CN110964705A

CN110964705A - Novel application of (R) -omega-transaminase mutant

Info

Publication number: CN110964705A
Application number: CN201911215847.0A
Authority: CN
Inventors: 黄�俊; 刘春燕; 梅乐和; 吕常江; 王宏鹏; 于林凯; 胡升; 赵伟睿; 蒋成君
Original assignee: Zhejiang Lover Health Science and Technology Development Co Ltd
Current assignee: Zhejiang Lover Health Science and Technology Development Co Ltd
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-04-07

Abstract

The application discloses an application of an (R) -omega-transaminase mutant, wherein the amino acid sequence of the (R) -omega-transaminase mutant is shown as SEQ ID No.1, 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone is used as a substrate, the (R) -omega-transaminase mutant is used as a catalyst, and a catalytic reaction is carried out in the presence of an amino donor to synthesize (R) -2, 3-dichlorophenethylamine or (R) -2, 5-dichlorophenethylamine. The method takes (R) -omega-transaminase as a biocatalyst to catalyze and generate 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone, and the product can be used as a drug intermediate to synthesize related drugs. The method has the advantages of strong stereoselectivity, simple preparation process and convenient extraction, and solves the problems of low optical purity, low yield, environmental pollution and the like widely existing in the synthesis of the chiral compound at present.

Description

Novel application of (R) -omega-transaminase mutant

Technical Field

The invention relates to the technical field of molecular biology, in particular to a new application of an (R) -omega-transaminase mutant, namely a biocatalysis method for synthesizing (R) -2, 3-dichlorophenethylamine or (R) -2, 5-dichlorophenethylamine.

Background

The chiral compound has a special stereo structure, and enantiomers of the chiral compound often show different physicochemical properties and physiological activities, and play an important role in the fields of new material development, new drug design, fine chemical synthesis and the like in recent years.

The PPARG gene belongs to the subfamily of nuclear receptor peroxisome proliferator-activated receptors (gamma) because of a gene encoding a peroxisome proliferator-activated receptor gamma. PPARG has different degrees of expression in tissues such as fat, liver, skeletal muscle, kidney, pancreas and the like, is heterodimerized with a retina-like receptor (9-cis-RXR) in vivo, is activated after being combined with a ligand, and activates or inhibits gene transcription through the action of a PPAR response element (PPREs) in a target gene promoter, thereby participating in various physiological and pathological processes in vivo, including differentiation of fat cells, inflammatory response, apoptosis, obesity, atherosclerosis, cancer and the like. In recent years, it has been found that full agonists such as rosiglitazone have significant side effects in clinical applications such as causing weight gain, sodium water retention which may lead to an increased risk of cardiovascular events and the development of osteoporosis. Recent research shows that non-agonist PPARG ligand (compound I, the structural formula is shown as formula (I)) can activate PPARG to regulate glycolipid metabolism and other related functions by inhibiting PPARG phosphorylation level, and does not cause PPARG full-agonist side effects.

Histone acetylation is catalyzed by Histone Acetyltransferase (HAT), resulting in an open autosomal structure that allows transcription of genes including tumor suppressor genes histone deacetylase inhibitors can inhibit deacetylation of histones, which has been shown to be an effective method for treating related cancers it has been found that histone acetylation is also regulated by bromoproteins, which are evolutionarily conserved bundles of about 110 amino acids, consisting of 4 α helices, binding to the acetyl lysine of acetylated proteins, these domains are present in many chromosome-related proteins, including HAT.

Therefore, inhibition or modulation of bromoproteins may provide a novel mode of pharmaceutical intervention for such diseases. Some new researches find that a compound can be combined with bromodomain protein and can generate an inhibition effect on the bromodomain protein, and the structure of the compound is shown as formulas (II) and (III):

disclosure of Invention

The invention provides a new application of an (R) -omega-transaminase mutant, which takes the transaminase as a catalyst to biologically synthesize (R) -2, 3-dichlorophenethylamine or (R) -2, 5-dichlorophenethylamine.

The application of the (R) -omega-transaminase mutant is characterized in that the amino acid sequence of the (R) -omega-transaminase mutant is shown in SEQ ID NO.1, 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone is used as a substrate, the (R) -omega-transaminase mutant is used as a catalyst, and under the condition of an amino donor, the (R) -2, 3-dichlorophenethylamine or the (R) -2, 5-dichlorophenethylamine is synthesized.

