Leucine dehydrogenase mutant and application thereof to synthesis of aromatic chiral amine

Abstract

The invention discloses a leucine dehydrogenase mutant and an application thereof to synthesis of aromatic chiral amine, and belongs to the technical field of enzyme engineering and microorganism engineering. Mutation is performed on the 70th lysine and the 263rd asparagine, and the 115th alanine, the 136th threonine and/or the 42rd leucine of leucine dehydrogenase (Leucine Dehydrogenase, LeuDH, EC 1.4.1.9 ) as shown in SEQID NO.1 of an amino acid sequence, so that the leucine dehydrogenase obtains catalytic activity to aromatic chiral ketone of benzylacetone, phenylacetone and the like.

Description

technical field [0001] The invention relates to a leucine dehydrogenase mutant and its application in the synthesis of aromatic chiral amines, and belongs to the technical fields of enzyme engineering and microbial engineering. Background technique [0002] Devalolol as a novel β-blocker and β 2 -Agonist drugs, with good hemodynamic effects and ideal antihypertensive effects, can improve the compliance of large arteries in antihypertensive treatment, and have a certain protective effect on blood vessels, especially suitable for mild to moderate Treatment of high-grade essential hypertension. [0003] Optically pure (R)-4-phenyl-2-butylamine is a key intermediate in the synthesis of Devalol. In view of the important position of devalolol in antihypertensive drugs, the synthesis of (R)-4-phenyl-2-butylamine also has great practical application value. [0004] At present, the amine dehydrogenase obtained due to the mutation of amino acid dehydrogenase can catalyze 4-phenyl-2...

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Problems solved by technology

[0005] But, there are still many problems in the above-mentioned method for synthesizing (R)-4-phenyl-2-butylamine by amine dehydrogenase, wherein, the most important problem is that existing amine dehydrogenase is to 4-phenyl-2- The catalytic activity of butanone is very low, which greatly affects the conversion rate of (R)-4-phenyl-2-butylamine by amine dehydrogenase

For example, Zhi Li et al. achieved a highly asymmetric reduction of 4-phenyl-2-butanone by mutating phenylalanine dehydrogenase from Rhodococcus sp.M4 to amine dehydrogenase Amination is (R)-4-phenyl-2-butylamine, and the optical purity (ee value) of its product exceeds 98%, but, the phenylalanine dehydrogenase double mutant K66Q / N262C that its mutation obtains is right The catalytic activity of 4-phenyl-2-butanone is only 6.2U / g, and the triple mutant of phenylalanine dehydrogenase K66Q / N262C / S149G, which is further mutated on the basis of the double mutant, is 4-phenylalanine dehydrogenase The catalytic activity of -2-butanone is only 8.8U / g; Schell et al. realized the conversion of 4- The highly asymmetric reductive amination of phenyl-2-butanone to (R)-4-phenyl-2-butanamine, however, the phenylalanine dehydrogenase double mutant K78S / N276L obtained by its mutation is sensitive to 4- The catalytic activity of phenyl-2-butanone is only 11.3U / g, and the four-mutant L50F / K78S / N276L / L306A of phenylalanine dehydrogenase obtained by further mutation on the basis of the double mutant is 4-phenyl The catalytic activity of -2-butanone is only 21.7U / g

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