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

Neustroev, Kirill N., Golubev, Alexander M, Sinnott, Michael L., Borriss, Rainer, Krah, Martin, Brumer III, Harry, Eneyskaya, Elena V., Shishlyannikov, Sergey, Shabalin1, Konstantin A., Peshechonov, Viacheslav T., Korolev, Vladimir G. and Kulminskaya, Anna A. (2006) Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants. Glycoconjugate Journal, 23 (7/8). pp. 501-511. ISSN 0282-0080

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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, 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-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

Item Type: Article
Subjects: Q Science > Q Science (General)
Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Schools: School of Applied Sciences
School of Applied Sciences > Biomolecular Sciences Research Centre
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Depositing User: Sara Taylor
Date Deposited: 14 Mar 2008 15:18
Last Modified: 07 Apr 2018 20:00
URI: http://eprints.hud.ac.uk/id/eprint/580

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