生物拆分制备(S)-α-乙基-2-氧-1-吡咯烷乙酸工艺研究

Preparation of(S)-alpha-ethyl-2-oxo-1-pyrrolidineacetic acid by microbial resolution

耐酪氨酸冢村氏菌(Tsukamurella tyrosinosolvens)E105可选择性催化(R,S)-α-乙基-2-氧-1-吡咯烷乙酸乙酯生物拆分制备左乙拉西坦关键手性中间体(S)-α-乙基-2-氧-1-吡咯烷乙酸.通过单因素实验和响应面法对菌株产酶培养条件进行优化,并考察含酶静息细胞催化制备(S)-α-乙基-2-氧-1-吡咯烷乙酸的反应过程.结果表明:最优产酶培养条件为装液量30%,接种量4%,发酵36h;培养基组成为葡萄糖17.70g/L,酵母膏16.53g/L,NH4Cl 9.5g/L,K2HPO42g/L,KH2PO41g/L,NaCl 0.6g/L,MgSO4·7H2O 0.5g/L,初始pH值7.32.在优化条件下,酶活力为19.33U/g,细胞干重达4.13g/L,分别较优化前提高了32.6%和60.1%.当细胞加量为30g/L,底物浓度60mmol/L,反应体系为300mL,拆分24h后,产率和ee值分别为48%和99%,(S)-α-乙基-2-氧-1-吡咯烷乙酸浓度达28.8mmol/L,较优化前提高了32.1%.

（ S ）-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid, a key chiral intermediate of Levetiracetam, was produced by microbial resolution of ethyl （R, S）-alpha-ethyl-2-oxo-1- pyrrolidineacetate using Tsukamurella sp. E105. Lipase-producing conditions for this strain were optimized by single factor experiments and response surface methodology, respectively. Then, the preparation of （S）-alpha-ethyl-2-oxo-l-pyrrolidineacetie acid catalyzed by resting cells of El05 was performed. The results showed that the optimal lipase-producing conditions were as follows： glucose 17.70 g/L, yeast extract 16.53 g/L, NH4C1 9.5 g/L, K2HPO4 2 g/L, KH2PO4 1 g/L, NaC1 0.6 g/L, MgSO4· 7H20 0 5 g/L, initial pH 7. 32, loaded volume 300 mL/L, inoculum size 4%, incubated for 36 h. Under the optimal conditions, the maximum enzyme activity of 19.33 U/g and 4. 13 g （DCW）/L of biomass were achieved, with an increase of 32. 6% and 60.1%, respectively, compared to the control. Resting cells of E105 were then used for the preparation of （S）-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid at 60 mmol/L of substrate concentration in 300 mL reaction system. The yield of 48% and ee value of 99% were obtained after reaction for 24 h. The final product concentration reached 28. 8 mmol/L, an increase of 32.1% in contrast to that before its optimization.