Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hy drolases, a family 9 carbohydrate-binding module (CBM), and two S-layer hom ologous (SLH) domains [Biosci, Biotechnol. Biochem,, 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn 10B and compared their hydrolytic activity toward xylan and some preparatio ns of plant cell walls; Xyn10B Delta CBM consists of a catalytic domain onl y, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulo se, ball-milled chitin, Sephadex G-25, and amyloseresin, A cellulose bindin g assay in the presence of soluble saccharides suggested that the CBM of Xy n10B had an affinity for even monosaccharides such as glucose, galactose, x ylose, mannose and ribose, Removal of the CBM from the enzyme negated its c ellulose- and xylan-binding abilities and severely reduced its enzyme activ ity toward insoluble xylan and plant cell walls but not soluble xylan. Thes e findings clearly indicated that the CBM of Xyn10B is important in the hyd rolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and t he ability to increase hydrolytic activity toward insoluble xylan.