亮氨酸脱氢酶催化2-酮丁酸生成L-2-氨基丁酸需要辅酶NADH参与,构建Escherichia coli (LeuDH/FDH),通过共表达亮氨酸脱氢酶和甲酸脱氢酶实现了辅酶NADH胞内循环再生。通过产酶条件优化,提高该菌株催化制备L-2-氨基丁酸的效率。结果表明,在5 L发酵罐上,在诱导温度为22 ℃、诱导剂乳糖质量浓度为8.0 g/L和诱导时间为17 h的条件下,亮氨酸脱氢酶和甲酸脱氢酶的酶活分别达到79.2 U/g和216.1 U/g,催化效果最佳。利用该菌株全细胞为催化剂,耦合苏氨酸脱氨酶,在1 L反应体系中进行了催化反应,L-苏氨酸质量浓度为180 g/L时,无需额外添加辅酶, 8 h反应后底物转化率达到99%,L-2-氨基丁酸e.e.值99.5%以上,时空产率19.3 g/(L·h)。该研究为建立高效、低成本的L-2-氨基丁酸工业化生产方法提供了基础。
A recombinant Escherichia coli (LeuDH/FDH) that co-expressed leucine dehydrogenase and formate dehydrogenase was constructed to allow intracellular coenzyme NADH regeneration, and the cultivation condition was optimized. The results demonstrated that the highest catalytic efficiency was achieved in a 5 L fermenter at 22 ℃ with 8.0 g/L lactose and inducted for 17 h. Under this condition, the activities of leucine dehydrogenase and formate dehydrogenase reached 79.2 U/g and 216.1 U/g, respectively. By using the whole cells and coupled with threonine deaminase in a 1 L bioreactor, the conversation rate of L-2-aminobutyric acid reached 99% with the yield of 19.3 g/(L·h) in 8 h from 180 g/L L-threonine without NADH added. The e.e. value of L-2-aminobutyric acid was over 99.5%. This provides an efficient and low-cost method for industrial production of L-2-aminobutyric acid.
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