title: Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment.
abstract: To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50-57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.
authors: Marine Biotechnology Research Center, Korea Ocean Research & Development Institute, P.O. Box 29, Ansan, 425-600, South Korea.
authors: Jeong Ho
authors: JH
authors: Jeon
authors: Hyun Sook
authors: HS
authors: Lee
authors: Jun Tae
authors: JT
authors: Kim
authors: Sang-Jin
authors: SJ
authors: Kim
authors: Sang Ho
authors: SH
authors: Choi
authors: Sung Gyun
authors: SG
authors: Kang
authors: Jung-Hyun
authors: JH
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D016000:Cluster Analysis; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D019015:Geologic Sediments; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D056910:Republic of Korea; D012492:Salts / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012492:Salts; D004950:Esterases