Identification and characterization of novel esterases from a deep-sea sediment metagenome |
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Authors: | Xiawei?Jiang Xuewei?Xu Yingyi?Huo Yuehong?Wu Xufen?Zhu Xinqi?Zhang Email author" target="_blank">Min?WuEmail author |
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Institution: | (1) College of Life Sciences, Zhejiang University, Room 209, 866 Yuhangtang Road, Hangzhou, 310058, China;(2) Laboratory of Marine Ecosystem and Biogeochemistry, State Oceanic Administration, Hangzhou, 310012, China;(3) Second Institute of Oceanography, State Oceanic Administration, Hangzhou, 310012, China; |
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Abstract: | A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine
novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes
in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic
enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and
characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured
bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6
revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5. |
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