【作者】 吴薇；

【导师】 杨立荣； 吴坚平；

【作者基本信息】 浙江大学 ， 生物化工， 2011， 硕士

【摘要】 普瑞巴林(Pregabalin),(S)-3-氨甲基-5-甲基己酸,是一种用于抗神经痛和癫痫症的辅助治疗的手性药物,其国际市场需求呈现较快增长之势。开展生物催化法制备普瑞巴林的研究,筛选拆分制备普瑞巴林的微生物,具有重要的应用前景和价值。(S)-2-羧乙基-3-氰基-5-甲基己酸乙酯((S)-CCMAE)是合成普瑞巴林的关键手性中间体。本文首先以外消旋体2-羧乙基-3-氰基-5-甲基己酸乙酯(CCMAE)为底物,从土壤中筛选得到一株具有自主知识产权的能够对映选择性水解(R)-CCMAE的假单胞菌(Pseudomonas) CGMCC No.4184。对其产酶条件及培养基进行了优化,使酶活从15.92 U/L提高到33.43 U/L。随后对假单胞菌CGMCCNo.4184静息细胞水解拆分底物的反应条件进行了研究,确定了较优反应条件为：30～35℃,pH 7.5-8.5。并成功利用微生物静息细胞转化法制备了ee值达99.8%的(S)-CCMAE,这与目前报道的生物拆分工艺均有所不同。研究还发现Tween-80和TritonX-100可明显提高对映选择率E值和酶活。最后利用海藻酸钠-PVA复合载体法制备了假单胞菌CGMCC No.4184的固定化细胞,对其技术参数和反应条件作了优化,并利用所制得的固定化细胞制备ee值99%以上的(S)-CCMAE。该固定化细胞能够较好地进行重复利用。本研究中筛选获得的高选择性R型水解酶可能是一种新酶,后续的基因克隆和表达、酶学性质研究工作正在进行中,为进一步改善酶的催化性能和拓展应用范围打下了良好的基础。更多还原

【Abstract】 Pregabalin, (S)-3-aminomethyl-5-methylhexanioc acid, is the 3-isobutyl substituent of GABA and a chiral drug that can be used as an adjuvant therapy for antineuralgic and anti-Epilepsy. The applications of pregabalin have grown markedly in recent years and are likely to expand in the coming years. Therefore, the studies on the preparation methods of optically active pregabalin and screening of biocatalysts are very important both for the knowledge on biocatalytic mechanism and its application.(S)-2-carboxyethyl-3-Cyano-5-methylhexanoic acid ethylester ((S)-CCMAE) is an important chiral intermediate of pregabalin. In this paper, firstly, using racemic CCMAE as the substrate, Pseudomonas sp. CGMCC No.4184 was isolated which can enantioselectively hydrolyze (R)-CCMAE. By optimization of the fermentation medium and the reaction conditions, the enzyme activity increased from 15.92 U/L to 33.43 U/L. Secondly, the resolution conditions by resting cells of Pseudomonas CGMCC No.4184 was investigated, and the results showed that the optimal temperature was 30-35℃and pH 7.5-8.5. After optimization, (S)-CCMAE were successfully prepared with ees of 99.8%. This method was different from all reportorial biotransformation process. Tween-80 and TritonX-100 were found to increase the enantioselectivity (E value) and enzyme activitiy. Finally, Sodium alginate-PVA-CaCl2 method was used to prepare the immobilized cells of Pseudomonas CGMCC No.4184. By optimization of the parameters and the reaction conditions, (S)-CCMAE (ees over 99%) were prepared and the immobilized cells have got good operational stability.In this study, the isolated hydrolase that has the high R-selectivity may be a noval enzyme. In order to improve the catalysis performance and exploit the application of this R-enantioselective hydrolyse, the study on gene cloning-expression and the enzymology characters would be carried out soon.更多还原