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Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: Cloning, expression and binding studies

Lookup NU author(s): Professor Harry Gilbert

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Abstract

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.


Publication metadata

Author(s): Abou Hachem M, Nordberg Karlsson E, Bartonek-Roxa E, Raghothama S, Simpson PJ, Gilbert HJ, Williamson MP, Holst O

Publication type: Article

Publication status: Published

Journal: Biochemical Journal

Year: 2000

Volume: 345

Issue: 1

Pages: 53-60

ISSN (print): 0264-6021

ISSN (electronic): 1470-8728

Publisher: Portland Press

URL: http://dx.doi.org/10.1042/0264-6021:3450053

DOI: 10.1042/0264-6021:3450053

PubMed id: 10600638


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