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Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis

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Title: Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis
Authors: Hamura, Ken Browse this author
Saburi, Wataru Browse this author →KAKEN DB
Matsui, Hirokazu Browse this author →KAKEN DB
Mori, Haruhide Browse this author →KAKEN DB
Keywords: Cellobiose phosphorylase
Glycoside hydrolase family 94
Oligosaccharide synthesis
Site-directed mutagenesis
Acceptor specificity
Issue Date: 20-Sep-2013
Publisher: Elsevier sci ltd
Journal Title: Carbohydrate research
Volume: 379
Start Page: 21
End Page: 25
Publisher DOI: 10.1016/j.carres.2013.06.010
PMID: 23845516
Abstract: Cellobiose phosphorylase (EC, CBP) catalyzes the reversible phosphorolysis of cellobiose to alpha-D-glucose 1-phosphate (Glc1P) and D-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent k(cat)/K-m values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-beta-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4 h reaction, the amounts of 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively. (C) 2013 Elsevier Ltd. All rights reserved.
Type: article (author version)
Appears in Collections:農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 森春英

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