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J. Microbiol. Biotechnol. 2009; 19(3): 277-285

Published online March 28, 2009

Copyright © The Korean Society for Microbiology and Biotechnology.

Cloning, Sequencing, and Expression of the Gene Encoding a Multidomain Endo-β-1,4-Xylanase from Paenibacillus curdlanolyticus B-6, and Characterization of the Recombinant Enzyme

Rattiya Waeonukul 1, Patthra Pason 1, Khin Lay Kyu 1, Kazuo Sakka 2, Akihiko Kosugi 3, Yutaka Mori 3 and Khanok Ratanakhanokchai 1*

School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkuntien, Bangkok 10150, Thailand, 1Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu 514-8507, Japan, 2Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukaba, Ibaraki 305-8686, Japan

Abstract

The nucleotide sequence of the Paenibacillus curdlanolyticus
B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of
3,828 nucleotides encoding a protein of 1,276 amino acids
with a predicted molecular mass of 142,726 Da. Sequence
analysis indicated that Xyn10A is a multidomain enzyme
comprising nine domains in the following order: three family
22 carbohydrate-binding modules (CBMs), a family 10 catalytic
domain of glycosyl hydrolases (xylanase), a family 9 CBM,
a glycine-rich region, and three surface layer homology
(SLH) domains. Xyn10A was purified from a recombinant
Escherichia coli by a single step of affinity purification on
cellulose. It could effectively hydrolyze agricultural wastes
and pure insoluble xylans, especially low substituted insoluble
xylan. The hydrolysis products were a series of short-chain
xylooligosaccharides, indicating that the purified enzyme was
an endo-β-1,4-xylanase. Xyn10A bound to various insoluble
polysaccharides including Avicel, α-cellulose, insoluble
birchwood and oat spelt xylans, chitin, and starches, and
the cell wall fragments of P. curdlanolyticus B-6, indicating that
both the CBM and the SLH domains are fully functioning
in the Xyn10A. Removal of the CBMs from Xyn10A
strongly reduced the ability of plant cell wall hydrolysis.
These results suggested that the CBMs of Xyn10A play an
important role in the hydrolysis of plant cell walls.

Keywords: Paenibacillus curdlanolyticus, multidomain xylanase, gene cloning, carbohydrate-binding module, surface layer homology