Unnatural amino acids and their derivatives are important chemical intermediates. Among them, L-tert-leucine is an important unnatural amino acid. It can be used for synthesizing pharmaceutical intermediates, as a chiral inducer for asymmetric synthesis and a resolving reagent for asymmetric resolution, and is a chiral precursor for synthesizing a variety of anti-AIDS drugs and hepatitis virus inhibitors. With the development of my country’s social economy and the enhancement of environmental protection awareness, the pharmaceutical and chemical industry not only needs to improve the production capacity of L-tert-leucine, but also needs to create a green and pollution-free production process. Therefore, the use of enzymatic biotransformation to produce L-tert-leucine is being paid more and more attention by relevant institutions. Divided from the production route, the methods for producing L-tert-leucine mainly include chemical reagent separation method, chiral source synthesis method, chemical synthesis method and biotransformation method. Among them, the resolution method is limited by the yield, the chiral source method is limited by the production capacity of natural products, and the cost of chemical synthesis method is high, so other synthetic methods have no successful large-scale application examples. The main method of leucine. In biotransformation method, utilize lipase to split racemate DL-tertiary leucine relevant technological research is more, such as Chinese patent CN201010210770.0 etc., but this route has the same defect as chemical splitting method, namely receive. rate cannot exceed 50%. Therefore, the main research directions of biological methods are currently focused on the route of reducing trimethylpyruvate to L-tert-leucine using L-leucine dehydrogenase, and using formate dehydrogenase to regenerate coenzyme NADH in situ, Ammonium formate is used to provide ammonia molecules and hydrogen atoms. As early as the 1990s, Germany’s Degussa Company used this route to combine with an ultrafiltration membrane reactor to produce L-tertiary leucine. Several patent applications have been disclosed for this route, such as Chinese patents CN201110116253.1, CN201110277030.3, etc. The main problems of its existence are as follows: 1. In fact, the L-tert-leucine dehydrogenase recognized by the academic community will be significantly inhibited by the substrate when the substrate concentration is too high, and the reaction rate will drop sharply (Enzyme Catalysis in Organic Synthesis EditionI, Wiley, 1186). ( J. Mol. Catal: B 1998, 5: 1-11). It can be seen that the above patent The method is inefficient or difficult to repeat. 2. Enzyme feeding amount is too much. Obviously, this is an inevitable consequence of problem I. For example, in the patent CN201110116253.1, the expensive coenzyme NAD feeding amount reaches 1% (w/w) of the substrate, and the economy is poor, and the concentration that the whole cell catalyst accounts for the reaction system is 80% (4g/5 mL) in the patent CN201110277030.3 3. There is no clear post-extraction process. Also due to the existence of problem I, its yield and yield cannot meet the needs of industrialized production. Based on the above three points, there is no report on the large-scale production of L-tert-leucine using this method. In order to solve the above issues, the item of the present creation is to offer a technique for enzymatically manufacturing L-tert-leucine. The technique has light preparation problems, is eco-friendly, has high return and also affordable, and also appropriates for commercial production. In order to attain the above purpose, the technical plan provided by the innovation is: a production approach of L-tertiary leucine, the technique makes use of L-leucine dehydrogenase as well as formate dehydrogenase to produce L-tertiary leucine, Its preparation response formula is:
a production approach of L-tertiary leucine, the technique makes use of L-leucine dehydrogenase as well as formate dehydrogenase to produce L-tertiary leucine, Its preparation response formula is: Wherein, LeuDH is leucine dehydrogenase; NADH is minimized nicotinamide adenine dinucleotide, particularly reductase coenzyme; NAD is oxidized nicotinamide adenine dinucleotide, particularly dehydrogenase coenzyme; FDH is formate dehydrogenase.
