【作者】 刘维明； 沈东； 黄和； 胡燚；

【Author】 Liu Weiming;Shen Dong;Huang He;Hu Yi;College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University;Pujiang College,Nanjing Tech University;

【机构】 南京工业大学生物与制药工程学院； 南京工业大学浦江学院；

【摘要】 L-叔亮氨酸(L-tert-leucine,L-Tle)是一种非蛋白原的手性氨基酸,在抗癌药、抗艾滋药、手性助剂和配体的合成中具有广泛的应用。本文构建了重组菌E.coli BL21(DE3)/LeuDH-FDH,实现了亮氨酸脱氢酶(LeuDH,来源于Bacillus sphaericus CGMCC 1.1677)和甲酸脱氢酶(FDH,来源于Candida boidinii)在E.coli中的共表达;研究了重组E.coli细胞破碎液催化三甲基丙酮酸(TMP)氨化还原制备L-Tle的反应工艺,考察了pH、温度、甲酸铵浓度、TMP浓度、细胞浓度及NAD+浓度等因素对反应的影响,优化了反应工艺条件。在优化后的条件下,即TMP 0.75 M,甲酸铵1.50 M,重组E.coli细胞30 g/L,NAD+0.25 g/L,pH 8.5,30 oC,反应15 h后转化率和ee值均大于99%,时空产率为157 g L-1 d-1。进一步采用流加底物的方式提高了底物浓度和反应效率,TMP终浓度可达1.5 M,反应6 h转化率和ee值均大于99%,时空产率高达786 g L-1 d-1,是已报道工艺的2.1-7.2倍[1-2]。反应工艺放大至1 L反应体系后可稳定运行,显示了良好的工业化应用前景。更多还原

【Abstract】 L-tert-leucine(L-Tle) is a nonproteinogenic amino acid and is widely used in the preparation of anticancer drugs, anti-AIDS drugs, chiral auxiliaries and ligands. Recombinant E. coli was constructed to coexpress LeuDH from Bacillus sphaericus CGMCC 1.1677 and FDH from Candida boidinii. L-Tle was prepared through reductive amination of TMP catalyzed by cell-free extracts of recombinant E. coli. Under the optimized reaction conditions, L-Tle was obtained in >99% ee with conversion and space-time yield of >99% and 157 g L-1 d-1, respectively. Substrate feeding strategy was addressed to increase the substrate concentration and thus improve the reaction efficiency. The continuous process was conducted at an overall TMP concentration of 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L-1 d-1, which was 2.1 to 7.2-fold as that of the previously described procedures[1-2]. Furthermore, the preparation was successfully operated on a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of L-Tle.更多还原