title: Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library.
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.
authors.affiliation: 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.
authors.forename: Chengzhang
authors.initials: C
authors.lastname: Fu
authors.forename: Yongfei
authors.initials: Y
authors.lastname: Hu
authors.forename: Feng
authors.initials: F
authors.lastname: Xie
authors.forename: Hui
authors.initials: H
authors.lastname: Guo
authors.forename: Elizabeth Jane
authors.initials: EJ
authors.lastname: Ashforth
authors.forename: Steven W
authors.initials: SW
authors.lastname: Polyak
authors.forename: Baoli
authors.initials: B
authors.lastname: Zhu
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015698:Genomic Library; D019015:Geologic Sediments / Q000737:chemistry / Q000382:microbiology*; D007700:Kinetics; D056186:Metagenomics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012623:Seawater / Q000382:microbiology*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004950:Esterases