title: Cloning and characterization of two functionally diverse lipases from soil metagenome.
abstract: A soil metagenomic library was constructed and two functionally diverse lipase genes, SMlipB and SMlipD, were screened by a function-driven approach and characterized. The optimal temperature for enzyme activity of SMlipB and SMlipD was 50°C and 30°C, respectively, and optimal pH was determined to be 7.0 and 9.0, respectively. Both enzymes exhibited broad substrate specificity and showed enhanced activity in the presence of SDS and Tween 20. The SMlipB enzyme was highly resistant to many organic solvents, especially isopropanol, ethanediol, DMSO, methanol and xylene, whereas SMlipD activity was inhibited in all the solvents except xylene. Sequence analysis revealed SMlipB consisted of an open reading frame of 1,212 bp and encoded for 404 amino acids. It contained the GXXGXD motifs, which are supposed to be involved in Ca(2+) binding in proteases and lipases, and an extreme C-terminal motif consisting of a negatively charged amino acid followed by four hydrophobic residues, essential for the secretion of metalloprotease, and belongs to lipase subfamily I.3. SMlipD contained 1,071 bp ORF and encoded for 357 amino acids. It contains Ca(2+) ion binding sites extending from amino acid 282 to 294 and two Cystein residues (218,308), proven necessary for forming a disulfide bridge and belongs to lipase subfamily1.2.
authors.affiliation: Department of Food Microbiology, Central Food Technological Research Institute CSIR, Myscore 570020, India. mahejibin@cftri.org
authors.forename: Mahejibin
authors.initials: M
authors.lastname: Khan
authors.forename: K
authors.initials: K
authors.lastname: Jithesh
authors.forename: R
authors.initials: R
authors.lastname: Mookambikay
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D054892:Metagenome; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D008049:Lipase