Production and characterisation of cross-linked enzyme crystals (CLECs®) for application as process scale biocatalysts

Cross-linked enzyme crystals (CLECs(R)) are a novel form of immobilised biocatalyst designed for application in large-scale biotransformation processes. In this work we review the production and characterisation of CLECs(R) prepared from three enzymes (yeast alcohol dehydrogenase I (YADHI), Candida rugosa lipase and cu-chymotrypsin) over a range of crystallisation and cross-linking conditions. Optimisation and control of the crystallisation process, with respect to crystal form and enzyme activity yield, was facilitated by the use of triangular crystallisation diagrams which allowed three parameters (e.g. protein concentration, precipitant concentration and pH) to be varied simultaneously. These diagrams showed regions, or 'crystallisation windows', in which particular crystal forms or optimal activity recoveries (up to 87%) could be obtained. They also identified conditions for reproducible scale-up of the lipase crystallisation from 0.5 to 500 mL scale. In order to evaluate the suitability of a particular batch of CLECs(R) for large-scale use, a hierarchy of standard tests is proposed. This is designed to expose key properties of the CLECs(R) relative to each other, and the free enzyme, and to minimise the number of experiments necessary to evaluate each batch of biocatalyst. In general, the CLECs(R) of each enzyme were found to be more resistant to harsh environmental conditions, such as extremes of temperature and DH and the presence of solvents or proteases, than the free enzymes. Cross-linking of the crystals with glutaraldehyde also yielded mechanically robust catalysts that could withstand the various forces associated with shear in agitated vessels and particle compression in repeated dead-end filtration cycles. The hierarchy of tests proposed here clearly indicated that many of the above properties were also dependent on both the crystal form and size, and the concentration of cross-linking reagent used. Accurate control of the crystallisation conditions used for CLEC(R) production is therefore vital as this will influence the suitability of the CLECs(R) for their end use.