利用产脂肪酶B的毕赤酵母工程菌生物拆分制备西司他丁关键手性中间体的体系优化

摘要

S （＋）2，2二甲基环丙烷甲酸（ S （＋） DMCPA）是合成西司他丁的关键手性中间体。构建的毕赤酵母工程菌生产的脂肪酶可高选择性催化外消旋2，2二甲基环丙烷甲酸乙酯（ DMCPE）不对称水解制备 S （＋） DMCPA。对该工程菌产胞外脂肪酶的产酶条件及生物拆分反应条件进行优化，以提高其催化效率。优化获得重组毕赤酵母最佳产酶条件：采用BMMY培养基，培养基初始pH 8�0，500 mL摇瓶装50 mL培养基，每隔4 h向培养基中补加终体积分数为1％的甲醇。培养192 h后，发酵液中脂肪酶比酶活为4�41 U／L，较优化前提高了90�9％。利用含脂肪酶的发酵上清液进行DMCPE的生物拆分反应，底物浓度为15 mmol／L，30℃反应36 h，S （＋） DMCPA的产率可达43�8％，e�e．值为99�8％。%S⁃(+)⁃2, 2⁃dimethylcyclopropane carboxylic acid ( S⁃(+)⁃DMCPA ) is a key chiral intermediate for the synthesis of cilastatin. A recombinant Pichia patoris can convert racemic ethyl⁃2,2⁃dimethylcyclopropane carboxylate ( DMCPE) to S⁃(+)⁃DMCPA with high enantioselectivity. In order to enhance its catalytic efficiency,the lipase⁃producing conditions and catalytic conditions were optimized. The optimal conditions for lipase production were as follows:the optimal loaded volume was 50 mL/500 mL, initial pH of fermentation medium was 8�0. Methanol was added into the medium to a final concentration of 1% ( V/V) at every 4 h intervals. Under the optimal conditions,the lipase activity of the recombinant reached 4�41 U/L within 192 h,with an increase of 90�9% compared to the control. The supernatant containing lipase was then used for biocatalytic resolution of DMCPE to S⁃(+)⁃DMCPA at 15 mmol/L substrate concentration at 30 ℃ for 36 h. Under above conditions,the best yield of 43�8% was obtained with enantiomeric excess (e�e.) value of 99�8%.