Rapid determination of enzyme purity by a microdialysis-based assay

Abstract

Microdialysis was shown to be useful as a fast on-line sampling method for determining the purity of starch hydrolysing enzymes. The enzymes were characterised using their hydrolytic properties. β-Amylases and pullulanases from different sources and/or manufacturers were investigated, with maltose, maltoheptaose, pullulan, and potato amylopectin starch (PAP) as substrates. The hydrolysis products were sampled via an on-line microdialysis probe and determined in a high-performance anion-exchange chromatographic (HPAEC) system. Comparison between the expected (theoretical) hydrolysis products with those obtained in the experiments made it possible to determine impurities in the enzymes. Two of the β-amylases and one pullulanase released unwanted hydrolysis products, indicating trace impurities in the enzyme preparation. Microdialysis sampling allows on-line sampling and eliminates separate sample preparation and clean-up steps. On-line microdialysis coupled to a HPAEC system is therefore a fast and simple technique for analysing enzyme hydrolysates.

References

1. D. J. Holme and H. Peck, Analytical Biochemistry, Longman Scientific and Technical, 2nd edn., 1993, p. 303.
2. S. Hizukuri and Y. Maehara, Carbohydr. Res., 1990, 206, 145.
3. D. J. Manners, Carbohydr. Polym., 1989, 11, 87.
4. K. S. Wong and J. Jane, J. Liq. Chrom. Rel. Technol., 1997, 20, 297.
5. I. Hanashiro, J.-I. Abe and S. Hizukuri, Carbohydr. Res., 1996, 283, 151.
6. Q. Zhu, R. Sjöholm, K. Nurmi and E. Bertoft, Carbohydr. Res., 1998, 309, 213.
7. K. Koch, R. Andersson and P. Åman, J. Chromatogr. A, 1998, 800, 199.
8. H. Fredriksson, J. Silvero, R. Andersson, A.-C. Eliasson and P. Åman, Carbohydr. Polym., 1998, 35, 119.
9. Q. Zhu and E. Bertoft, Int. J. Biol. Macromol., 1996, 21, 131.
10. P. A. M. Steeneken, A. C. Tas, A. J. J. Woortman and P. Sanders, Spec. Publ.-R. Soc. Chem., 1997, 205, 153.
11. M. Gohdes and P. Mischnick, Carbohydr. Res., 1998, 309, 109.
12. U. K. Laemmli, Nature, 1970, 227, 680.
13. D. Wu and F. E. Regnier, J. Chromatogr., 1992, 608, 349.
14. T. Palmer, Understanding Enzymes, Prentice Hall/Ellis Horwood, 4th edn., 1995, p. 300.
15. F. W. Willenbrock, D. C. A. Neville, G. S. Jacob and P. Scudder, Glycobiology, 1991, 1, 223.
16. K. Tyagarajan, J. G. Forte and R. R. Townsend, Glycobiology, 1996, 6, 83.
17. N. Torto, T. Buttler, L. Gorton, G. Marko-Varga, H. Stålbrand and F. Tjerneld, Anal. Chim. Acta, 1995, 313, 15.
18. C. M. Zook and W. R. LaCourse, Anal. Chem., 1998, 70, 801.
19. T. Lu, W. R. LaCourse and J. Jane, Starch/Stärke, 1997, 49, 505.
20. K. Koizumi, Y. Kubota, T. Tanimoto and Y. Okada, J. Chromatogr., 1989, 464, 365.
21. C. E. Lunte, D. Scott and P. Kissinger, Anal. Chem., 1991, 63, 773A.
22. N. Torto, T. Laurell, L. Gorton and G. Marko-Varga, Anal. Chim. Acta, 1999, 379, 281.
23. N. Torto, G. Marko-Varga, L. Gorton, H. Stålbrand and F. Tjerneld, J. Chromatogr. A, 1996, 725, 165.
24. T. Laurell and T. Buttler, Anal. Methods Instrum., 1995, 2, 197.
25. N. Nelson, J. Biol. Chem., 1944, 153, 375.
26. M. Somogyi, J. Biol. Chem., 1952, 195, 19.
27. J. J. Marshall and W. J. Whelan, J., Anal. Biochem., 1973, 52, 642.
28. M. Florkin and E. Stotz, Comprehensive Biochemistry, Elsevier, 3rd edn., 1973, vol. 13, p. 212.
29. H. F. Zobel, Starch/Stärke, 1988, 40, 44.
30. P. Hofvander, P. T. Persson, A. Tallberg and O. Wikström, Amylogene HB, Swedish Pat. 9004096-5, Sweden, 1992.
31. A. Guilbot and C. Mercier, The Polysaccharides, ed. G. Aspinall, Academic Press, 1985, vol. 3, p. 232.