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

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          Abstract

          Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus beta-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 beta(1-4) and beta(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl beta-laminarin oligosaccharides of degree of polymerisation 2-8 were synthesized enzymatically. Catalytic efficiency values, k (cat)/K (m), 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-beta-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 beta-glucopyranosyl-beta-1,3-D-glucopy- ranosyl-beta-1,3-D-glucopyranose and beta-glucopyranosyl-beta-1, 3-D-glucopyranosyl-beta-1,3-D-glucpyranosyl-beta-1,3-D- glucopyranoxside.

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          Author and article information

          Journal
          Glycoconj. J.
          Glycoconjugate journal
          Springer Nature
          0282-0080
          0282-0080
          Nov 2006
          : 23
          : 7-8
          Affiliations
          [1 ] Molecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science, Gatchina, 188300, Russia.
          Article
          10.1007/s10719-006-6733-0
          17006642
          99cf5d2d-a0d7-465d-b3be-4edec76b4acf
          History

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