title: Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability.
abstract: An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.
authors.affiliation: Microbial Enzyme, RU, Korea Research Institute of Bioscience & Biotechnology, Yusong, Taejon.
authors.forename: M H
authors.initials: MH
authors.lastname: Kim
authors.forename: H K
authors.initials: HK
authors.lastname: Kim
authors.forename: J K
authors.initials: JK
authors.lastname: Lee
authors.forename: S Y
authors.initials: SY
authors.lastname: Park
authors.forename: T K
authors.initials: TK
authors.lastname: Oh
mesh_terms: D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D013696:Temperature
chemical_list: D017931:DNA Primers; D008049:Lipase; D002118:Calcium