利用纤维床生物反应器制备左乙拉西坦关键手性中间体

Preparation of levetiracetam key chiral intermediate with fibrous-bed bioreactor(FBB)

利用纤维床生物反应器固定耐酪氨酸冢村氏菌(Tsukamurella tyrosinosolvens)E105细胞不对称水解(R,S)-α-乙基-2-氧代-1-吡咯烷乙酸乙酯制备左乙拉西坦关键手性中间体(S)-α-乙基-2-氧代-1-吡咯烷乙酸.考察了载体的化学修饰、缓冲液pH和离子强度等对棉纤维载体吸附细胞的影响,确定了最优的菌体吸附条件;在此基础上研究了纤维床生物反应器细胞动态吸附动力学以及利用纤维床生物反应器固定化细胞不对称水解(R,S)-α-乙基-2-氧代-1-吡咯烷乙酸乙酯制备左乙拉西坦关键手性中间体(S)-α-乙基-2-氧代-1-吡咯烷乙酸反应的过程曲线、细胞负载量对产率和ee值的影响,继而,考察了纤维床反应器中固定化细胞的重复利用性能.结果表明:在1.115mol/L,pH7.0的磷酸钾缓冲液中,以聚乙烯亚胺修饰棉纤维为固定化载体,细胞最大吸附量可达185mg/g;细胞在纤维床生物反应器上的动态吸附符合一级动力学,动力学方程为Ln(C/C0)=-0.947t;以细胞负载浓度为50g/L的纤维床生物反应器进行不对称水解反应,反应36h后产率和ee值分别为43.2%和98.2%,且纤维床反应器固定化细胞具有一定的重复利用性能.研究结果为纤维床反应器与发酵罐偶联进行半连续化制备左乙拉西坦手性中间体奠定了一定的理论基础.

This study was designed to utilize the FBB-immobilized Tsukamurella tyrosinosolvens E105 cells to catalyze the hydrolysis of （R, S）-α-ethyl-2-oxo-l-pyrrolidine acetic acid ethyl ester to generate desired （S）-α-ethyl-2-oxo-l-pyrrolidine acetic acid, a key chiral intermediate of levetiracetam. Firstly, the adsorption process was analyzed by batch experiments in terms of impact factors, such as chemical modification of the carrier, buffer pH and ionic strength, aimed to determine the optimum adsorption conditions. On the basis of the optimum adsorption conditions, time course of FBB-immobiIized cells catalyzed hydrolysis of （R, S）-α-ethyl-2-oxo-l-pyrrolidine acetic acid ethyl ester to generate desired （S）-α-ethyl-2-oxo-l-pyrrolidine acetic acid and the effect of adopted cell mass on the yield and the product ee value were investigated. Finally, the reuse performance of the FBB immobilized cells was studied. The results showed that the highest cell adsorption capacity was obtained at the PEI modified cotton fiber under 1. 115 tool/L, pH 7.0 potassium phosphate buffer with 185 mg/g. The cells adsorption process followed first-order kinetics and the obtained equation was Ln （C/C0）=-0. 947t. The yield and the product ee value of the asymmetric hydrolysis reaction reached 43.2% and 98.2% respectively after 36 h when dry cell concentration was 50 g/L in FBB. The immobilized cells have a comparable advantage in the reuse experiments for preparation of levetiracetam. To some extent, this research lay a foundation for semi-continuous production of the levetiracetam chiral intermediate using fibrous-bed bioreactor coupling with fermentation cylinder.