Thermostable Lipase ofBacillus stearothermophilus: High-level Production, Purification, and Calcium-dependent Thermostability
- 1 January 2000
- journal article
- Published by Oxford University Press (OUP) in Bioscience, Biotechnology, and Biochemistry
- Vol. 64 (2) , 280-286
- https://doi.org/10.1271/bbb.64.280
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...Keywords
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