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

A technology of leucine dehydrogenase and mutants, applied in applications, enzymes, oxidoreductases, etc., can solve problems such as low catalytic activity and affect the conversion rate of 4-phenyl-2-butylamine, and achieve catalytic activity high effect

Active Publication Date: 2020-01-07
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

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

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Preparation and expression of different leucine dehydrogenase mutants

[0085] Specific steps are as follows:

[0086] Chemically synthesize the gene of leucine dehydrogenase encoding amino acid sequence as shown in SEQ ID NO.1 (the nucleotide sequence of the gene is as shown in SEQ ID NO.2); With reference to the literature "Chen F F, Liu Y Y, Zheng G W , etal.Asymmetric Amination of Secondary Alcohols by using a Redox-Neutral Two-Enzyme Cascade[J].ChemCatChem,2015,7(23):3838-3841."The 70th lysine of leucine dehydrogenase Mutation to serine, asparagine at position 263 to leucine, so that leucine dehydrogenase obtains the activity of aliphatic ketones (the 70th position here corresponds to the 77th position in the literature, and the 263rd position here is the Corresponding to the 270th position in the literature), obtain the leucine dehydrogenase mutant (amine dehydrogenase) whose amino acid sequence is shown in SEQ ID NO.3 and the leucine whose encoding am...

Embodiment 2

[0112] Example 2: Catalytic activity of different leucine dehydrogenase mutants to 4-phenyl-2-butanone

[0113] Specific steps are as follows:

[0114] in NH 4 Cl-NH 4 Add 4-phenyl-2-butanone (5mmol / L) and NADH (0.2mmol / L) to OH buffer (1mol / L, pH 9.0) to obtain a reaction system; keep the reaction system at 30°C for 2min Add 20ul of wild type, mutant A115G, T136A, A115G / T136A, A115G / T136A / L42A, A115G / T136A / T45A, A115G / T136A / T114A, A115G / T136A / E116A and A115G / T136A / L42G obtained in Example 1 Concentrate pure enzyme to start the reaction, the control group does not contain concentrated enzyme solution, and other components are the same; the reaction is carried out at 30°C for 5min, and the absorbance change at 340nm is recorded every 10s to obtain wild type, mutant A115G, T136A, A115G / Catalytic activity of T136A, A115G / T136A / L42A, A115G / T136A / T45A, A115G / T136A / T114A, A115G / T136A / E116A and A115G / T136A / L42G on 4-phenyl-2-butanone; catalytic activity (U / g) = Ew × V × 1000 / 62...

Embodiment 3

[0116] Embodiment 3: the catalytic activity of different leucine dehydrogenase mutants to phenylacetone

[0117] Specific steps are as follows:

[0118] Add NH in Tris-HCL buffer (100mmol / L, pH 9.0) 4Cl (1mol / L), phenylacetone (5mmol / L) and NADH (0.2mmol / L) to obtain a reaction system; after incubating the reaction system at 30°C for 2min, add 20ul of the wild type and mutant obtained in Example 1 The concentrated pure enzymes of A115G / T136A, A115G / T136A / L42A, and A115G / T136A / L42G start the reaction. The control group does not contain concentrated enzyme solution, and the other components are the same; the reaction is carried out at 30°C for 5 minutes, and the temperature at 340nm is recorded every 10s. The absorbance change of wild type, mutant A115G / T136A, A115G / T136A / L42A, A115G / T136A / L42G is obtained the catalytic activity of phenylacetone; Catalytic activity (U / g)=Ew×V×1000 / 6220 / L ×The protein concentration of the concentrated enzyme solution; where, Ew is the absorbanc...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P13/00C12P13/02C12N15/70C12N1/21C12R1/19
CPCC12N9/0016C12N15/70C12P13/001C12P13/02C12Y104/01009
Inventor 穆晓清徐岩吴涛
Owner JIANGNAN UNIV
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