Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies.
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 Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-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.
Abou Hachem
M
M
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, SE-221 00 Lund, Sweden.
Nordberg Karlsson
E
E
Bartonek-Roxâ
E
E
Raghothama
S
S
Simpson
P J
PJ
Gilbert
H J
HJ
Williamson
M P
MP
Holst
O
O
D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D011489:Protein Denaturation; D013379:Substrate Specificity; D013816:Thermodynamics; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase