Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment.

Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33-58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant proteins in Escherichia coli. The purified enzymes had optimum temperatures of 30-35°C and the cold-activity property. Among them, one enzyme showed lipase activity by preferentially hydrolyzing p-nitrophenyl palmitate and p-nitrophenyl stearate and high salt resistance with up to 4 M NaCl. Our research demonstrates the feasibility of developing novel lipolytic enzymes from marine environments by the combination of functional metagenomic approach and protein expression technology.

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.

Functional proteolytic complexes of the human mitochondrial ATP-dependent protease, hClpXP.

Human mitochondrial ClpP (hClpP) and ClpX (hClpX) were separately cloned, and the expressed proteins were purified. Electron microscopy confirmed that hClpP forms heptameric rings and that hClpX forms a hexameric ring. Complexes of a double heptameric ring of hClpP with hexameric hClpX rings bound on each side are stable in the presence of ATP or adenosine 5'-(3-thiotriphosphate) (ATPgammaS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPgammaS-dependent peptidase activity. hClpXP cannot degrade lambdaO protein or GFP-SsrA, specific protein substrates recognized by Escherichia coli (e) ClpXP. However, eClpX interacts with hClpP, and, when examined by electron microscopy, the resulting heterologous complexes are indistinguishable from homologous eClpXP complexes. The hybrid eClpX-hClpP complexes degrade eClpX-specific protein substrates. In contrast, eClpA can neither associate with nor activate hClpP. hClpP has an extra C-terminal extension of 28 amino acids. A mutant lacking this C-terminal extension interacts more tightly with both hClpX and eClpX and shows enhanced enzymatic activities but still does not interact with eClpA. Our results establish that human ClpX and ClpP constitute a bone fide ATP-dependent protease and confirm that substrate selection, which differs between human and E. coli ClpX, is dependent solely on the Clp ATPase. Our data also indicate that human ClpP has conserved sites required for interaction with eClpX but not eClpA, implying that the modes of interaction with ClpP may not be identical for ClpA and ClpX.