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Future of enzymology: Immobilised enzymes

Dr Girish Walavalkar examines the emerging technology of immobilised enzymes and explores how it can be put to use in the textile industry.

Enzymes are the catalysts of all reactions in living systems. The reactions are catalysed in the ‘active sites’ of globular, enzymatic proteins. The enzymatic proteins are composed of amino acids with a variety of side chains ranging from non-polar to acidic, basic and neutral polar, aliphatic and aromatic compounds. The side chains allow for a globular, three dimensional enzymatic protein to create ’active sites’ which facilitate all ranges of microenvironments for catalysis.

Major advances in microbial technology and genetics have brought about a broad range of enzymatic applications in various industries. The estimated value of world enzyme market is presently about US $ 1.3 billion and it has been forecasted to rise to about US $ 2 billion by year 2005. Detergents (37%), textiles (12%), starch (11%), baking (8%), and animal feed (8%) are the main industries, which use about 75% of industrially produced enzymes.

Enzymatic processes have been increasingly incorporated in textiles over the past years. Cotton, wool, flax, etc, are natural materials used in textiles that can be processed with enzymes. Enzymes have been used for desizing, scouring, polishing, washing, degumming, peroxide degradation in bleaching baths, as well as for depolarisation of dye house wastewaters, bleaching of released dyestuff and inhibiting dye transfer. Furthermore many new applications are under development, such as natural and synthetic fiber modification, enzymatic dyeing and finishing.

A unique feature of enzyme molecule action is its reusability. To exploit this commercially, a new technology called enzyme immobilisation is being developed rapidly. Scientifically, immobilisation of an enzyme has been defined as “the imprisonment of an enzyme in the distinct phase that allows exchange with, but is separated from, the bulk phase in which substrate, activator or inhibitor molecules are dispersed and monitored.”

When enzymes are used in soluble form, they retain some activity after reaction, which cannot be commercially recovered for reuse and is wasted. This remaining residual activity contaminates the product and its removal may involve extra purification costs. In order to eliminate this wastage and improve productivity, simple and economic methods that enable the enzyme to be separated from the reaction product need to be used. The easiest way of achieving this is by separating the enzyme and the product during the reaction using a two-phase system - one phase containing the enzyme and the other containing the product. The enzyme is imprisoned within its phase, allowing its reuse or continuous use but preventing it from contaminating the product. The other molecules, including the reactants, are able to move freely between the two phases. This is known as ‘immobilisation’ and may be achieved by fixing the enzyme to or within some other material. The term ‘immobilisation’ does not necessarily mean that the enzyme cannot move freely within its particular phase, although this is often the case.

A wide variety of insoluble materials, usually inert polymeric or inorganic materials, may be used to immobilise the enzymes. Immobilisation of enzymes often incurs additional expenses and is only undertaken if there is a sound economic or process advantage as compared to free (soluble) enzymes. Some of the current popular uses of immobilised enzymes in the food industry are as Lactase (from which we get lactose free milk and whey) and Lipase (coca butter and substituents).

Methods of immobilisation

Immobilisation methods may be subdivided into the following groups, depending upon the physical relationship of the enzyme catalyst to the polymer matrix:

Adsorption

Adsorption of an enzyme to a polymer material probably represents the easiest method of immobilisation. A range of non-specific or specific binding forces may be employed, for example, electrostatic, hydrophobic interactions, or affinity bonding to specific ligands attached to the polymer. This type of method has a number of potential advantages:

i) Mild conditions and ease of preparation of the immobilised material (simply stir polymer and enzyme together)

ii) Availability of ready prepared polymer material suitable for use in column reactors

iii) Potential for regeneration of the immobilised catalyst

iv) Minimum limitations for access of the substrate to the bound enzyme

v) Application to whole cells or organelles.

Covalent bonding

There are two ways of covalently bonding an enzyme to a polymer. The first is by activating the polymer with a reactive group. The second is by using a bi-functional reagent as a bridge between the enzyme and the polymer. The principle groups on enzymes, through which coupling with the polymer occurs, are hydroxyl and amino groups, and to a lesser extent, sulphydryl

groups. This raises one of the major problems with the technique: the enzyme may often be inactivated by virtue of conformational change consequent to reaction or reaction with a group at the active site of enzyme.

