Immobilization of thermophilic enzymes in miniaturized flow reactors

Abstract

The exploitation of enzymes for biotransformation reactions for the production of new and safer drug intermediates has been the focus of much research. While a number of enzymes are commercially available, their use in an industrial setting is often limited to reactions that are cost-effective and they are rarely investigated further. However, the development of miniaturized flow reactor technology has meant that the cost of such research, once considered cost- and time-inefficient, would be much less prohibitive. The use of miniaturized flow reactors for enzyme screening offers a number of advantages over batch enzyme assay systems. Since the assay is performed on a miniaturized scale, enzyme, substrate and cofactor quantities are significantly reduced, thus reducing the cost of laboratory-scale investigations. Since flow reactors use microfluidic systems, where the substrate and products flow out of the system, the problems of substrate inhibition and product inhibition encountered by some enzymes are avoided. Quite often, enzymes fulfil a single-use function in biotransformation processes; however, enzyme immobilization allows enzyme reuse and often helps to increase enzyme stability. We have used an aminoacylase enzyme with potential use for industrial biotransformation reactions and have successfully immobilized it in miniaturized flow reactors. This L-aminoacylase is from the thermophilic archaeon Thermococcus litoralis. Two approaches to enzyme immobilization have been examined, both involving enzyme cross-linking. The first reactor type has used monoliths, to which the enzyme was attached, and the second contained previously cross-linked enzyme trapped using frits, in the microfluidic channels. Two different microreactor designs were used in the investigation: microreactor chips for the monoliths and capillary flow reactors for the cross-linked enzyme. These systems allowed passage of the substrate and product through the system while retaining the aminoacylase enzyme performing the catalytic conversion. The enzyme has been successfully immobilized and used to produce stable biocatalytic microreactors that can be used repeatedly over a period of several months.