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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

Authors 

Myung Hee KimHyung Kwoun KimByung Chul OhTae Kwang Oh

Publisher 

The Korean Society for Applied Microbiology

Issue Date 

2000

Citation 

Journal of Microbiology and Biotechnology, vol. 10, no. 6, pp. 764-769

Keywords 

bacillus stearothermophilusG275E mutant lipaselipasemolecular evolutionsubstrate specificityglycinetriacylglycerol lipaseamino acid substitutionenzyme specificitygeobacillus stearothermophilus

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.

ISSN 

1017-7825

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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