CN118109432A - Amine dehydrogenase mutant, single plasmid double enzyme co-expression system and application thereof in chiral amine synthesis - Google Patents

Abstract

The invention discloses an amine dehydrogenase mutant, a single plasmid double enzyme co-expression system and application thereof in chiral amine synthesis, wherein the amine dehydrogenase mutant comprises the following components: the amino acid sequence of the mutant V294A/N208K is shown as SEQ ID No.4 by taking amine dehydrogenase TvAmDH with the amino acid sequence shown as SEQ ID No.2 as a template, substituting valine at 294 th site with alanine and substituting asparagine at 208 th site with lysine. The invention obtains an amine dehydrogenase mutant with enhanced catalytic activity through reasonable design, has asymmetric reductive amination activity on various aromatic carbonyl compounds, and also greatly improves the catalytic activity on coenzyme NADH. The amine dehydrogenase mutant has good application prospect in preparing corresponding chiral amine by catalyzing asymmetric reductive amination of aromatic carbonyl compounds, and has higher application value.

Description

Amine dehydrogenase mutant, single plasmid double enzyme co-expression system and application thereof in chiral amine synthesis

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to an amine dehydrogenase TvAmDH mutant, a single plasmid double enzyme co-expression system constructed by the same and application of the same in chiral amine synthesis.

Background

Chiral amines are a class of compounds that play an important role in a number of fields, such as pharmaceutical synthesis and chemical engineering. It is counted that nearly 40% of commonly used small molecule drugs contain chiral amine precursors such as sitagliptin (antidiabetic agent) and dortevir (anti-hiv agent). Traditional synthesis of chiral amines often requires the use of toxic chemicals and expensive metal catalysts, and is difficult to synthesize products with high optical purity, and other problems, resulting in industrial unsustainable synthesis. In contrast, the bioenzyme synthesis can effectively overcome the problems, and gradually becomes an ideal alternative way for industrial synthesis.

In a series of enzymes for synthesizing chiral amine, amine dehydrogenase can take cheap inorganic ammonia as an amino donor, and prochiral ketone is asymmetrically synthesized into chiral amine without byproducts, so that the amine dehydrogenase is one of the first paths for chiral amine enzymatic synthesis. The Bommarius team successfully creates a novel amine dehydrogenase from amino acid dehydrogenases by mutating two key amino acid residues combined with carboxyl groups in a substrate active cavity, so that a catalytic substrate of the enzyme is converted from amino acid to ketone compounds, and an important basis is provided for the subsequent creation of more novel amine dehydrogenases.

Thereafter, researchers have made a number of significant efforts to expand the range of amine dehydrogenase substrates and to increase their catalytic activity by means of protein engineering. In terms of broadening the substrate range, chen Feifei et al succeeded in identifying two key contributing residues in the active pocket that affect substrate binding, enlarging the active pocket, enabling the novel amine dehydrogenases to utilize large volumes of fatty ketone substrate that are not convertible by the wild type. The Mu Xiaoqing subject group et al successfully identified five important sites affecting the catalytic activity of aromatic ketones by using a substrate walking strategy, and obtained multiple rounds of iteration on multiple high-activity mutants. Although these successful reports lay a strong foundation for the research of amine dehydrogenases, the catalytic activity towards aromatic carbonyl compounds is still maintained at a low level, severely limiting the use of such enzymes, and thus the development of more amine dehydrogenases with high reductive amination activity is required.

Disclosure of Invention

The invention aims to provide an amine dehydrogenase TvAmDH mutant, a single plasmid double enzyme coexpression system and application thereof in chiral amine synthesis, so as to solve the problem that the existing amine dehydrogenase TvAmDH has low catalytic activity on aromatic carbonyl compounds.

Specifically, the invention relates to an amine dehydrogenase TvAmDH mutant of a thermophilic lactobacillus source (Thermoactinomyces vulgaris), a coding gene thereof, and application of a double-enzyme expression body containing the gene in preparing chiral amine by catalyzing asymmetric reductive amination of carbonyl compounds and ammonia molecules, wherein the leucine dehydrogenase has improved catalytic activity on 24 compounds in 25 different aromatic carbonyl compounds, and has important significance in green catalytic synthesis of chiral amine compounds.

