L-tert-leucine (L-Tle) is an important chiral amino acid, which can be used to produce HIV or HCV protease inhibitors. Scientists developed enzymatic reductive amination to produce L-Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) based on cofactor regeneration. It greatly improved the yield and optical purity of L-Tle. However, owing to different activity of LeuDH and FDH, the NADH consumption would be faster than its regeneration. Therefore, external NADH is necessary. Many methods have been used to solve this problem, but none of them made a difference. Our project devised a method that regulating the enzyme activity of LeuDH by changing RBS of leudh. And fdh and leudh were borne in a plasmid. This idea controls copy numbers and transformation efficiency, and the only variable is RBS efficiency of leudh. With the different efficiency of RBS, we can get different enzyme activity of LeuDH. And from the experimental results, we obtained the suitable RBS efficiency and the best catalytic efficiency. Finally, the desired L-Tle was obtained with >99% conversion and a high enantioselectivity of >99% e.e. value.
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