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 Escherichi a coli and purified by affinity chromatography, Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-s wollen cellulose but not to Avicel or crystalline cellulose. Binding to ins oluble substrates was significantly enhanced by the presence of Na+ and Ca2 + ions. The binding affinities for soluble polysaccharides were tested by a ffinity 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 specificit ies. Binding to short oligosaccharides was measured by NMR; both modules bo und with similar affinities. The binding of the modules was shown to be dom inated 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 aff inity for both insoluble xylan and amorphous cellulose.