The (R) - ω -transaminase mutant (transaminase mutant L118T) was obtained by mutating the 118 th leucine (codon TTG) of a wild-type Aspergillus terreus (Aspergillus terreus) ω -transaminase to threonine (codon AGG). The nucleotide sequence of the (R) -omega-transaminase mutant is shown in SEQ ID NO. 2.

Optionally, the amino donor is sec-butylamine.

2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone is taken as an amino acceptor, sec-butylamine is taken as an amino donor, amino from the amino donor is transferred to the amino acceptor by transaminase catalysis, and (R) -2, 3-dichlorophenethylamine or (R) -2, 5-dichlorophenethylamine is obtained by reaction.

Optionally, the mass ratio of the substrate to the catalyst is 50-100: 1.

optionally, the molar ratio of the amino donor reagent to the substrate is 50mM: 0-30 mM.

Optionally, the pH of the catalytic reaction system is 6.5-8.5.

Optionally, the temperature of the catalytic reaction is 20-50 ℃; the time of catalytic reaction is 18-36 h.

Optionally, the stirring speed in the reaction process is 100-500 rpm.

Alternatively, the (R) - ω -transaminase mutant is expressed from the recombinant plasmid pET-28a- ω -TA. The original vector of the recombinant plasmid is pET-28 a.

The method generates (R) -2, 3-dichlorophenethylamine (non-agonist PPARG ligand intermediate) and (R) -2, 5-dichlorophenethylamine (R1-L1-N-R2) drug intermediate by biocatalysis, and the drug intermediate reacts with other compounds to generate the compounds (II) and (III). Compared with a chemical synthesis method, the chiral amine compound is synthesized by utilizing (R) -omega-transaminase biocatalysis, the synthesis condition is mild, the operation is simple, and the separation and extraction are easy.

Drawings

FIG. 1 is a SDS-PAGE analysis result of the transaminase L118T mutant (threonine with leucine mutation at position 118) prepared in example 1.

FIGS. 2-a-2-e are ultra-high performance liquid chromatograms of transaminase catalyzing substrate 2, 3-dichloroacetophenone to 2, 3-dichlorophenethylamine under different reaction conditions (example 2-example 5).

FIGS. 3-a to 3-e are ultra-high performance liquid chromatograms of transaminase catalyzing substrate 2, 5-dichloroacetophenone to generate 2, 5-dichlorophenethylamine under different reaction conditions (example 2 to example 5).

FIGS. 4-a to 4-b are graphs of the products after derivatization (example 2 to example 5) (4-a: FDAA-R-2, 3-dichlorophenethylamine; 4-b: R-2, 3-dichlorophenethylamine).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

1. Experimental Material

(1) LB culture medium: 10g/L tryptone (from Oxoid), 5g/L yeast powder (from Oxoid),10g/L sodium chloride (from Biotechnology engineering Co., Ltd. (Shanghai)), pH 7.0. LB solid medium: 2 percent (mass ratio) of agar powder is added into the LB liquid culture medium.

(2) Sodium chloride, glycerol, calcium chloride, imidazole, glacial acetic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, pyridoxal 5-phosphate, coomassie brilliant blue protein concentration assay kit, Ni-NTA chromatography media, isopropyl- β -D-thiogalactoside (IPTG), kanamycin sulfate, coenzyme pyridoxal phosphate (PLP) DNA, and protein Marker were purchased from bio-engineering limited (shanghai).

(3) The strain is as follows: in the application, omega-aminotransferase gene (omega-TA gene) (SEQ ID NO.2) of omega-aminotransferase gene (omega-TA gene) with codon optimization (namely, leucine codon TTG corresponding to the 118 th position of amino acid sequence is mutated into threonine codon AGG) is entrusted to general biological system (Anhui) limited company for whole gene synthesis, pET-28a plasmid is used as a cloning vector in the gene synthesis service, and the enzyme cutting sites are NdeI and HindIII respectively. The constructed recombinant plasmid pET-28 a-omega-opt-TA (transaminase mutant L118T plasmid) is transferred into E.coli BL21(DE3) to obtain recombinant bacteria.