The aim of the invention can be achieved by the following technical scheme:

According to a first aspect of the present invention, based on structural design of the enzyme molecule, an amine dehydrogenase mutant having catalytic activity on aromatic carbonyl compounds is obtained, which is: the amino acid sequence of the mutant V294A/N208K is shown as SEQ ID No.4 by taking amine dehydrogenase TvAmDH with the amino acid sequence shown as SEQ ID No.2 as a template, substituting valine at 294 th site with alanine and substituting asparagine at 208 th site with lysine.

According to a second aspect of the present invention, there is provided a gene encoding the amine dehydrogenase mutant.

According to a third aspect of the present invention, there is provided a recombinant vector comprising the coding gene.

According to a fourth aspect of the present invention, there is provided a recombinant expression transformant comprising the recombinant expression vector.

According to a fifth aspect of the present invention, there is provided a method for constructing a single plasmid double enzyme co-expression system, comprising the steps of: 1) Extracting and amplifying plasmids pET-28a-V294A/N208K, plasmid pET-28a-bmgdh and a vector plasmid pET-28a (+), wherein the plasmid pET-28a-V294A/N208K is constructed by a vector plasmid pET-28a and a mutant V294A/N208K according to claim 1, and the plasmid pET-28a-bmgdh is constructed by a vector plasmid pET-28a and glucose dehydrogenase GDH with an amino acid sequence shown as SEQ ID NO. 6; 2) Carrying out double-section fusion on the two sections of target gene fragments purified in the step 1); 3) And respectively carrying out enzyme digestion on the plasmid pET-28a-V294A/N208K, the plasmid pET-28a-bmgdh and the vector pET-28a (+) by using endonucleases, and sequentially connecting the plasmids to transform host escherichia coli BL21 (DE 3), so as to construct the single-plasmid double-enzyme coexpression system.

According to a sixth aspect of the present invention, there is provided a single plasmid double enzyme co-expression system constructed according to the above construction method.

In particular, amine dehydrogenase TvAmDH is a class of nicotinamide-coenzyme-dependent oxidoreductases, so that it is necessary to provide a coenzyme from the outside to drive the redox reaction. Cofactor regeneration is achieved by constructing a glucose dehydrogenase (bmgdh) and amine dehydrogenase TvAmDH co-expression system in order to increase the efficiency of coenzyme utilization and thereby reduce costs.

According to a seventh aspect of the invention, the invention also provides a preparation method of the amine dehydrogenase TvAmDH mutant, which comprises freeze-dried bacterial powder of a single plasmid double enzyme co-expression system, crude enzyme liquid and pure enzyme.

According to a preferred embodiment of the invention, the method comprises the following steps: the recombinant cells are coated on a solid flat plate, cultured for 12 hours in a constant temperature incubator at 37 ℃, single colonies are picked up into a 5mL LB culture medium test tube, kanamycin solution with one thousandth concentration is added, and the test tube is placed in a constant temperature shaker at 37 ℃ for shake culture for 8 hours. And transferring 500 mu L of bacterial liquid into 50mL of TB liquid culture medium, adding kanamycin solution with one thousandth concentration, and culturing for 2-2.5h until the OD600 of the cells reaches 0.6-0.8. The cultured bacterial liquid is added with one ten thousandth concentration of inducer IPTG (final concentration is 0.1 mM) for induction, and then the shake flask is placed in a constant temperature incubator at 20 ℃ and 200rpm for 12 hours of incubation. After balancing the bacterial solutions, centrifuging (4 ℃,800 rpm,10 min), discarding the supernatant, adding 15mL of physiological saline, washing for 2 times, discarding the supernatant, refrigerating for 6 hours, and freeze-drying by using a vacuum freeze dryer to obtain freeze-dried enzyme powder.

