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

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 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.

Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

“…6B). Their activities also were comparable to the activities of previously characterized carboxyl esterases from Mediterranean deep-sea locations (average, 54.5 U mg Ϫ1 [3]) and other marine deep-sea sediment (ϳ1.70 to 560 U mg Ϫ1 [58][59][60]). The specific activities of these enzymes also were in the range of that observed for most similar proteins (34 to 39% sequence identity) that have been characterized (150 to 2,500 U mg Ϫ1 [61,62]).…”

A Short Review on Various Screening Methods to Isolate Potential Lipase Producers: Lipases-the Present and Future Enzymes of Biotech Industry

“…This approach also overcomes the problem of cultivation of microbes as majority of the micro organisms are not to cultivation (Handelsman et al, 1998). There were so many recent reports proving the novelty of this approach in the identification of true lipases (Jiang et al, 2006;Lee et al, 2006;Elend et al, 2007;Jeon et al, 2009;Liu et al, 2009;Meilleur et al, 2009;Glogauer et al, 2011).…”

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

“…Most of the previously studied lipases have temperature optima in the range of 30 to 60C, 382 although some extreme lipases were also found that exhibited high activity even at low or 383 high temperatures (Jeon et al, 2009;Joseph et al, 2008;Kulkarni & Gadre, 1999;Nawani & 384 Kaur, 2007). Adjustment of the temperatures for optimal lipase performance, depending on 385 application, can be achieved through the use of stabilizers such as ethylene glycol, sorbitol, or 386 glycerol (Franken et al, 2010;Gupta et al, 2004).…”