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Screening for novel lipolytic enzymes from uncultured soil microorganisms

  • Genomics and Proteomics
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Abstract

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C4) but not p-nitrophenyl palmitate (C16).

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Acknowledgements

We thank E.H. Lee for technical assistance with the subcloning. This research was supported by a grant (MG02-0101-0030202-0) to S.-W. Lee from the Microbial Genomics and Application Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea.

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Authors and Affiliations

  1. Biological Function Research Team, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu, Daejeon, 305-343, South Korea

    Seon-Woo Lee, He Kyoung Lim, Jin-Cheol Kim, Gyung Ja Choi & Kwang Yun Cho

  2. Fine and Bio-Chemical Process R & D Center, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu, Daejeon, 305-343, South Korea

    Keehoon Won

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  1. Seon-Woo Lee
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  2. Keehoon Won
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  3. He Kyoung Lim
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  4. Jin-Cheol Kim
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Correspondence to Seon-Woo Lee.

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Lee, SW., Won, K., Lim, H.K. et al. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl Microbiol Biotechnol 65, 720–726 (2004). https://doi.org/10.1007/s00253-004-1722-3

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  • DOI: https://doi.org/10.1007/s00253-004-1722-3

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