(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

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

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 No.4184的产酶条件进行了研究,结果表明该菌产酶最适温度为30℃,最适pH为7.4。通过单因素试验、Plackett-Burman试验和响应面法研究并优化了产酶培养基组成,使酶活提高到33.43 U/L,比初始酶活(15.92 U/L)提高了2.1倍。利用静息细胞催化水解底物,当底物浓度和静息细胞干重分别为5 g/L和8 g/L时,在pH 7.4的磷酸缓冲液中反应48 h(温度30℃,摇床转速200 r/min),转化率为56.3%,底物对映体过量值达到98.3%,较好地实现了拆分制备(3S)-2-羧乙基-3-氰基-5-甲基己酸乙酯的目标。

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 eep 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 ees 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.