(3R)-2-羧乙基-3-氰基-5-甲基己酸乙酯水解酶产生菌的筛选与产酶条件优化

以2-羧乙基-3-氰基-5-甲基己酸乙酯为唯一碳源,采用手性气相法检测,筛选到一株能产对映选择性水解酶的假单胞菌Pseudomonas CGMCC No.4184.该菌产的水解酶能优先水解R型底物产生(3R)-2-羧乙基-3-氰基-5-甲基己酸,产物对映体过量值达到90%以上.对菌株Pseudomonas CGMCC ...

Strain Screening and Culture Condition Optimization for the Enantioselectively Hydrolysis of(3R) - 2-Carboxyethyl-3-Cyano-5-Methylhexanoic Acid Ethyl Ester

Using (R, S)-2-carboxyethyl-3-cyano-5-methyl-hexanoic acid ethylester (rac-CCMAE) as sole carbon source, Pseudomonas sp. CGMCC No. 4184 was isolated which can enantioselectively hydrolyze (R)-CCMAE to (3R)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (3R-CCMA) with ee_p of over 90%. The optimal temperature and pH for the production of hydrolase by Pseudomonas CGMCC No. 4184 were 30℃ and 7.4. Single-factor experiment, Plackett-Burman design and response surface methodology were applied subsequently to optimize the fermentation medium. After optimization, the enzyme activity increased from 15.92U/L to 33.43 U/L (about 2.1 fold). By using resting cell catalysis system, (S)-CCMAE were successfully prepared with the conversion of 56.3% and ee_s of 98.3% after 48 h under the condition of 5g/L CCMAE, 8g/L (cell dry weight) resting cells, 20ml 0.1mol/L phosphate buffer (pH 7.4), 30℃ and 200r/min.