The Immobilization Of Enzymes

Pineapple is great source of bromelain which is a protease used in the immobilized of enzymes.
Photo courtesy of Monika Schröder through pixabay.com

The immobilization of enzymes is a biochemical engineering process for fixing enzymes to a matrix or membrane system with a view to generating a bioreactor. The processes of creating immobilized enzymes are very similar to immobilizing cells and indeed many researchers and academics treat them within the same sphere.

One appropriate and simple definition for enzyme immobilization is that it is a process of confining an enzyme in a distinct phase from another phase which contains substrates and products. It means that enzymes (and indeed cells) are physically restrained to a defined region in space whilst they retain their catalytic activity. It means they can be continuously and repeatedly used.

The Advantages Of Immobilizing Enzymes

Enzymes when immobilised are protected from degradation and deactivation which means they have improved stability. They are certainly more stable with improved stability to raised temperatures and to changes in pH.

These enzymes are also retained in reactors and the products created are also free of any contamination by enzymes. It also means the enzymes can be recycled and used repetitively.

From a commercial point of view, there are cost savings and benefits in reaction efficiency. In some applications that are used as controlled release agents. 

There is a capability of stopping the catalysis rapidly by removing the enzyme from the reaction solution. Likewise, the reaction can be started up usually just as quickly. The applications of many enzymes means they can produce a multienzyme reaction system. 

The kinetics of an immobilised enzyme are usually very different. The pH optimum and the temperature optimum for catalysis are usually altered to a certain degree of benefit. The Km using Lineweaver-Burke kinetics often increases and in some cases 10 to 100-fold higher. Immobilization means the enzymes can be operated in more challenging conditions such as altered pH, higher processing temperatures and so on.

Materials used for immobilizing enzymes include gels and ion exchange materials. Ion exchange resins include anion and cation resins but the former is usually preferred for fixing enzymes. To ensure an enzyme is firmly linked requires some form of chemical linking without damaging the enzyme. Glutaraldehyde is a very commonly used linking agent but there are others such as trichlorotriazine (Chellapandia & Sastry, 1996). In this example an alkaline protease was covalently linked to activated nylon.

A typical system for fixing a robust enzyme like a protease say would be to mix the enzyme with a 10 per cent glutaraldehyde in a buffer solution. The enzyme is then chemically linked through covalent bonding to the immobilization material. Glutaraldehye is an aggressive linking agent and can damage activity so it is usually hoped that the properties of the immobilized enzyme is not altered with too much detriment.

Immobilization Of Enzymes: Applications

A vast array of enzymes, probably nearly all of them have been immobilized for some purpose or other. The most common commercial types immobilized include proteases, peptide hydrolases, β‐galactosidase, invertase, and glucoamylase dehydrogenases etc. Some enzymes are less robust probably because of the processes used for immobilization and these include  lipase, hexokinase, glucose‐6‐phosphate dehydrogenase, and xanthine oxidase. In one example (Ohmiya et al., 1978) this latter group was actually deactivated.

In many examples, enzymes were immobilized so they could be examined more carefully. It soon became apparent that their kinetic properties were altered so much so that they became valuable commercial entities.

Proteases

Proteases are used to hydrolyse proteins. These can be used commercially for removing proteinaceous dirt from clothes, cleaning process equipment, breaking down proteins in foods for nutrition and so on. Rennet is a good example of a well known protease with an acidic pH optimum for example whilst alkaline proteases are commonly employed in detergents.

Alkaline and acidic proteases are one of the most important classes of commercial enzymes. Proteases are also extremely robust when it comes to immobilization compared to others.  An alkaline protease and an acidic one (rennet) were immobilised on an anion exchange resin for example such as Dowex MWA-1 (mesh size 20-50) using 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5) (Ohmiya et al., 1978). In their example, the properties of the proteases were not damaged in the process based on retention of activity. Kinetically, the Km value increased tenfold in both cases as a result which is often the situation as the active site is altered. Both immobilised proteases were more stable than the free based equivalent when their activity was challenged at a temperature such as  60°C. 

A similar case in stability benefit was seen for an immobilized alkaline protease linked to activated nylon (Chellapandian & Sastry, 1996).

References

Chellapandian, M., Sastry, C.A. (1996) Covalent linking iof alkaline protease on trichlorotriazine activated nylon. Bioprocess Eng. 15 (2) pp. 95-98

Ohmiya, K., Tanimura, S., Kobayashi, T., Shimizu, S. (1978) Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde. Biotech. Bioeng. 20 (1) pp. 1-15 (Article)

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