Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome |
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Authors: | Jeong Ho Jeon Jun-Tae Kim Yun Jae Kim Hyung-Kwoun Kim Hyun Sook Lee Sung Gyun Kang Sang-Jin Kim Jung-Hyun Lee |
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Institution: | (1) Marine Biotechnology Center, Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul, 425-600, South Korea;(2) Division of Biotechnology, The Catholic University of Korea, 43-1 Yokkok 2-dong, Wonmi-gu, Bucheon, Gyeonggido, 420-743, South Korea |
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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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Keywords: | Lipase Metagenome Deep-sea sediment |
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