According to a preferred embodiment of the present invention, a crude enzyme solution is obtained, the method comprising the steps of: the recombinant cells are coated on a solid flat plate, cultured for 12 hours in a constant temperature incubator at 37 ℃, single colonies are picked up into a 5mL LB culture medium test tube, kanamycin solution with one thousandth concentration is added, and the test tube is placed in a constant temperature shaker at 37 ℃ for shake culture for 8 hours. And transferring 500 mu L of bacterial liquid into 50mL of TB liquid culture medium, adding kanamycin solution with one thousandth concentration, and culturing for 2-2.5h until the OD600 of the cells reaches 0.6-0.8. The cultured bacterial liquid is added with one ten thousandth concentration of inducer IPTG (final concentration is 0.1 mM) for induction, and then the shake flask is placed in a constant temperature incubator at 20 ℃ and 200rpm for 12 hours of incubation. After balancing these bacterial solutions, centrifuging (4 ℃,800 rpm,10 min), discarding the supernatant, adding 15mL of physiological saline to wash for 2 times, discarding the supernatant, adding sodium phosphate buffer (100 mM, pH 8.0) according to the mass of the wet bacterial cells in proportion for resuspension, crushing by an ultrasonic cytobreaker, centrifuging the suspension bacterial solution after crushing (4 ℃,800 rpm,10 min), and pouring out the supernatant on ice to obtain a crude enzyme solution.

According to a preferred embodiment of the invention, the pure enzyme is obtained, said method comprising the steps of: after the crude enzyme solution is obtained, the enzyme with His tag is purified by nickel column affinity chromatography, and the specific operation steps are as follows:

the device comprises: the peristaltic pump, nickel column and detector were connected by tubing and the nickel column was placed on ice.

Balance: the nickel column was rinsed with 5 column volumes of sterile water to empty the column of 20% ethanol, and then equilibrated with 3 column volumes of equilibration liquid while zeroing the detector readings.

Loading: the enzyme solution after the disruption and centrifugation was filtered and filtered to remove impurities by using a 0.22 μm filter head, and after the detector showed stable numbers, the enzyme solution was loaded at a flow rate of 1 mL/min.

Eluting: the system was first re-equilibrated with 20mM imidazole solution, the hetero proteins were removed with 50mM imidazole solution, and then the target proteins adsorbed on the nickel column were eluted with 75mM,100mM,150mM,200mM,250mM,375mM and 500mM imidazole solutions in this order in an amount of 3 column volumes to obtain a collection solution with different gradients.

Preservation column: after the elution, the column was passed through 3 column volumes of equilibration liquid, sterile water and 20% ethanol in sequence, and stored at 4 ℃.

Ultrafiltration concentration: SDS-PAGE analysis was performed on the collected eluate to determine the appropriate elution gradient. The gradient of the target protein was concentrated by ultrafiltration (4,500 rpm,20 min) using an ultrafiltration tube, and repeated several times until the protein solution was concentrated to about 1 mL.

Desalting: and adding 9mL of C solution for ultrafiltration to about 1mL, and repeating for three times until imidazole is diluted by 1,000 times so as to avoid toxic action of imidazole on enzyme.

And (3) preserving: the purified enzyme after concentration and desalination is evenly mixed with precooled 75% glycerol in a proportion of 1:1, and the mixture is preserved at the temperature of minus 40 ℃ for standby.

The method for measuring the enzyme activity comprises the following steps: the change in absorbance at 340nm (ε= 6,220M-1 cm-1) of NADH was measured by a microplate reader. One enzyme activity unit (U) is defined as: under certain reaction conditions, 1. Mu. Mol NADH was consumed in 1 minute. The specific activity (U/mg) of the enzyme was defined as: the enzyme activity per mg protein. The enzyme activity measurement system (200. Mu.L, 30 ℃) contained: a suitable amount of enzyme solution, substrate acetophenone (10 mM), NADH (0.5 mM), ammonium chloride/ammonia buffer (1M, pH 9.5). The procedure set for the microplate reader was as follows: the temperature was set at 30℃and the reaction system was first stirred for 10 seconds to homogenize, then the absorbance change at 340nm was measured over 5 minutes and read every 10 seconds.

According to an eighth aspect of the present invention there is also provided the use of the amine dehydrogenase mutant or the recombinant amine dehydrogenase mutant catalyst for catalyzing asymmetric reductive amination of aromatic carbonyl compounds to produce the corresponding chiral amine.

The invention provides a method for preparing corresponding chiral amine by asymmetrically reductive amination of aromatic carbonyl compounds by using the single plasmid double enzyme coexpression system or the amine dehydrogenase TvAmDH mutant.

