Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library |
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Authors: | Chengzhang Fu Yongfei Hu Feng Xie Hui Guo Elizabeth Jane Ashforth Steven W Polyak Baoli Zhu Lixin Zhang |
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Institution: | (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;(2) South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, People’s Republic of China;(3) Graduate School of the Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China;(4) School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide, South Australia, 5005, Australia;(5) Institute of Microbiology, Chinese Academy of Sciences, No. 8 Zhongguancun Bei er tiao, Haidian District, Beijing, 100190, People’s Republic of China; |
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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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