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

TitleTransferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants
Publication TypeJournal Article
Year of Publication2006
AuthorsNeustroev, KN, Golubev, AM, Sinnott, ML, Borriss, R, Krah, M, Brumer, H, Eneyskaya, EV, Shishlyannikov, S, Shabalin, KA, Peshechonov, VT, Korolev, VG, Kulminskaya, AA
JournalGLYCOCONJUGATE JOURNAL
Volume23
Pagination501-511
Date PublishedNOV
Type of ArticleArticle
ISSN0282-0080
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.

DOI10.1007/s10719-006-6733-0