Crosslinking

Crosslinking of the enzyme to itself by reaction with a bi-functional reagent with the possible inclusion of an inert protein is essentially an extension of covalent bonding techniques. This brings with it all the associated advantages and problems. The bi-functional reagent most often used is glutaraldehyde. The technique is cheap and simple, but is not used often with pure enzymes because it yields very little bulk of immobilised enzyme with a very high intrinsic activity.

Entrapment

Entrapment of biocatalysts within a polymer matrix is in principle easy to perform. The biocatalyst is dissolved in a solution of the polymer precursors and polymerisation is initiated. The major advantages of this type of method are simplicity, mild conditions of use, preparation of immobilised biocatalyst, and usefulness for immobilising cell preparations. However, there is a need to keep the average pore size of the gel as high as possible to prevent excessive diffusion. Thus, due to the limitation and the variability of pore sizes in such gels, there may be great leakage of the biocatalyst from the gel, particularly with low molecular weight enzymes.

Encapsulation

Encapsulation of biocatalysts by enclosing a droplet of solution of biocatalyst in a semipermeable membrane capsule has, to date, largely been restricted to medical application. This technique is simple and cheap, but to be effective, the biocatalyst must be stable in solution.

The choice of immobilisation methods is based largely upon empirical factors. A few general rules exist, along with obvious considerations such as substrate molecular weight, size and inherent stability of enzyme molecule, requirement for a specific physical form of the immobilised enzyme, etc. There is usually, however, little alternative to the try-it-and-see principle.

Immobilised enzymes in textile industry

Immobilised enzyme systems are used where they offer cost advantages to users on the basis of total manufacturing costs. The plant size needed for continuous process is two orders of magnitude smaller than that required for batch process using free enzymes. The capital costs are therefore considerably smaller and a plant may be prefabricated cheaply off-site. Immobilised enzymes offer greatly increased productivity on the enzyme weight basis and also often provide process advantages.

Different supports and methods are constantly being tried to immobilise various industrial enzymes to get increased activity and stability in various applications. In the textile industry, high water and energy costs in wet processing imposed the implementation of closed-loop processes and recycling of the textile liquors. For the adequate processing of textile materials, it is necessary to apply various additives. The removal of hydrogen peroxide or hydrolysed dyestuff after the bleaching and dyeing processes are the two major areas were the washing liquors can be recycled by means of an appropriate enzymatic treatment like with catalases, laccases, peroxidases, and azoreductases.

These treatments can be carried out with enzymes in both free and immobilised form. Usually, low concentration of the free enzymes is sufficient to achieve the desired effect. However, the catalyst is not recoverable and stability problems may arise from the high temperature and alkalinity of the bleaching, washing or dyeing liquors. The main disadvantage of using free enzymes is that the protein remains in the recycled effluent that is intended to be reused for dyeing. The dyeing is carried out mostly at temperatures where proteins undergo denaturation. The transition from globular to random coil conformation of the protein occurs during denaturation at elevated temperature. This thermally initiated process increases the hydrophobic dye-protein interactions. The denaturated protein binds dye from the dyeing solution and thereby decreases its concentration. The presence of denaturated protein does not alter the dye exhaustion kinetics, but reduces the final exhaustion level. Use of immobilised enzymes will avoid the problem described above.

Presently, the technology of immobilised enzymes is going through a phase of evolution and maturation. Evolution is reflected in the ever-broadening range of applications of immobilised enzymes. Maturation is mirrored in the development of the theory of how immobilised enzymes function and how the technique of immobilisation is related to their primary structure through the formation and configuration of their three dimensional structure. There still remains much room for the development of useful processes and materials based on this hard-won understanding. Immobilised enzymes will clearly be more widely used in the future. This is just the beginning of the immobilised enzyme technology era.

(Dr Girish Walavalkar heads Resil Chemicals’ enzymes division, Resil Biotech, based in Bangalore).