According to a preferred embodiment of the invention: the system for catalyzing the asymmetric reductive amination reaction of the carbonyl compound by the amine dehydrogenase TvAmDH mutant comprises a proper amount of enzyme solution, different aromatic carbonyl compounds (10 mM), NADH (0.5 mM) and an ammonium chloride/ammonia buffer solution (1M, pH 9.5). The procedure set for the microplate reader was as follows: the temperature was set at 30℃and the reaction system was first stirred for 10 seconds to homogenize the reaction, and then the change in absorbance was measured over 5 minutes, reading every 10 seconds.

In the invention, in order to realize the efficient synthesis of the aromatic carbonyl compound, based on the structural design of the amine dehydrogenase TvAmDH, the valine at 294 site is firstly replaced by alanine based on the structural design of the protein, so that the steric hindrance of an active pocket is reduced, and the catalytic efficiency of the enzyme on non-natural substrates is improved. Then, based on the V294A mutant, the coenzyme binding efficiency is further modified by adopting a consensus sequence strategy, the 208 site is subjected to saturation mutation, and the asparagine at the 208 site is replaced by lysine, so that the double-point mutant V294A/N208K of the amine dehydrogenase TvAmDH is obtained. Kinetic parameters show that the catalytic efficiency of the finally obtained double-point mutant V294A/N208K on alpha-acetophenone is improved by 15 times, and the catalytic efficiency of the finally obtained double-point mutant V294A/N208K on coenzyme NADH is improved by 10 times. The mutant shows different degrees of improvement (1.2-4.8 times) on the catalytic activity of 24 compounds in 25 aromatic carbonyl compounds, and shows the potential of the mutant in chiral aromatic amine synthesis.

In conclusion, through reasonable design, the invention obtains the amine dehydrogenase TvAmDH mutant with enhanced catalytic activity, has asymmetric reductive amination activity on various aromatic carbonyl compounds, and also greatly improves the catalytic activity on coenzyme NADH. The invention also constructs a single plasmid double enzyme co-expression system of the amine dehydrogenase TvAmDH mutant and the glucose dehydrogenase, improves the mass transfer efficiency of the coenzyme and reduces the dosage of the coenzyme. Therefore, the method reduces the reaction cost, has good application prospect in preparing corresponding chiral amine by catalyzing asymmetric reductive amination of the aromatic carbonyl compound, and has great application value.

Drawings

FIG. 1 is an SDS-PAGE protein gel electrophoresis of an amine dehydrogenase TvAmDH and glucose dehydrogenase single plasmid dual enzyme co-expression system, wherein M is a protein molecular weight standard reagent, 1 is a cell disruption supernatant of an amine dehydrogenase TvAmDH mutant, 2 is a glucose dehydrogenase cell disruption supernatant, and 3 is a cell disruption supernatant of a single plasmid dual enzyme co-expression system;

FIG. 2 is a schematic representation of the results of an "alanine scan" of 11 potential active sites in an amine dehydrogenase active pocket;

FIG. 3 is a heat map of the results of saturation mutagenesis, wherein a redder color represents a more pronounced increase in activity and a bluer color represents a decrease in activity;

FIG. 4 is a coenzyme binding domain consensus sequence analysis;

FIG. 5 is a schematic representation of the results of a superimposed double point mutant reaction based on the V294A mutant;

FIG. 6 is a temperature optimized schematic of the enzymatic scale preparation of chiral amine drug intermediates, with an optimal temperature of 30 ℃;

FIG. 7 is a schematic illustration of pH optimisation for enzymatic scale preparation of chiral amine drug intermediates, with an optimum pH of 9.5;

FIG. 8 is a schematic illustration of the optimization of group solvents for the enzymatic scale preparation of chiral amine drug intermediates, the most preferred group solvent being dimethyl sulfoxide (DMSO);

FIG. 9 is a schematic representation of the optimization of cofactor NADH concentration for the enzymatic scale preparation of chiral amine drug intermediates, with an optimal concentration of 0.3mM;

FIG. 10 shows the reaction mechanism of amine dehydrogenase and glucose dehydrogenase in catalyzing asymmetric reductive amination of aromatic carbonyl compounds to produce the corresponding chiral amines.

