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- Title
Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases.
- Authors
Huiying Luo; Yang, Jun; Jiang Li; Pengjun Shi; Huoqing Huang; Yingguo Bai; Yunliu Fan; Bin Yao
- Abstract
We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-β- d-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 °C, maintained more than 60% of maximal activity when assayed at pH 1.5–4.0, and had good thermal stability at 60 °C and remained stable at pH 1.0–6.0. The enzyme activity was enhanced in the presence of Ni2+ and β-mercaptoethanol and inhibited by some metal irons (Hg2+, Cu2+, Pb2+, Mn2+, Li+, and Fe3+) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4- O-methyl- d-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg−1, respectively. The apparent Km and Vmax values for beechwood xylan were 5.6 mg ml−1 and 3,622 μmol min−1 mg−1, respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.
- Subjects
XYLANASES; GLYCOSIDASES; GLYCOSYLATION; PICHIA pastoris; GENETIC engineering; HYDROGEN-ion concentration; ANTISENSE DNA; CLONE cells; ENTEROBACTERIACEAE; MICROBIOLOGY
- Publication
Applied Microbiology & Biotechnology, 2010, Vol 86, Issue 6, p1829
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-009-2410-0