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Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.

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Acknowledgments

We thank Dr. Simon Shih for helpful discussions. This work was supported in part by grants from the National 863 Project (2007AA09Z443), the Key Project for International Cooperation (2007DFB31620), the National Natural Science Foundation of China (30973665, 30700015, 30873129, 30901849, 30911120484, 81011120046, and 30911120483), the National Key Technology R&D Program (2007BAI26B02), the CAS Pillar Program (KSCX2-YW-R-164), and from the Important National Science & Technology Specific Projects (2008ZX09401-05, 2009ZX09302-004). L. Z. received funding from the Hundred Talents Program.

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Authors and Affiliations

  1. Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People’s Republic of China

    Chengzhang Fu, Yongfei Hu, Feng Xie, Hui Guo, Elizabeth Jane Ashforth, Baoli Zhu & Lixin Zhang

  2. South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, People’s Republic of China

    Lixin Zhang

  3. Graduate School of the Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Chengzhang Fu, Yongfei Hu, Feng Xie & Hui Guo

  4. School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide, South Australia, 5005, Australia

    Steven W. Polyak

  5. Institute of Microbiology, Chinese Academy of Sciences, No. 8 Zhongguancun Bei er tiao, Haidian District, Beijing, 100190, People’s Republic of China

    Lixin Zhang

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  1. Chengzhang Fu
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  2. Yongfei Hu
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  3. Feng Xie
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  4. Hui Guo
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  5. Elizabeth Jane Ashforth
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  6. Steven W. Polyak
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  7. Baoli Zhu
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Correspondence to Baoli Zhu or Lixin Zhang.

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Chengzhang Fu and Yongfei Hu contributed equally to this article.

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Fu, C., Hu, Y., Xie, F. et al. Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library. Appl Microbiol Biotechnol 90, 961–970 (2011). https://doi.org/10.1007/s00253-010-3079-0

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  • DOI: https://doi.org/10.1007/s00253-010-3079-0

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