Detailed Description

The invention is further described below in conjunction with specific embodiments. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are, unless specified otherwise, conventional in the art or are in accordance with the experimental methods recommended by the manufacturers of the kits and instruments.

Example 1 construction of Single plasmid double enzyme Co-expression Strain of amine dehydrogenase TvAmDH and glucose dehydrogenase

The novel amine dehydrogenase TvAmDH is constructed by using leucine dehydrogenase derived from actinomycetes at high temperature (Thermoactinomyces vulgaris, accession number ATCC 27868) as shown in SEQ ID NO.1 as a female parent and designing primers for the pseudo mutation sites, wherein the amino acid sequence of the amine dehydrogenase TvAmDH is shown in SEQ ID NO.2, and the upstream and downstream primer sequences (SEQ ID NO. 7-10) are shown in the following Table 1.

TABLE 1

Firstly, extracting plasmids of the novel amine dehydrogenase TvAmDH, glucose Dehydrogenase (GDH) with an amino acid sequence shown as SEQ ID NO.6 (from bacillus megatherium, bacillus megateriumde Bary) and a vector pET-28a (+), wherein the specific operation method is as follows: a small amount of the bacterial liquid was inoculated into 5mL of LB medium (containing 50. Mu.g/mL of kanamycin) and cultured in a shaker at 37℃and 200rpm for 10-12 hours. After the culture is finished, the thalli are collected by centrifugation, plasmids are extracted according to the steps on the instruction book of the kit, and the plasmids are preserved at the temperature of minus 20 ℃ for standby after the concentration is measured. The primers required for PCR amplification of the gene of interest (SEQ ID NOS.11-14, as shown in Table 2 below) were designed using PRIMER PREMIER software and appropriate cleavage sites were introduced. The TSINGKE MASTER Mix enzyme is used for carrying out PCR amplification on the target gene, and the plasmid is transferred into an expression host escherichia coli BL21 (DE 3) for the expression of the subsequent recombinase after sequencing.

TABLE 2

Example 2 design of amine dehydrogenase TvAmDH by protein engineering means

In order to obtain an amine dehydrogenase TvAmDH with a more efficient catalytic efficiency on aromatic carbonyl compounds, we have chosen to study alpha-acetophenone as a representative substrate, and then the conversion of the novel amine dehydrogenase at a substrate concentration of 50mM for 12 hours was only 40%. The novel amine dehydrogenase TvAmDH was engineered with leucine dehydrogenase as the initial protein scaffold, and has a better suitability for aliphatic substrates, thus requiring further design by protein engineering techniques. To maintain the hydrophobicity of the active pocket, the potential active site of 11 residues in the active pocket was determined by "alanine scanning" and the results were shown in FIG. 2 to be higher for five single point mutants of E114A, T119A, N145A, V291A, V A than TvAmDH, with the best performing mutant V294A conversion reaching 65%. Based on this, we selected these five sites for saturation mutagenesis, resulting in a total of 8 single point mutants with higher conversion than TvAmDH, including E114V, T119A, T134S, N145P, V291G, V294G, V A, with the best mutant being V294A, as shown in FIG. 3, which is a 27% improvement over TvAmDH. In addition, the amine dehydrogenase performs a catalytic function dependent on the cofactor NADH, by consensus sequence analysis of the coenzyme binding region, as shown in FIG. 4, these peripheral residues are highly conserved and most of the peripheral residues occupy the major conserved amino acid types. And 208 has more amino acids, which shows that the site has greater plasticity. Thus, based on the V294A mutant, a saturation mutation was performed at position 208. As shown in FIG. 5, the double point mutant V294A/N208K had a reaction conversion rate of 73% which was higher than that of the single point mutant V294A. Then we further carried out asymmetric reductive amination of 25 aromatic carbonyl compounds with the obtained double point mutant V294A/N208K, which showed higher catalytic activity for 24 compounds than the novel amine dehydrogenase TvAmDH. The obtained mutants were: a derivative protein formed by substituting valine at 294 of an amino acid sequence shown as SEQ ID No.2 with alanine, wherein a new amino acid sequence is shown as SEQ ID No. 3; further, the 208 th asparagine of the amino acid sequence shown as SEQ ID No.3 is replaced by lysine to form derivative protein, and the new amino acid sequence is shown as SEQ ID No. 4.

