Substitution of glycine 275 by glutamate (G275E) in lipase of Bacillus stearothermophilus affects its catalytic activity and enantio- and chain length specificity

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Title
Substitution of glycine 275 by glutamate (G275E) in lipase of Bacillus stearothermophilus affects its catalytic activity and enantio- and chain length specificity
Author(s)
Myung Hee Kim; Hyung Kwoun Kim; Byung Chul Oh; Tae Kwang Oh
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 10, no. 6, pp. 764-769
Publication Year
2000
Abstract
The lipase gene (lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution; S113 (AGC→AGT), L252 (TTG→TTA), and G275E (GGA→GAA). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity (Vmax) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was 68°C and it showed a typical Ca2+-dependent thermostability, properties of which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.
Keyword
bacillus stearothermophilusG275E mutant lipaselipasemolecular evolutionsubstrate specificity
ISSN
1017-7825
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
Division of Biomedical Research > Metabolic Regulation Research Center > 1. Journal Articles
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