Cross-linked protein crystal technology in bioseparation and biocatalytic applications

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Doctoral thesis (article-based)
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47, [33]
Technical biochemistry report, Teknillisen biokemian tiedote, 8
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 carrier-immobilized ENA5His column was prepared to study the effect of bare chemical cross-linking. 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 D- and 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). The cross-linking of XI crystals improved their stability towards organic solvents. This can be exploited in the production of D-fructose and possibly also other sugars. Adding acetone to the reaction mixture enhanced the production of D-fructose from D-glucose catalyzed by CLXIC in terms of product yield and reaction rate. The D-fructose equilibrium concentration increased from 49 % to 64 % when increasing the acetone concentration from 0 % to 90 % in the reaction mixture at 50 °C. In 50 % acetone the fructose production rate was more than doubled compared with that in a pure buffer solvent. Acetone seems to have only a minor role in the inactivation of cross-linked crystals. The residual activity of CLXIC was 70-80 % of the initial activity after a 24-h incubation at 50 °C in a buffer solution (pH 7.2) containing 10-90 % acetone. In the soluble form, the activity of XI decreased by 75 % after incubation in 50 % acetone for 24 h at 50 °C.
cross-linked protein crystal, protein crystallization, bioseparation, biocatalysis
Other note
  • Vuolanto A., Kiviharju K., Nevanen T. K., Leisola M. and Jokela J., 2003. Development of cross-linked antibody Fab fragment crystals for enantioselective separation of a drug enantiomer. Crystal Growth & Design 3, number 5, pages 777-782.
  • Vuolanto A., Leisola M. and Jokela J., 2004. Enantioselective affinity chromatography of a chiral drug by crystalline and carrier-bound antibody Fab fragment. Biotechnology Progress 20, number 3, pages 771-776.
  • Vuolanto A., Uotila S., Leisola M. and Visuri K., 2003. Solubility and crystallization of xylose isomerase from Streptomyces rubiginosus. Journal of Crystal Growth 257, numbers 3-4, pages 403-411.
  • Vuolanto A., Pastinen O., Schoemaker H. E. and Leisola M., 2002. C-2 epimer formation of tetrose, pentose and hexose sugars by xylose isomerase. Biocatalysis and Biotransformation 20, number 4, pages 235-240.
  • Vilonen K., Vuolanto A., Jokela J., Leisola M. and Krause A. O. I., 2004. Enhanced glucose to fructose conversion in acetone with xylose isomerase stabilized by crystallization and cross-linking. Biotechnology Progress, in press.
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