EXAMPLE 3 construction of an amine dehydrogenase TvAmDH mutant

The primers were designed for the pseudo-mutation sites separately using the plasmids of the single plasmid double enzyme co-expression system as the female parent, and the upstream and downstream primer sequences (SEQ ID NOS.15-18) are shown in Table 3 below.

TABLE 3 Table 3

The PCR reaction system is shown in Table 4 below.

TABLE 4 Table 4

PCR reaction conditions: pre-denaturation at 95℃for 3min; denaturation at 98℃for 5s, annealing at 58℃for 15s, extension at 72℃for 5min, 30 cycles of this stage; finally, final extension is carried out for 5min at 72 ℃; preserving at 4 ℃.

After the completion of PCR amplification and positive by agarose gel electrophoresis, 0.5. Mu. LDpnI enzyme was added to specifically cleave off methylated template DNA, and the mixture was left under a metal bath at 37℃for 3 hours. Transformation, picking up bacteria and sequencing can be performed subsequently.

Example 4 Induction and expression of Single plasmid double enzyme Co-expression System

The recombinant cells are coated on a solid flat plate, cultured for 12 hours in a constant temperature incubator at 37 ℃, single colonies are picked up into a 5mLLB culture medium test tube, kanamycin solution with one thousandth concentration is added, and the test tube is placed in a constant temperature shaker at 37 ℃ for shake culture for 8 hours. And transferring 500 mu L of bacterial liquid into 50mL of TB liquid culture medium, adding kanamycin solution with one thousandth concentration, and culturing for 2-2.5h until the OD600 of the cells reaches 0.6-0.8. The cultured bacterial liquid is added with one ten thousandth concentration of inducer IPTG (final concentration is 0.1 mM) for induction, and then the shake flask is placed in a constant temperature incubator at 20 ℃ and 200rpm for 12 hours of incubation. After balancing these bacterial solutions, centrifuging (4 ℃,800 rpm,10 min), discarding the supernatant, adding 15mL of physiological saline to wash for 2 times, discarding the supernatant, adding sodium phosphate buffer (100 mM, pH 8.0) according to the mass of the wet bacterial cells in proportion for resuspension, crushing by an ultrasonic cytobreaker, centrifuging the suspension bacterial solution after crushing (4 ℃,800 rpm,10 min), and pouring out the supernatant on ice to obtain a crude enzyme solution. Wherein, the SDS-PAGE protein gel electrophoresis diagram of the single plasmid double enzyme co-expression system of the amine dehydrogenase TvAmDH is shown in figure 1.

EXAMPLE 5 preparation of chiral amines by reductive amination of aromatic carbonyl Compounds catalyzed by amine dehydrogenase TvAmDH mutant

Analytical grade reductive amination is established by taking aromatic carbonyl compounds as substrates, and a reaction system (1 mL) comprises: substrate (50 mM), NADH (0.5 mM), methanol (5% v/v), lyophilized powder of the monogranzyme coexpression strain (10 g/L), ammonium chloride/ammonia buffer (1M, pH 9.5).

The specific operation is as follows: firstly weighing the freeze-dried bacterial powder into an EP tube, then placing the rest components into a beaker for uniform mixing, adjusting the pH to 9.5 by using a pH meter, taking 1mL of reaction liquid, adding the reaction liquid into the freeze-dried bacterial powder, fully and uniformly mixing, and reacting the reaction mixture for 24 hours at the temperature of 30 ℃ and the speed of 250 rpm. After the reaction was completed, 50. Mu.L of NaOH solution (10M) was added thereto and mixed by shaking to quench the reaction. Subsequently, the specific activities of amine dehydrogenase TvAmDH and its mutant V294A/N208K for various aromatic carbonyl compounds were measured, and the results are shown in Table 5 below.

TABLE 5

Example 6 enzymatic Scale preparation of chiral amine drug intermediates

The o-fluoro acetophenone is an aromatic carbonyl compound with wide application, and the reduction product o-fluoro phenethylamine is an important chiral intermediate, can be used for synthesizing a hypertension regulator, and has higher application value. In order to realize the scheme optimization of the enzymatic scale preparation of the chiral amine drug intermediate, the conditions of optimal reaction temperature, pH, group solvent, cofactor concentration and the like are explored, and the results are shown in figures 6-9. The optimal temperature is 30 ℃; the optimal pH is 9.5; the most preferred group solvent is Dimethylsulfoxide (DMSO); the optimal concentration of cofactor NADH is 0.3mM.

