Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases |
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Authors: | Huiying Luo Jun Yang Jiang Li Pengjun Shi Huoqing Huang Yingguo Bai Yunliu Fan Bin Yao |
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Institution: | (1) Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People’s Republic of China;(2) Department of Biology, East China Institute of Technology, Fuzhou, Jiangxi, 344000, People’s Republic of China;(3) Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China; |
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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 K
m and V
max 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. |
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