title: High level expression of thermostable lipase from Geobacillus sp. strain T1.
abstract: A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
authors.affiliation: Enzyme and Microbial Technology Research Group, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, Serdang, Malaysia.
authors.forename: Thean Chor
authors.initials: TC
authors.lastname: Leow
authors.forename: Raja Noor Zaliha Raja Abdul
authors.initials: RN
authors.lastname: Rahman
authors.forename: Mahiran
authors.initials: M
authors.lastname: Basri
authors.forename: Abu Bakar
authors.initials: AB
authors.lastname: Salleh
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D008049:Lipase