Then, under the optimal reaction condition, in a 10mL reaction system, the single plasmid double enzyme co-expression system catalyzes o-fluoro acetophenone with the concentration of 50mM and 100mM, and for a 50mM substrate, the conversion rate reaches 90% in 1 hour, and the reaction is completely converted in 8 hours; for 100mM substrate, a conversion of >99% and a stereoselectivity of >99% (R) can also be achieved at 12 hours.

The above description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can be changed in various ways, namely, any simple, equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent. The present invention is not described in detail in the conventional art.

Claims (10)

1. An amine dehydrogenase mutant, characterized in that the amine dehydrogenase mutant is: the amino acid sequence of the mutant V294A/N208K is shown as SEQ ID No.4 by taking amine dehydrogenase TvAmDH with the amino acid sequence shown as SEQ ID No.2 as a template, substituting valine at 294 th site with alanine and substituting asparagine at 208 th site with lysine.

2. A nucleic acid encoding the amine dehydrogenase mutant according to claim 1, wherein the nucleic acid is represented by SEQ ID No. 5.

3. A recombinant expression vector comprising the nucleic acid sequence of claim 2.

4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.

5. The construction method of the single plasmid double enzyme co-expression system is characterized by comprising the following steps:

1) Extracting and amplifying plasmids pET-28a-V294A/N208K, plasmid pET-28a-bmgdh and a vector plasmid pET-28a (+), wherein the plasmid pET-28a-V294A/N208K is constructed by a vector plasmid pET-28a and a mutant V294A/N208K according to claim 1, and the plasmid pET-28a-bmgdh is constructed by a vector plasmid pET-28a and glucose dehydrogenase GDH with an amino acid sequence shown as SEQ ID NO. 6;

2) Carrying out double-section fusion on the two sections of target gene fragments purified in the step 1);

3) And respectively carrying out enzyme digestion on the plasmid pET-28a-V294A/N208K, the plasmid pET-28a-bmgdh and the vector pET-28a (+) by using endonucleases, and sequentially connecting the plasmids to transform host escherichia coli BL21 (DE 3), so as to construct the single-plasmid double-enzyme coexpression system.

6. A single plasmid double enzyme coexpression system constructed by the construction method of claim 5.

7. A recombinant amine dehydrogenase mutant catalyst prepared from the recombinant expression transformant of claim 4, wherein the recombinant amine dehydrogenase mutant catalyst comprises lyophilized powder, crude enzyme solution or pure enzyme.

8. Use of the amine dehydrogenase mutant of claim 1 or the recombinant amine dehydrogenase mutant catalyst of claim 7 for catalyzing asymmetric reductive amination of an aromatic carbonyl compound to produce a corresponding chiral amine.

9. The use according to claim 8, wherein the aromatic carbonyl compound has the structural formula:

10. A method for preparing a corresponding chiral amine by asymmetric reductive amination of an aromatic carbonyl compound using the amine dehydrogenase mutant of claim 1 or the recombinant amine dehydrogenase mutant catalyst of claim 7, comprising:

1) An analytical grade reductive amination reaction is established by taking an aromatic carbonyl compound as a substrate, and a 1mL reaction system comprises: 40-60 mM substrate, 0.4-0.6 mMNADH, 5% v/v methanol, and 1M ammonium chloride/ammonia buffer at pH 9.5;

2) Firstly, taking a proper amount of recombinant amine dehydrogenase mutant catalyst into an EP tube, then placing the rest components into a beaker, uniformly mixing, using a pH meter to adjust the pH to 9.5, taking 1mL of reaction liquid, adding the reaction liquid into the heavy histamine dehydrogenase mutant catalyst, fully and uniformly mixing, reacting the reaction mixture for 20-30 hours at 28-32 ℃ under the condition of 250rpm, and adding 50 mu L of 10MNaOH solution after the reaction is finished, vibrating and uniformly mixing to quench the reaction.