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Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment

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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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Acknowledgments

This work was supported by the KORDI in-house program (PE98513) and the Marine and Extreme Genome Research Center program and the Development of Biohydrogen Production Technology Using Hyperthermophilic Archaea program of the Ministry of Land, Transport, and Maritime Affairs, Republic of Korea.

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Authors and Affiliations

  1. Marine Biotechnology Research Center, Korea Ocean Research & Development Institute, P.O. Box 29, Ansan, 425-600, South Korea

    Jeong Ho Jeon, Hyun Sook Lee, Jun Tae Kim, Sang-Jin Kim, Sung Gyun Kang & Jung-Hyun Lee

  2. National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Seoul National University, Seoul, 151-921, South Korea

    Jeong Ho Jeon & Sang Ho Choi

  3. Department of Marine Biotechnology, University of Science and Technology, Daejeon, 305-333, South Korea

    Hyun Sook Lee, Sang-Jin Kim, Sung Gyun Kang & Jung-Hyun Lee

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  1. Jeong Ho Jeon
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  2. Hyun Sook Lee
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  3. Jun Tae Kim
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  4. Sang-Jin Kim
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  5. Sang Ho Choi
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  6. Sung Gyun Kang
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  7. Jung-Hyun Lee
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Correspondence to Jung-Hyun Lee.

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Fig. S1

SDS-PAGE of the purified EstKT4, EstKT7, and EstKT9. M: Molecular size markers, T: whole cell extracts, S: soluble fractions of cell extracts, P: enzymes purified using metal-affinity column chromatography (DOC 114 kb)

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Jeon, J.H., Lee, H.S., Kim, J.T. et al. Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment. Appl Microbiol Biotechnol 93, 623–631 (2012). https://doi.org/10.1007/s00253-011-3433-x

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  • DOI: https://doi.org/10.1007/s00253-011-3433-x

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