title: A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters.
abstract: BACKGROUND
  Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. However, since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud.
  
  
  RESULTS
  Based on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48-79 % similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which has 316 amino acids with a predicted molecular weight of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity: 1389 U mg(-1)) at 45 °C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 °C, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90 % of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concentrations (up to 3.0 M). These results demonstrate a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concentrations in non-aqueous media.
  
  
  CONCLUSIONS
  This manuscript reports unprecedented alcohol tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high organic solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. wygao@mail.ecust.edu.cn.
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. 403773076@qq.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. bioclf@163.com.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. fsea.lurker@gmail.com.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. 1032044926@qq.com.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. gaobei@ecust.edu.cn.
authors.forename: Bei
authors.initials: B
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. hlwang@ecust.edu.cn.
authors.forename: Hualei
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. dzhwei@ecust.edu.cn.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D002999:Clone Cells; D019295:Computational Biology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D004950:Esterases / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D015723:Gene Library; D019015:Geologic Sediments / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D008066:Lipolysis / Q000187:drug effects / Q000235:genetics; D056186:Metagenomics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D012623:Seawater; D017422:Sequence Analysis, DNA; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013649:Taste; D013696:Temperature
chemical_list: D003902:Detergents; D004952:Esters; D007477:Ions; D008670:Metals; D012997:Solvents; D004950:Esterases