Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome.
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 degrees C, respectively, and rEML1 displayed more than 50% activity at 5 degrees 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 > or = 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.
Jeon
Jeong Ho
JH
Marine Biotechnology Center, Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul 425-600, South Korea.
Kim
Jun-Tae
JT
Kim
Yun Jae
YJ
Kim
Hyung-Kwoun
HK
Lee
Hyun Sook
HS
Kang
Sung Gyun
SG
Kim
Sang-Jin
SJ
Lee
Jung-Hyun
JH
D002210:Caprylates / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D019015:Geologic Sediments / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D000069463:Olive Oil; D010802:Phylogeny; D010938:Plant Oils / Q000378:metabolism; D011136:Polysorbates / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000378:metabolism
D002210:Caprylates; D004791:Enzyme Inhibitors; D000069463:Olive Oil; D010938:Plant Oils; D011136:Polysorbates; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; C003637:tricaprylin; D017830:Octoxynol; D008049:Lipase