title: Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization.
abstract: An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55 degrees C. It was highly stable in the temperature range of 30-65 degrees C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37 degrees C in 0.1% Chaps and Triton X-100.
authors.affiliation: Department of Chemistry, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors.forename: S
authors.initials: S
authors.lastname: Sinchaikul
authors.forename: B
authors.initials: B
authors.lastname: Sookkheo
authors.forename: S
authors.initials: S
authors.lastname: Phutrakul
authors.forename: F M
authors.initials: FM
authors.lastname: Pan
authors.forename: S T
authors.initials: ST
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003902:Detergents; D008049:Lipase