Cross-linked Protein Crystal Technology in Bioseparation and Biocatalytic Applications

Chemical cross-linking of protein crystals form an insoluble and active protein matrix. Cross-linked protein crystals (CLPCs) have many excellent properties including high volumetric activity and stability. In this thesis CLPC technology was studied in bioseparation and biocatalytic applications. A novel immunoaffinity separation material, cross-linked antibody crystals (CLAC), was developed in this thesis for enantiospecific separation of a chiral drug, finrozole. Previously, the preparation of an antibody Fab fragment ENA5His capable of enantiospecific affinity separation of the chiral drug has been described. However, in a carrier-immobilized form ENA5His suffered from poor stability in the presence of a high concentration of organic solvents needed to release the bound drug. In this study crystallization conditions for ENA5His were found by means of small-scale vapor diffusion experiments. Crystallization was further scaled up to 10 ml batch crystallization with a 70% protein yield. Glutaraldehyde cross-linking modified the ENA5His crystals into an insoluble form producing a CLAC matrix. The CLAC matrix packed in a column separated pure enantiomers from the racemic mixture of the drug. The CLAC matrix was totally stable at the elution conditions enabling reuse of the immunoaffinity column. However, the specific drug enantiomer binding capacity of CLAC was only 50% of the corresponding capacity of carrier-immobilized ENA5His. Also a cross-linked carrierimmobilized ENA5His column was prepared to study the effect of bare chemical crosslinking. Surprisingly, the cross-linked immobilized ENA5His was as active as the native immobilized ENA5His and simultaneously stable against the denaturing effect of methanol. Xylose isomerase (XI) is a widely used enzyme in industry as a result of its ability to catalyze isomerization of D-glucose to D-fructose. Previously it has been shown that XI accepts all of the pentose sugars and many hexose sugars as isomerization substrates. In the present study novel tetrose isomerizations and C-2 epimerizations with both the Dand L-forms of the sugars by an industrial xylose isomerase (XI) from Streptomyces rubiginosus were described. Furthermore, the results showed that the real equilibrium of XI catalyzed reactions is not between two isomers but between a ketose and its two aldose isomers. These findings together with previous results show that XI can be used as a catalyst for production of a variety of sugars. Obtaining homogeneous enzyme crystals is critical to their application as catalysts in a cross-linked form. In this study a method for the production of homogeneous crystals of XI was developed. Firstly, the XI crystal solubility was measured with respect to precipitant salt concentration, temperature, and pH. Secondly, based on the results of the solubility study, a process for the production of uniform XI crystals of different size classes was developed. Crystals with average crystal sizes between 12 μm and 360 μm were produced. XI crystals were further cross-linked by glutaraldehyde and L-lysine to prepare insoluble cross-linked xylose isomerase crystals (CLXIC).