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Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome

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Abstract

To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25°C, respectively, and rEML1 displayed more than 50% activity at 5°C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of ≥8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome.

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Acknowledgement

This work was supported by KORDI in-house program (PE98230) and the Marine and Extreme Genome Research Center Program, Ministry of Marine Affairs and Fisheries, Republic of Korea.

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Authors and Affiliations

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

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

  2. Division of Biotechnology, The Catholic University of Korea, 43-1 Yokkok 2-dong, Wonmi-gu, Bucheon, Gyeonggido, 420-743, South Korea

    Hyung-Kwoun Kim

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  1. Jeong Ho Jeon
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Correspondence to Jung-Hyun Lee.

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Jeon, J.H., Kim, JT., Kim, Y.J. et al. Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol 81, 865–874 (2009). https://doi.org/10.1007/s00253-008-1656-2

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  • DOI: https://doi.org/10.1007/s00253-008-1656-2

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