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Applied Microbiology and Biotechnology

, Volume 86, Issue 6, pp 1829–1839 | Cite as

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

  • Huiying Luo
  • Jun Yang
  • Jiang Li
  • Pengjun Shi
  • Huoqing Huang
  • Yingguo Bai
  • Yunliu Fan
  • Bin YaoEmail author
Biotechnologically Relevant Enzymes and Proteins

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.

Keywords

Acidic xylanase Glycosyl hydrolase family 30 Bispora sp. MEY-1 Pichia pastoris 

Notes

Acknowledgements

This work was supported by the National High Technology Research and Development Program of China (863 program, Grant No. 2007AA100601), Key Program of Transgenic Plant Breeding (2008ZX08003-002), and the Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-42-G2-05). We thank Dr. Kenji Fukuda of Obihiro University of Agriculture and Veterinary Medicine, Japan, for his generosity to provide substrate pustulan.

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Huiying Luo
    • 1
  • Jun Yang
    • 1
  • Jiang Li
    • 2
  • Pengjun Shi
    • 1
  • Huoqing Huang
    • 1
  • Yingguo Bai
    • 1
  • Yunliu Fan
    • 3
  • Bin Yao
    • 1
    Email author
  1. 1.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research InstituteChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.Department of BiologyEast China Institute of TechnologyFuzhouPeople’s Republic of China
  3. 3.Biotechnology Research InstituteChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China

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