High level expression of thermostable lipase from Geobacillus sp. strain T1.
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.
Leow
Thean Chor
TC
Enzyme and Microbial Technology Research Group, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, Serdang, Malaysia.
Rahman
Raja Noor Zaliha Raja Abdul
RN
Basri
Mahiran
M
Salleh
Abu Bakar
AB
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
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D008049:Lipase