Journal of Biochemistry Advance Access originally published online on July 15, 2009
Journal of Biochemistry 2009 146(5):633-641; doi:10.1093/jb/mvp108
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Characterization and Application of Carbohydrate-binding Modules of β-1,3-xylanase XYL4
1Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581; 2Department of life Science, Faculty of Bioresources, Mie University, Mie 514-8507; and 3Bio-Architecture Center, Kyushu University, Fukuoka 812-8581, Japan
*To whom correspondence should be addressed. Tel: +81 92 642 2898, Fax: +81 92 642 2907, E-mail: makotoi{at}agr.kyushu-u.ac.jp
Received April 3, 2009; Accepted June 19, 2009
 |
Abstract |
|---|
β-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 β-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble β-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble β-1,3-xylan, but not to β-1,4-xylan, Avicel, β-1,4-mannan, curdlan, chitin or soluble glycol-β-1,3-xylan. Both the enzyme and the binding activities for insoluble β-1,3-xylan but not soluble glycol-β-1,3-xylan were enhanced by NaCl in a concentration-dependent manner, indicating that the CBMs of XYL4 bound to β-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 β-1,3-xylan-binding proteins including β-1,3-xylanase from the extract of a turban shell, Turbo cornutus.
Key Words: β-1,3-xylan, β-1,3-xylanase, carbohydrate-binding module, deletion mutant, glycoside hydrolase
Abbreviations: AlcCBM, CBM of Alcaligenes sp. strain XY-234; BSA, bovine serum albumin; CBB, Coomassie brilliant blue; CBM(s), carbohydrate-binding module(s); CM, catalytic module; GH, glycoside hydrolase; IPTG, isopropyl β-D-thiogalactoside; PCR, polymerase chain reaction; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electophoresis; TBS, Tris-buffered saline; T-TBS, TBS containing 0.02% Tween 20; TLC, thin-layer chromatography; XYL4, β-1,3-xylanase from Vibrio sp. strain AX-4