Cloning, expression, and characterization of a family 52 beta-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1.
A lambda 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 beta-xylosidase gene. In the nucleotide sequence analysis, we found a 2190-bp open reading frame (ORF) named xysB. XysB protein is similar to some beta-xylosidases, which are categorized in the glycosyl hydrolase family 52. Another ORF (xyg), that showed similarity to the family 67 alpha-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 beta-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 degrees C and pH 6.0. It was stable below 40 degrees C and pH 5-8. The Km and Vmax were calculated to be 0.34 mM and 33 nmol x min(-1) x microg(-1), respectively. This enzyme also showed transglycosylation activity against X3 and produced X4 and X5.
Suzuki
T
T
Molecular Genetics Research Center, Gifu University, Japan. suzuki@cc.gifu-u.ac.jp
Kitagawa
E
E
Sakakibara
F
F
Ibata
K
K
Usui
K
K
Kawai
K
K
D000333:Aeromonas / Q000201:enzymology; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D014990:Xylans; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; C026579:exo-1,4-beta-D-xylosidase