Glycoconjugate Journal

, Volume 23, Issue 7, pp 501–511

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Authors

  • Kirill N. Neustroev
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
  • Alexander M. Golubev
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
  • Michael L. Sinnott
    • Department of Chemical and Biological SciencesUniversity of Huddersfield, Queensgate
  • Rainer Borriss
    • AG Bakteriengenetik, Institut fur BiologieHumboldt Universitt Berlin Chausseestrasse 117
  • Martin Krah
    • AG Bakteriengenetik, Institut fur BiologieHumboldt Universitt Berlin Chausseestrasse 117
  • Harry BrumerIII
    • Department of Biotechnology, Royal Institute of Technology (KTH)AlbaNova University Centre
  • Elena V. Eneyskaya
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
  • Sergey Shishlyannikov
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
  • Konstantin A. Shabalin
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
  • Viacheslav T. Peshechonov
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
  • Vladimir G. Korolev
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
    • Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of Science
Article

DOI: 10.1007/s10719-006-6733-0

Cite this article as:
Neustroev, K.N., Golubev, A.M., Sinnott, M.L. et al. Glycoconj J (2006) 23: 501. doi:10.1007/s10719-006-6733-0

Abstract

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, kcat/Km, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside.

Keywords

LaminarinaseRhodothermus marinusp-nitrophenyl β-laminarin oligosaccharidesTransglycosylation

Copyright information

© Springer Science + Business Media, LLC 2006