Characterization and Application of Carbohydrate-binding Modules of -1,3-xylanase XYL4

Abstract

beta-1,3-Xylanase from Vibrio sp. strain AX-4 (XYL4) is a modular enzyme composed of an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules (CBMs) belonging to family 31 in the C-terminal region. To investigate the functions of these three modules, five deletion mutants lacking individual modules were constructed. The binding assay of these mutants showed that a repeating unit of the CBM was a non-catalytic beta-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble beta-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble beta-1,3-xylan, but not to beta-1,4-xylan, Avicel, beta-1,4-mannan, curdlan, chitin or soluble glycol-beta-1,3-xylan. Both the enzyme and the binding activities for insoluble beta-1,3-xylan but not soluble glycol-beta-1,3-xylan were enhanced by NaCl in a concentration-dependent manner, indicating that the CBMs of XYL4 bound to beta-1,3-xylan through hydrophobic interaction. This property of the CBMs was successfully applied to the purification of a recombinant XYL4 from the cell extracts of Escherichia coli transformed with the xyl4 gene and the detection of beta-1,3-xylan-binding proteins including beta-1,3-xylanase from the extract of a turban shell, Turbo cornutus.