The site-directed mutagenesis primers corresponding to the omega-transaminase mutant L118T are as follows:

L118T-F：5’-GGGATGCATTTGTTGAAACCATAGTCACCCGCGGTC-3’(SEQ ID NO.3)；

L118T-R：5’-GACCGCGGGTGACTATGGTTTCAACAAATGCATCCC-3’(SEQ ID NO.4)。

and (3) PCR system:

and (3) PCR reaction conditions:

98℃1min

72℃7min

keeping the temperature at 4 DEG C

And (3) carrying out enzymolysis on the PCR reaction product for 2h at 37 ℃ by using Dpn I to eliminate a male parent template, transforming the enzymolysis product into a chemically competent cell E.coli DH 5 α by adopting a heat shock method, and coating an LB solid plate containing kanamycin (50 mu g/mu L) with a transformation solution to obtain a site-specific mutagenesis library.

(4) The buffer solution formula comprises:

20mmol/L elution buffer: 50mmol/L sodium dihydrogen phosphate, 300mmol/L sodium chloride, 20mmol/L imidazole, pH 8.0; 50mmol/L elution buffer: 50mmol/L sodium dihydrogen phosphate, 300mmol/L sodium chloride, 50mmol/L imidazole, pH 8.0; 100mmol/L elution buffer: 50mmol/L sodium dihydrogen phosphate, 300mmol/L sodium chloride, 100mmol/L imidazole, pH 8.0; 250mmol/L elution buffer: 50mmol/L sodium dihydrogen phosphate, 300mmol/L sodium chloride, 250mmol/L imidazole, pH 8.0.

Gel staining solution: 45.4mL of methanol, 9.2mL of glacial acetic acid, 0.05g of Coomassie Brilliant blue R250 and 100mL of deionized water; gel decolorant liquid: 5mL of methanol and 7.5mL of glacial acetic acid are dissolved in 100mL of deionized water; electrophoresis buffer solution: 3.03g Tris, 14.4g glycine, 1g SDS, with deionized water to volume of 1L, 0.22 μm filter paper suction filtration, 4 degrees C cold storage.

2. Expression and purification of mutant L118T transaminase

Adding 2 μ L plasmid of transaminase mutant L118T into 50 μ L E.coli BL21 competent cells, mixing, standing on ice for 30min, hot beating in 42 deg.C water bath for 90s, quickly returning to ice bath for 3min, adding 600 μ L LB culture medium, and recovering at 37 deg.C and 180r/min for 50 min. mu.L of the culture solution was spread evenly on LB solid plates (containing 50. mu.g/mL kanamycin), incubated at 37 ℃ for 30min in an incubator, and the plates were inverted for overnight culture.

A single colony was inoculated into a tube containing 5mL of LB liquid medium and cultured overnight at 37 ℃ at 200 r/min. Inoculating the cultured bacterial liquid at an inoculation amount of 1% (volume ratio) into 100mL LB medium (tryptone 10g, yeast powder 5g, sodium chloride 10g, pH 7.0 adjusted) containing 50. mu.g/mL kanamycin, culturing at 37 ℃ and 180r/min to OD₆₀₀When the value is 0.4-0.6, adding a proper volume of IPTG (final concentration is 0.5mmol/L), then carrying out induced culture for 18h under the conditions of 16-30 ℃ and 150-200 r/min, and collecting the thalli.

Washing the collected thalli twice by using a phosphate buffer solution, re-suspending by using a cell breaking buffer solution with the volume of 10% of fermentation liquor, and breaking cells by using ultrasonic waves, wherein the working conditions of ultrasonic cell breaking are as follows: the power is 300W, the work time is 3s, the pause time is 6s, and the ultrasound time is 8 minutes. And centrifuging the cell-breaking solution at 12000r/min and 4 ℃ for 30min, and collecting supernatant to obtain the crude omega-transaminase solution containing the mutant L118T transaminase.

And (3) separating and purifying the obtained crude enzyme liquid by adopting Ni-NTA affinity chromatography. And (3) carrying out sampling, washing and eluting, collecting eluent, and dialyzing to remove small molecules to obtain the pure enzyme. After appropriate dilution, the concentration of the pure enzyme was determined by the Coomassie Brilliant blue method.

The specific purification steps are as follows:

(1) equilibration of Ni-NTA affinity chromatography column: 3 column volumes each of 20% ethanol, deionized water and 20mM imidazole buffer;

(2) loading: the crude enzyme solution is absorbed by a syringe and filtered by a 0.45 mu m filter membrane, and the recombinant protein with 6 histidine labels is specifically combined with the filler.

