Enhanced activity of Thermomyces lanuginosus lipase by site-saturation mutagenesis for efficient biosynthesis of chiral intermediate of pregabalin
| Autor: | Yu-Guo Zheng, Hong-Ye Ma, Ren-Chao Zheng, Li-Tao Ruan, Xiao-Ling Tang |
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| Rok vydání: | 2016 |
| Předmět: |
Environmental Engineering
biology 010405 organic chemistry Stereochemistry Chemistry Mutagenesis Mutant Biomedical Engineering Wild type Bioengineering 010402 general chemistry 01 natural sciences 0104 chemical sciences Kinetic resolution chemistry.chemical_compound Biosynthesis Biochemistry biology.protein Specific activity Lipase Saturated mutagenesis Biotechnology |
| Zdroj: | Biochemical Engineering Journal. 113:12-18 |
| ISSN: | 1369-703X |
| DOI: | 10.1016/j.bej.2016.05.007 |
| Popis: | Thermomyces lanuginosus lipase (TLL) variants with enhanced activity for kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) were constructed by site-saturation mutagenesis. Single mutant S83T and double mutant S58L/S83T exhibited 2.69 and 5.46-fold improvement in their specific activity for CNDE over the wild type TLL. The catalytic efficiency of S83T and S58L/S83T mutants were significantly increased, with k cat / K m values of 11.3 and 27.3 mM −1 min −1 , which was 2.97 and 7.18 times higher than that of the wild type. The whole cell catalysis of 3 M CNDE by E sch e richia coli harboring mutant S58L/S83T (5% w/v) resulted in 44.8% yield and >96% ee P within 24 h. These encouraging results demonstrated the great potential of the modified TLL for efficient production of ( S )-2-carboxyethyl-3-cyano-5-methylhexaoic acid used as chiral intermediate for pregabalin. |
| Databáze: | OpenAIRE |
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