Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants
2006 (English)In: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 23, no 08-jul, 501-511 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2006. Vol. 23, no 08-jul, 501-511 p.
laminarinase, Rhodothermus marinus, p-nitrophenyl beta-laminarin oligosaccharides, transglycosylation, bacillus 1,3-1,4-beta-d-glucan 4-glucanohydrolases, oligosaccharide synthesis, enzymatic-synthesis, transglycosylation activity, aspergillus-niger, beta-glucosidase, alpha-amylase, active-site, mechanism, (1->3)-beta-d-glucan
IdentifiersURN: urn:nbn:se:kth:diva-16010DOI: 10.1007/s10719-006-6733-0ISI: 000240855200005ScopusID: 2-s2.0-33749165434OAI: oai:DiVA.org:kth-16010DiVA: diva2:334052
QC 201005252010-08-052010-08-05Bibliographically approved