(3) Cleaning: 2-3 column volumes of 50mM imidazole buffer, and detecting whether the impure protein is removed by using Bradford solution;

(4) and (3) elution: 5mL of 100mM elution buffer and 5mL of 250mM elution buffer;

(5) and (3) column preservation: 20mM imidazole buffer, deionized water and 20% ethanol each 3 column volumes, finally stored in 20% ethanol.

3. SDS-PAGE analysis of transaminase mutant L118T

The purified protein is detected by SDS-PAGE discontinuous electrophoresis, and the specific steps are as follows:

preparing glue: 12% of separation gel and 5% of concentrated gel. The formulation is shown in table 1.

TABLE 1 SDS-PAGE protein electrophoretic separation gel and concentrated gel formulations

Sample treatment: mixing the crude enzyme solution 40 μ L and 5 Xprotein sample buffer solution 10 μ L, and boiling water bath for 10 min.

Loading: protein Marker 5. mu.L, sample 10. mu.L.

Electrophoresis conditions: the voltage is 170V, electrophoresis is carried out for about 45min, and the electrophoresis is stopped when the bromophenol blue indicator moves to a position about 1cm away from the lower end edge of the gel.

Dyeing: the staining solution is immersed in the gel, heated in a microwave oven for 1min, and stained on a shaking table for 25 min.

And (3) decoloring: and (4) recovering the dyeing liquid, replacing the decoloring liquid, and replacing the decoloring liquid once per hour until the protein band is clear.

SDS-PAGE analysis of transaminase mutant L118T (i.e. (R) - ω -transaminase mutant) is shown in FIG. 1, and the protein molecular weight is close to the theoretical molecular weight of the wild-type enzyme of 36.1 kDa. The amino acid sequence is shown as SEQ ID NO. 1.

The (R) -2, 3-dichlorophenethylamine and (R) -2, 5-dichlorophenethylamine were synthesized by biocatalysis using the above-described (R) - ω -transaminase mutants as described in examples 2 to 5 below:

example 2

(R) - ω -transaminase mutants catalyze the transfer of amino groups from amino donors to 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone: the substrate solution is prepared by phosphate buffer solution (50mmol/L, pH 8.0), 1mL reaction system comprises 50mM sec-butylamine, 30mM amino acceptor ketone (the substrate ketone is 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone), 0.1mM PLP, 0.1mg/mL (R) -omega-transaminase mutant, the catalytic reaction is carried out for 30h at 30 ℃, and the blank control replaces the (R) -omega-transaminase mutant by buffer solution.

Adding 2% of Marfey chiral derivative reagent (the Marfey reagent and amino groups in chiral molecules of reaction products are subjected to substitution reaction to obtain an optical isomer containing two or more chiral centers through derivation, conveniently and quickly analyzing the optical purity of the derived prochiral compound by analytical means such as HNMR, HPLC and the like), and deriving for 2h at 40 ℃. And after derivatization is finished, adding 2M HCl to terminate derivatization, extracting a product with ethyl acetate, drying the product by using a nitrogen blowing instrument, redissolving the product with acetonitrile, filtering the product with a filter membrane, and then placing the product in a sample bottle.

Example 3

(R) - ω -transaminase mutants catalyze the transfer of amino groups from amino donors to 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone: the substrate solution is prepared by phosphate buffer solution (50mmol/L, pH 8.0), 1mL reaction system comprises 50mM sec-butylamine, 1mM amino acceptor ketone (the substrate ketone is 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone), 0.1mM PLP, 0.1mg/mL (R) -omega-transaminase mutant, the catalytic reaction is carried out for 30h at 30 ℃, and the blank control replaces the (R) -omega-transaminase mutant by buffer solution.

Adding 2% of Marfey chiral derivatization reagent into the reacted mixed solution, and performing derivatization for 2h at 40 ℃. After the derivatization was complete, the derivatization was stopped by adding 2M HCl. And extracting the product with ethyl acetate, blowing the product with a nitrogen blowing instrument, re-dissolving the product with acetonitrile, filtering the product with a filter membrane, and then placing the product in a sample bottle.

