Effect of chrystallisation and crosslinking conditions on the catalytic properties of crystalline enzymes

Cross-linked enzyme crystals (CLEC®s) are a novel form of immobilised biocatalyst designed for application in large-scale biotransformation processes. In this work systematic tools for the production and characterisation of CLEC®s were developed. These were subsequently used to determine how the crystallisation and crosslinking conditions during CLEC® manufacture influenced the process characteristics of the resultant biocatalyst preparations. A range of enzymes was used in this study which included Candida rugosa lipase, α-chymotrypin and a secondary alcohol dehydrogenase. Optimisation and control of the enzyme crystallisation process was facilitated by the use of triangular 'screening windows' which allowed three parameters (e.g. protein concentration, precipitant concentration and salt) to be varied simultaneously. These diagrams identified regions, or 'crystallisation windows', in which particular crystal forms or optimal specific activity recoveries (up to 87% in the case of lipase) could be obtained. They also identified conditions for reproducible scale-up of the crystallisation process. Crosslinking conditions were also investigated with a range of different crosslinking reagents and conditions. Glutaraldehyde was found to be the most generic and successful crosslinking reagent while others, such as phthalaldehyde, could be used but the resultant CLEC®s exhibited little activity. The effect of crystallisation and crosslinking conditions on the catalytic properties of crystalline enzymes was subsequently investigated using a hierarchy of standard tests. The hierarchy is designed to expose key properties of the CLEC®s relative to each other, and the free enzyme, and to minimise the number of experiments necessary to evaluate each batch of biocatalyst. The crystallisation conditions used for CLEC® production were found to significantly affect both the catalytic and mechanical properties of the CLEC®s. Different CLEC® preparations were found to be suited to particular processing environments such as extremes of temperature or the presence of organic solvents. Accurate control of the crystallisation conditions used for CLEC® production is therefore vital, as this will influence the suitability of the CLEC®s for their end use. Regarding the effects of crosslinking reagent, a higher concentration of glutaraldehyde (up to 10% v/v) was found to benefit all the properties of lipase CLEC®s. Phthalaldehyde crosslinked CLEC®s were generally found to be less well suited to process applications than glutaraldehdye crosslinked ones. Finally the feasibility of a continuous process for the production of CLEC®, utilising stirred tank crystallisation and crosslinking reactors, was demonstrated. The rig showed the feasibility of being able to more accurately control the size of the crystals obtained which was not possible with the previous batch crystallisations