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Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II

  • Preeti Bachhawat
  • Saroj Mishra
  • Yukti Bhatia
  • V. S. Bisaria
Article

Abstract

The biosynthetic activity of yeast Pichia etchellsii β-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate: 1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethylacetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

Index Entries

β-Glucosidase Pichia etchellsii cellooligosaccharides alkyl glucosides monoterpenyl glucosides 

References

  1. 1.
    Vic, G. and Thomas, D. (1993), Tetrahedron Lett. 33, 4567–4570.CrossRefGoogle Scholar
  2. 2.
    Gusakov, A. V., Sinitsyn, A. P., Klesov, A. A., and Goldshteins, G. K. (1984), Biokhimiya 49, 1110–1120.Google Scholar
  3. 3.
    Yan, T. R. and Liau, J. C. (1998), Biotechnol. Lett. 20, 591–594.CrossRefGoogle Scholar
  4. 4.
    Christakopoulos, P., Kakos, D., Macris, B. J., Goodenough, P. W., and Bhat, M. K. (1994), Biotechnol. Lett. 16, 587–592.CrossRefGoogle Scholar
  5. 5.
    Dong, W., Yinbo, Q. U., and Peiji, G. (1996), J. Gen. Appl. Microbiol. 42, 363–369.Google Scholar
  6. 6.
    Kuriyama, K., Tsuchiya, K., and Murui, T. (1995), Biosci. Biotechnol. Biochem. 59, 1142–1143.CrossRefGoogle Scholar
  7. 7.
    Bhatia, Y., Mishra, S., and Bisaria, V. S. (2002), CRC Crit. Rev. Biotechnol. 22, 375–407.CrossRefGoogle Scholar
  8. 8.
    Vulfson, E. N., Patel, R., Beecher, J. E., Andrews, A. T., and Law, B. A. (1990), Enzyme Microb. Technol. 12, 950–954.CrossRefGoogle Scholar
  9. 9.
    Kiwada, H., Nimura, H., Fujisaki, Y., Yamada, S., and Kato, Y. (1985), Chem. Pharm. Bull. 33, 753–759.PubMedGoogle Scholar
  10. 10.
    Nguyen, D. T., Lerch, H., Zemann, A., and Bonn, G. (1997), Chromatographia 46, 113–121.CrossRefGoogle Scholar
  11. 11.
    Kyoko, K. (1996), J. Chromatogr. A 720, 119–126.CrossRefGoogle Scholar
  12. 12.
    Simms, P. J., Haines, R. M., and Hicks, K. B. (1993), J. Chromatogr. A 648, 131–137.CrossRefGoogle Scholar
  13. 13.
    Malá, S., Dvoakova, H., Hrabal, R., and Králová, B. (1999), Carbohydr. Res. 322, 209–218.PubMedCrossRefGoogle Scholar
  14. 14.
    Trinel, P. A., Lepage, G., Jouault, T., Strecker, G., and Poulain, D. (1997), FEBS Lett. 416, 203–206.PubMedCrossRefGoogle Scholar
  15. 15.
    Lea, J., Granter, M. S., Heyrand, A., Carmen, L., Petkowicz, O., Rinaudo, M., and Reicher, F. (1995), Inst. J. Biol. Macromol. 17, 13–19.CrossRefGoogle Scholar
  16. 16.
    Shuji, Y. (1996), J. Chromatogr. A. 732, 141–156.Google Scholar
  17. 17.
    Masuda, J., Nishimura, Y., and Tonegawa, M. (1999), J. Chromatogr. A 845, 401–408.CrossRefGoogle Scholar
  18. 18.
    Brunt, K. (1982), J. Chromatogr. A 246, 145–151.CrossRefGoogle Scholar
  19. 19.
    Hodjson, J. (1999), Bio/Technology 9, 149–150.Google Scholar
  20. 20.
    Sartori, J., Potthast, A., Ecter, A., Sixta, H., Rusenau, T., and Kosma, P. (2003), Carbohydr. Res. 338, 1209–1216.PubMedCrossRefGoogle Scholar
  21. 21.
    Wallecha, A. and Mishra, S. (2003), Biochim. Biophys. Acta 1649, 74–81.PubMedGoogle Scholar
  22. 22.
    Pandey, M. and Mishra, S. (1997), Gene 190, 45–51.PubMedCrossRefGoogle Scholar
  23. 23.
    Bhatia, Y., Mishra, S., and Bisaria, V. S. (2002), Appl. Biochem. Biotechnol. 102–103, 367–379.PubMedCrossRefGoogle Scholar
  24. 24.
    Kannan, T., Loganathan, D., Bhatia, Y., Mishra, S., and Bisaria, V. S. (2004), Biocatalysis Biotransformation 22, 1–7.CrossRefGoogle Scholar
  25. 25.
    Geurtsen, R., Cote, F., Hahn, G., and Booms, G. J. (1999), J. Org. Chem. 64, 7828–7835.CrossRefGoogle Scholar
  26. 26.
    Sethi, B., Jain, M., Chowdhary, M., Soni, Y., Bhatia, Y., Sahai, V., and Mishra, S. (2002), Biotechnol. Bioprocess Eng. 7, 43–51.CrossRefGoogle Scholar
  27. 27.
    Nossal, N. G. and Heppel, L. A. (1996), J. Biol. Chem. 241, 3055–3062.Google Scholar
  28. 28.
    Bhatia, Y., Mishra, S., and Bisaria, V. S. (2004), Appl. Microbiol. Biotechnol., submitted.Google Scholar
  29. 29.
    Gunata, Z., Vallier, M. J., Sapis, J. C., Baumes, R., and Bayonove, C. (1994), Enzyme Microb. Technol. 16, 1055–1058.CrossRefGoogle Scholar
  30. 30.
    Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956), Anal. Chem. 28, 350–354.CrossRefGoogle Scholar
  31. 31.
    Pandey, M. and Mishra, S. (1995), J. Ferment. Bioeng. 80, 446–453.CrossRefGoogle Scholar
  32. 32.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951), J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  33. 33.
    Vroeman, S., Heldens, J., Boyd, C., Henrisaat, B., and Keen, N. J. (1995), Mol. Gen. Genet. 246, 465–477.CrossRefGoogle Scholar
  34. 34.
    Marri, L., Valentini, S., and Venditti, D. (1995), FEMS Microbiol. Lett. 128, 135–138.PubMedGoogle Scholar
  35. 35.
    Painbeni, E., Valles, S., Poliana, J., and Flors, A. (1992), J. Bacteriol. 174, 3087–3091.PubMedGoogle Scholar
  36. 36.
    Sethi, B., Mishra, B., and Bisaria, V. S. (1999), Biotechnol. Bioprocess Eng. 4, 189–194.Google Scholar
  37. 37.
    Hrmova, M., Petrakova, E., and Biely, P. (1991), J. Gen. Microbiol. 137, 541–547.PubMedGoogle Scholar
  38. 38.
    Loewenberg, J. R. (1984), Arch. Microbiol. 137, 53–57.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Preeti Bachhawat
    • 1
  • Saroj Mishra
    • 1
  • Yukti Bhatia
    • 1
  • V. S. Bisaria
    • 1
  1. 1.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiHauz-KhasIndia

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