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Title 

Thermostable lipase of Bacillus stearothermophilus: high-level production, purification, and calcium-dependent thermostability

Authors 

Myung Hee KimHyung Kwoun KimJung Kee LeeSun Yang ParkTae Kwang Oh

Publisher 

Japan Society for Bioscience, Biotechnology and Agrochemistry

Issue Date 

2000

Citation 

Bioscience Biotechnology and Biochemistry, vol. 64, no. 2, pp. 280-286

Keywords 

lipaseBacillus stearothermophilustryptophan fluorescence emissioncalcium-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°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°C and it was stable up to 55°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°C in the presence of calcium ions but at 58°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.

ISSN 

0916-8451

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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