino group of matrix and amino group of enzymes.
Covalent bonding method of enzyme immobilisation forms the strong linkage between enzyme and matrix. There is no leakage and desorption problem. Disadvantage of this method is chemical modification leads to confirmational changes in enzyme.
Entrapment
In this method enzymes are entrapped inside the porous matrix, which are stabilised by covalent or non-covalent bonds. Pore size of matrix is adjusted with concentration of polymer used. Commonly used matrix for enzyme immobilisation is polyacrylamide gel, cellulose triacetate, agar, gelatin, carrageenan, alginate, etc.
Methods of entrapment
- Inclusion in the gels: Enzyme is trapped inside the gel.
- Inclusion in fibres: Enzymes are trapped on fibres made up of support materials.
- Inclusion in microcapsules: Enzyme trapped in microcapsules formed by monomer mixture such as polyamine and calcium alginate.
Copolymerization or cross linking
In this method enzyme immobilisation is directly linked by covalent bonds between various groups of enzymes via polyfunctional reagents. Commonly used polyfunctional reagents are glutaraldehyde and diazonium salt. In this method there is no matrix or support medium is involved. This technique is cheap and simple and widely used in commercial preparations and industrial applications. The advantage of this method is that the polyfunctional reagent used for cross linking the enzyme may denature or structurally modified the enzyme leading to loss of catalytic properties.
Encapsulation
This technique of enzyme immobilisation is done by enclosing the enzyme in a membrane capsule. The capsule is made up of semi-permeable membrane like nitro cellulose or nylon. The effectiveness of this method depends upon the stability of enzyme inside the capsule. Using this technique, large quantity of enzymes can be immobilised by encapsulation.
Applications of enzyme immobilisation
- Immobilised enzymes are widely used in the diagnosis and treatment of many diseases. For example, fast diagnostic kit like ELISA.
- They can be used to overcome inborn metabolic disorder by the supply of immobilised enzymes.
- The use of immobilised enzymes allow biotechnologist to increase the efficiency of different enzymes such as proteases for cell or organelle lysis, Horse Radish Peroxidase (HRP) in blotting experiments.
- Immobilised enzymes are widely used in commercial production of antibiotics, beverages, amino acids and secondary metabolites.
Summary
Enzyme immobilisation technique involves fixing of enzymes on the solid supports to enhance their stability, reusability and functionality. There are five techniques of enzyme immobilisation, which includes adsorption, covalent bonding, entrapment, cross linking, encapsulation. These methods aim to retain the catalytic activity of enzyme while improving their operational characteristics. The applications of immobilised enzymes include biocatalysis in pharmaceuticals, food processing and environmental remediation.
Frequently asked questions
What are the 5 methods of enzyme immobilisation?
There are five techniques of enzyme immobilisation, which includes adsorption, covalent bonding, entrapment, cross linking, encapsulation.
What do you mean by enzyme immobilisation?
Enzyme immobilisation technique involves fixing of enzymes on the solid supports to enhance their stability, reusability and functionality.
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