Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

Masashi Kiyohara, Keishi Sakaguchi, Kuniko Yamaguchi, Toshiyoshi Araki, Takashi Nakamura, Makoto Ito

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

Original languageEnglish
Pages (from-to)949-957
Number of pages9
JournalBiochemical Journal
Volume388
Issue number3
DOIs
Publication statusPublished - Jun 15 2005

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Vibrio
Cloning
Molecular Cloning
Bacteria
Carbohydrates
Enzymes
Hydrolysis
Glycoside Hydrolases
Oligosaccharides
Mannans
Nucleophiles
Glycols
Xylose
Enzyme activity
Substrates
Viperidae
Genes
Open Reading Frames
Sequence Analysis
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4. / Kiyohara, Masashi; Sakaguchi, Keishi; Yamaguchi, Kuniko; Araki, Toshiyoshi; Nakamura, Takashi; Ito, Makoto.

In: Biochemical Journal, Vol. 388, No. 3, 15.06.2005, p. 949-957.

Research output: Contribution to journalArticle

Kiyohara, Masashi ; Sakaguchi, Keishi ; Yamaguchi, Kuniko ; Araki, Toshiyoshi ; Nakamura, Takashi ; Ito, Makoto. / Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4. In: Biochemical Journal. 2005 ; Vol. 388, No. 3. pp. 949-957.
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