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...

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Bibliographic Details
Published in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2004-01, Vol.68 (1), p.96-103
Main Authors: Leow, T.C. (Universiti Putra Malaysia, Serdang), Rahman, R.N.Z.R.A, Basri, M, Salleh, A.B
Format: Article
Language:English
Subjects:
Amino Acid Sequence
AMINO ACID SEQUENCES
Amino Acid Substitution
Bacillaceae - enzymology
Bacillaceae - genetics
BACTERIA
Base Sequence
Biological and medical sciences
cloning
Cloning, Molecular
Conserved Sequence
Enzyme Stability
ENZYMES
ESTERASES
Fundamental and applied biological sciences. Psychology
GENE EXPRESSION
Gene Expression Regulation, Bacterial
Gene Expression Regulation, Enzymologic
GENETIC ENGINEERING
Geobacillus
Geobacillus sp
Glutathione S-transferase (GST) fusion protein
Glutathione Transferase - genetics
Glutathione Transferase - metabolism
Hydrogen-Ion Concentration
Lipase - genetics
Lipase - metabolism
MOLECULAR CLONING
Molecular Sequence Data
overexpression
PEPTIDES
Polymerase Chain Reaction
Promoter Regions, Genetic
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
RECOMBINATION
thermostable lipase
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Description
Summary: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°C and pH 9, respectively. It was stable up to 65°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.
ISSN:0916-8451
1347-6947
DOI:10.1271/bbb.68.96