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Feature

Stop! Enzymes At Work

Immobilised enzymes are used in applications as diverse as artificial kidneys and clear fruit juice.

Dr Helen Petach and Dr Bill Henderson

Enzymes are Nature's catalysts, used to speed up a myriad of chemical reactions in living things. They carry out these reactions with extraordinary speed and selectivity when compared to other synthetic catalysts. Enzymes can be isolated -- and even genetically engineered -- for use in an ever-increasing number of practical applications, such as the lipase enzymes added to laundry detergents to break up greasy stains on clothing, ismerase enzymes used in the food industry to convert glucose to fructose, and pectinases which clarify fruit juices and wines by degrading pectins.

Enzymes naturally function in the highly regulated, stable ionic strength, neutral pH of the living cell. It is the extraction of enzymes to the harsh external environment that creates difficulties. The extraction process is often lengthy and expensive, and since the recovered enzyme often suffers from low stability, it is usually a very precious material. Thus, enzyme catalysts need to be recoverable and reusable to render their practical application worthwhile.

The technique of enzyme immobilisation -- attaching an enzyme to an insoluble, polymeric "support" -- prevents enzyme loss to the solution. An immobilised enzyme is easy to use, since at the end of the enzyme-catalysed reaction, the enzyme on the insoluble support is simply filtered off and recycled. Alternatively a column of the supported enzyme can be made, with the solution of the reactant simply passed through, allowing the product solution to be collected at the bottom of the column. As an added benefit, the supported enzyme often shows greater thermal stability and longevity than the related solution version.

Numerous methods are available to immobilise an enzyme. These range from simply physically absorbing it to the surface of a polymeric support, to entrapping it within a polymer. However, one of the most promising methods involves attaching the enzyme to the support by means of a strong chemical bond. In this way, the enzyme is prevented from leaching off the support, giving a highly stable immobilised system. The ability to efficiently immobilise the enzyme urease, which hydrolyses urea to carbon dioxide and water, is of great importance in the development of artificial kidneys.

Our own interests in the area of enzyme immobilisation lie with using a special type of phosphorus chemical, called a hydroxymethyl phosphine, as the link which attaches the enzyme to the insoluble support. These chemicals are commercially available and form extremely strong links between the enzyme and the support. The enzyme immobilised in this way can be used for a long time with little loss in catalytic power. Hydroxymethyl phosphines comprise one component in a flexible polymer film able to bind enzymes directly, without the need for any other coupling agent. Enzymes immobilised on these supports also show very high activities and stabilities.

One of the disadvantages of immobilising an enzyme is that often the immobilised enzyme has a lower activity than the same enzyme in solution, basically because the enzyme is too closely bound to the support, restricting its mobility. We are investigating the attachment of enzymes to the support using a very long, but flexible "molecular anchor", thus allowing the enzyme to move around much more freely, and so behave more like it was in solution. The enzyme still retains the attachment to the insoluble support, preventing it from being washed away, thus combining the best features of both solution and immobilised enzymes.

For medical applications of immobilised enzymes it is highly desirable to use a support which is biologically compatible. A very promising support material is the polymer chitosan, a polymeric amino sugar, readily obtained from chitin -- a material which New Zealand has an abundance of, since it is a waste product of the seafood industry, found in crab shells and the like.

One application that chitin has found in the area of immobilised enzymes is as a support for the enzyme papain (extracted from papayas), used as a clearing agent in the brewing industry. Similarly, the enzyme penicillin acylase has been bound to chitosan for the synthesis of penicillin derivatives such as ampicillin. The biocompatability of chitosan for medical usage has been thoroughly demonstrated, and our research has shown that it is possible to prepare very stable immobilised enzymes on chitosan using hydroxymethyl phosphines.

The continuing practical application of enzymes as catalysts is ever more likely as activity and stability are maintained upon immobilisation.

Dr Bill Henderson is a lecturers in inorganic chemistry at the University of Waikato.
Dr Helen Petach and Dr Bill Henderson are lecturers in biological sciences and chemistry, respectively, at the University of Waikato.