The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichiacoli 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.
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Research Article|
December 17 1999
Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies
Maher ABOU HACHEM;
Maher ABOU HACHEM
1
*Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
1To whom correspondence should be addressed (e-mail maher.abou-hachem@;biotek.lu.se).
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Eva NORDBERG KARLSSON;
Eva NORDBERG KARLSSON
*Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
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Eva BARTONEK-ROXÅ;
Eva BARTONEK-ROXÅ
*Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
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Srinivasrao RAGHOTHAMA;
Srinivasrao RAGHOTHAMA
†Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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Peter J. SIMPSON;
Peter J. SIMPSON
†Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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Harry J. GILBERT;
Harry J. GILBERT
‡Department of Biological and Nutritional Sciences, University of Newcastle, Newcastle upon Tyne NE1 7RU, U.K.
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Michael P. WILLIAMSON;
Michael P. WILLIAMSON
†Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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Olle HOLST
Olle HOLST
*Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
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Publisher: Portland Press Ltd
Received:
May 05 1999
Revision Received:
September 02 1999
Accepted:
October 14 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 345 (1): 53–60.
Article history
Received:
May 05 1999
Revision Received:
September 02 1999
Accepted:
October 14 1999
Citation
Maher ABOU HACHEM, Eva NORDBERG KARLSSON, Eva BARTONEK-ROXÅ, Srinivasrao RAGHOTHAMA, Peter J. SIMPSON, Harry J. GILBERT, Michael P. WILLIAMSON, Olle HOLST; Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies. Biochem J 1 January 2000; 345 (1): 53–60. doi: https://doi.org/10.1042/bj3450053
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