Example 4

(R) - ω -transaminase mutants catalyze the transfer of amino groups from amino donors to 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone: the substrate solution was prepared with phosphate buffer (50mmol/L, pH 8.0), 1mL of reaction system included 50mM sec-butylamine, 5mM amino acceptor ketones (substrate ketones 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone), 0.1mM PLP, 1mg/mL of (R) - ω -transaminase mutant, catalytic reaction was performed at 30 ℃ for 20h, and the blank control replaced the (R) - ω -transaminase mutant with buffer solution.

Example 5

(R) - ω -transaminase mutants catalyze the transfer of amino groups from amino donors to 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone: the substrate solution is prepared by phosphate buffer solution (50mmol/L, pH 8.0), 1mL of reaction system comprises 50mM sec-butylamine, 10mM amino acceptor ketone (the substrate ketone is 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone), 0.1mM PLP, 1mg/mL of (R) -omega-transaminase mutant, the catalytic reaction is carried out for 20h at 50 ℃, and the blank control replaces the (R) -omega-transaminase mutant by buffer solution.

Adding 1% of Marfey chiral derivatization reagent into the reacted mixed solution, and performing derivatization for 2h at 40 ℃. After the derivatization was complete, the derivatization was stopped by adding 2M HCl. And extracting the product with ethyl acetate, blowing the product with a nitrogen blowing instrument, re-dissolving the product with acetonitrile, filtering the product with a filter membrane, and then placing the product in a sample bottle.

Examples 2-5 UPLC-MS analysis method and results of reaction products:

ultra-high performance liquid chromatography analysis conditions: the chromatographic column is ACQUITY UPLC BEH C18 column (2.1X 100mm, 1.7 μm) with a column temperature of 40 deg.C; the sample injection amount is 1 mu L; the mobile phase is as follows: phase a (water + 0.1% formic acid), phase B (acetonitrile + 0.1% formic acid).

Analysis conditions of the Xevo G2-XS mass spectrometer: electrospray ion source, anion mode; the wavelength range of the photodiode array detector is 190-400 nm; the cone hole voltage is 40V, the collision energy is 20-55V, and the mass-to-charge ratio range of mass spectrum scanning is 50-1200 Da.

The (R) -omega-transaminase mutant catalyzes a substrate of 2, 3-dichloroacetophenone to generate 2, 3-dichlorophenethylamine under different reaction conditions (example 2 to example 5): the reaction system was 1mL, in which the amino group donor was 50mM, PLP was 0.1mM, and pH was 8.0. a, blank control, the other reaction conditions are the same, and the catalyst (R) -omega-transaminase mutant is not added; b 0.1mg/mL (R) - ω -transaminase mutant catalyzed 30mM 2, 3-dichloroacetophenone at 30 ℃ for 30h (example 2); c 0.1mg/mL (R) - ω -transaminase mutant catalyzed 1mM2, 3-dichloroacetophenone at 30 ℃ for 30h (example 3); d 1mg/mL (R) - ω -transaminase mutant at 30 ℃ catalyzes 5mM 2, 3-dichloroacetophenone for 20h (example 4); e 1mg/mL (R) - ω -transaminase mutant at 50 ℃ catalyzes the reaction of 10mM 2, 3-dichloroacetophenone for 20h (example 5).

The (R) -omega-transaminase mutant catalyzes a substrate 2, 5-dichloroacetophenone to generate the 2, 5-dichloroacetophenone under different reaction conditions (example 2-example 5): the reaction system is 1mL, wherein the amino donor sec-butylamine is 50mM, the PLP is 0.1mM and the pH is 8.0. a, blank control, the other reaction conditions are the same, and the catalyst (R) -omega-transaminase mutant is not added; b 0.1mg/mL (R) - ω -transaminase mutant catalyzed 30mM 2, 5-dichloroacetophenone at 30 ℃ for 30h (example 2); c 0.1mg/mL (R) - ω -transaminase mutant catalyzed 1mM2, 5-dichloroacetophenone at 30 ℃ for 30h (example 3); d 1mg/mL (R) - ω -transaminase mutant at 30 ℃ catalyzes 5mM 2, 5-dichloroacetophenone for 20h (example 4); e 1mg/mL (R) - ω -transaminase mutant at 50 ℃ catalyzes the reaction of 10mM 2, 5-dichloroacetophenone for 20h (example 5).

