BBB : Vol. 65 (2001) , No. 3 pp.487-494

Cloning, Expression, and Characterization of a Family 52 β-Xylosidase Gene (xysB) of a Multiple-xylanase-producing Bacterium, Aeromonas caviae ME-1

Tohru SUZUKI¹⁾, Emiko KITAGAWA²⁾, Fukumitsu SAKAKIBARA²⁾, Keiji IBATA²⁾, Kengo USUI²⁾ and Keiichi KAWAI²⁾

1) Molecular Genetics Research Center
2) Department of Biotechnology, Faculty of Agriculture, Gifu University

(Received March 27, 2000)
(Accepted November 10, 2000)

A λ phage genomic library of Aeromonas caviae ME-1, a multiple-xylanase-producing bacterium, was screened for xylan degradation activities. We isolated one clone, B65, which had weak xylanase activity, by the DNS method, but gave no visible bands on zymogram assay using SDS-xylan-PAGE. Based on TLC analyses of enzymatic products and some glycosidase assays using p-nitrophenyl substrates, we established that pB65 encodes a β-xylosidase gene. In the nucleotide sequence analysis, we found a 2190-bp open reading frame (ORF) named xysB. XysB protein is similar to some β-xylosidases, which are categorized in the glycosyl hydrolase family 52. Another ORF (xyg), that showed similarity to the family 67 α-glucuronidase, was also found downstream of the xysB gene. The xysB ORF and its promoter region were cloned into the pT7-Blue vector and the transformant cells had β-xylosidase activity. The relative molecular mass were estimated to be 75 kDa by SDS-PAGE and 159 kDa by gel filtration. These data showed that XysB has a dimeric structure of 80, 697 Da subunits. This enzyme showed optimal activity at 50°C and pH 6.0. It was stable below 40°C and pH 5-8. The Km and Vmax were calculated to be 0.34 mM and 33 nmol·min^-1·μg^-1, respectively. This enzyme also showed transglycosylation activity against X₃ and produced X₄ and X₅.

Key words:

Aeromonas caviae ME-1; β-Xylosidase; xysB; glycosyl hydrolase family 52; α-glucuronidase