Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment |
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Authors: | Jeong Ho Jeon Hyun Sook Lee Jun Tae Kim Sang-Jin Kim Sang Ho Choi Sung Gyun Kang Jung-Hyun Lee |
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Institution: | (1) Marine Biotechnology Research Center, Korea Ocean Research & Development Institute, P.O. Box 29, Ansan, 425-600, South Korea;(2) National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Seoul National University, Seoul, 151-921, South Korea;(3) Department of Marine Biotechnology, University of Science and Technology, Daejeon, 305-333, South Korea; |
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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. |
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