The detection result of the converted reaction solution by ultra performance liquid chromatography is shown in fig. 2, and fig. 2 is an ultra performance liquid chromatogram of (R) - ω -transaminase mutant catalyzing substrate 2, 3-dichloroacetophenone to generate 2, 3-dichlorophenethylamine under different reaction conditions: in the ultra-high performance liquid chromatogram generated by using 2, 3-dichloroacetophenone as a substrate in figure 2-a and 2, 5-dichloroacetophenone as a substrate in figure 3-a without the reaction of the (R) -omega-transaminase mutant, when the retention time is 1.26min, a peak of m/z 271.0516 appears, which is a peak of a Marfey chiral derivative reagent (FDAA). At a retention time of 1.77min, a peak of m/z 324.1318 appeared, and this reaction peak was found to be mainly FDAA-2-Butylamine.

FIGS. 2-b-2-e (2, 3-dichloroacetophenone + catalyst) and FIGS. 3-b-3-e (2, 5-dichloroacetophenone + catalyst) are ultra-high performance liquid chromatograms of the product of the reaction of (R) - ω -transaminase mutant with 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone, respectively. In contrast to FIG. 2-a, the peak at retention time 2.81 in FIGS. 2-b-2-e of m/z440.0527 appeared to be the peak for FDAA-2, 3-dichlorophenethylamine.

The peak at m/z440.0527 appeared at retention time 2.81 in FIGS. 3-b-3-d, the substrate conversion was low due to the catalytic environmental effects in FIG. 3-e, the product formed had a smaller response relative to the substrate, but a peak at m/z440.0527 was still found depending on the molecular weight, and m/z440.0527 was the peak for FDAA-2, 5-dichlorophenethylamine.

Mass spectrometry was performed on the compound at the m/z440.0527 peak in FIGS. 2 b-2-e and the compound at the m/z440.0527 peak in FIGS. 3 b-3-e, respectively, and the results are shown in FIGS. 4-a-4-b, which are primary mass spectrograms of the product after biocatalytic reaction and derivatization. FIG. 4-a is a primary mass spectrum of the compound with the peak m/z440.0527 in FIGS. 2-b-2-e, and FIG. 4-b is a primary mass spectrum of the compound with the peak m/z440.0527 in FIGS. 3-b-3-e. In the primary mass spectra of FIGS. 4-a and 4-b, molecular ion fragment peaks of M/z440.0527 ([ M-2H ] -), M/z442.0520([ M ] -) and M/z 443.0555([ M + H ] -) appear. Further verification that the peaks are produced by the compounds 2, 3-dichlorophenethylamine and 2, 5-dichlorophenethylamine, the (R) - ω -transaminase mutant used herein is R-chiral selective, and the catalytic production is (R) -2, 3-dichlorophenethylamine and (R) -2, 5-dichlorophenethylamine.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Zhejiang science and technology institute

Novel application of <120> (R) -omega-transaminase mutant

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<213> Artificial sequence (Artificial sequence)

<400>4

gaccgcgggt gactatggtt tcaacaaatg catccc 36

Claims

The application of the (R) -omega-transaminase mutant is characterized in that 2, 3-dichloroacetophenone or 2, 5-dichloroacetophenone is used as a substrate, the (R) -omega-transaminase mutant is used as a catalyst, and a catalytic reaction is carried out in the presence of an amino donor to synthesize (R) -2, 3-dichlorophenethylamine or (R) -2, 5-dichlorophenethylamine, wherein the amino acid sequence of the (R) -omega-transaminase mutant is shown in SEQ ID NO. 1.
2. The use according to claim 1, wherein the mass ratio of the substrate to the catalyst is 50-100: 1.
3. Use according to claim 1, characterized in that the amino donor is sec-butylamine.
4. The use according to claim 1, wherein the molar ratio of amino donor to substrate is 50mM:1 to 30 mM.
5. The use according to claim 1, wherein the catalytic reaction system has a pH of 6.5 to 8.5.
6. The use according to claim 1, wherein the temperature of the catalytic reaction is 20 to 50 ℃; the time of catalytic reaction is 18-36 h.
7. The use of claim 1, wherein the stirring speed during the catalytic reaction is 100-500 rpm.
8. The use according to claim 1, wherein the (R) - ω -transaminase mutant is expressed from the recombinant plasmid pET-28a- ω -TA.