Reductive aminase mutant and application thereof in synthesis of chiral alkylated benzyl propylamine

Abstract

The invention discloses a reductive aminase mutant and application of the reductive aminase mutant in synthesis of chiral alkylated benzyl propylamine. The invention relates to a mutant protein of carbonyl reductase, which is characterized in that the mutant protein has one or more alternative mutations in sites 85, 87, 128, 134, 162, 197, 219, 234 and 235 on the basis of an amino acid sequence as shown in SEQ ID NO: 1. On the basis of analysis of the crystal structure of reductive aminase M5 in the early stage, stereoselective transformation is carried out through site-specific mutagenesis, combinatorial mutation and the like, an efficient mutant for synthesizing an alkylated benzyl propylamine S-enantiomer is obtained, and a series of chiral amines with the selectivity larger than 99% S are synthesized with different amine donors such as benzyl acetone and benzylamine as substrates. And the method has important significance on industrial green synthesis of the chiral amine.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and enzyme engineering, and relates to a reductase mutant and its application in the synthesis of chiral alkylated benzylpropylamine. Background Art

[0002] In the case where a stereocenter is contained in R2, using a rare earth metal dibenzoyl catalyst, asymmetric intermolecular Markovnikov addition of a simple amine to an unactivated olefin can be achieved, with an ee value up to 61% and a de value up to 73%; low optical purity (S)-α-methyl-N-(benzyl)amphetamine can be synthesized. The original synthesis requires high-temperature and high-pressure reaction conditions during the reaction process, is not suitable for industrial production; uses expensive metal catalysts, is prone to environmental pollution and is not easy to remove, etc.; the optical purity of the product is low.

[0003] Chiral amine structural units widely exist in drug molecules for clinical applications. Their enantiomers can cause differences in the biological activities of drugs. Therefore, the optically pure synthesis of chiral amine drugs is very important. Compared with chemical methods, the enzymatic synthesis of chiral amine molecules has the advantages of economy, high efficiency, environmental friendliness, etc. Reductases discovered in recent years have been widely studied due to their atom economy advantages in catalyzing equimolar substrates. However, natural enzymes often have problems such as narrow substrate spectra, low substrate loading, and poor stereoselectivity, making them difficult to be applied industrially. Moreover, the directed evolution of natural enzymes to industrially applicable enzymes generally requires large-scale mutant screening, with low efficiency. Benzylpropylamine is an important functional group in many drugs, such as dilevalol and labetalol for treating hypertension. Based on the optimal mutant M5 of the engineered reductase with high selectivity and high conversion rate for piperidineamine obtained in the early stage of the laboratory, it was found that it has good catalytic activity for benzylacetone, with a substrate loading reaching 15 g / L, but the stereoselectivity is poor, and the ee value is only 12% (R). Summary of the Invention

[0004] The present invention obtained a reductase mutant V1 through semi-rational design, and its DNA and amino acid sequences are shown in SEQ ID NO.3-4. The present invention also provides the application of the reductase mutant V1 in the reaction of synthesizing alkylbenzylpropylamine from a ketone and an amine, wherein the ketone is benzylacetone; the amine is wherein, R2 is , , , or x is an integer from 1 to 10.

[0005] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions: The present invention provides a mutant protein of carbonyl reductase, wherein the mutant protein has one or more substitution mutations at positions 85, 87, 128, 134, 162, 197, 219, 234, and 235 based on the amino acid sequence shown in SEQ ID NO: 1.

[0006] Specifically, based on the amino acid sequence shown in SEQ ID NO: 1, it has a mutation at any one of the following positions: phenylalanine at position 85 is mutated to cysteine or threonine; phenylalanine at position 87 is mutated to tryptophan; tyrosine at position 128 is mutated to alanine or glycine; phenylalanine at position 134 is mutated to valine, isoleucine, or leucine; histidine at position 162 is mutated to alanine; valine at position 197 is mutated to threonine or serine; tyrosine at position 219 is mutated to isoleucine or phenylalanine; proline at position 234 is mutated to arginine; valine at position 235 is mutated to isoleucine.

[0007] Preferably, based on the amino acid sequence shown in SEQ ID NO: 1, it has a mutation at any one of the following positions: a combined mutation at positions 85 and 87; a combined mutation at positions 85, 87, and 128; a combined mutation at positions 85, 87, and 134; a combined mutation at positions 85, 87, 134, and 197; a combined mutation at positions 85, 87, 134, 197, and 219.

[0008] The present invention further provides a nucleic acid encoding the mutant protein described above.

[0009] The present invention also provides an expression vector containing the nucleic acid described above.

[0010] The present invention provides a recombinant host bacterium containing the nucleic acid or the expression vector described above.

[0011] The present invention also provides the use of the mutant protein described above in the synthesis of S-boswellic acid.

[0012] The present invention further provides a method for reducing 1-C-(β-D-xylopyranosyl)-acetone, which includes the steps of: Contacting the mutant protein described above with the reaction substrate 1-C-(β-D-xylopyranosyl)-acetone, adding a coenzyme recycling system for catalytic reaction, thereby obtaining the S-configured boswellic acid; after the reaction is completed, concentrating under reduced pressure, and separating and purifying the S-boswellic acid.

[0013] Specifically, the pH of the catalytic reaction system is 6.0 - 8.0; the substrate concentration is 80 - 240 g / L; the reaction time is 10 - 48 hours; Preferably, the pH of the catalytic reaction system is pH 6.0 - 7.0; the substrate concentration is 120 - 200 g / L; the reaction time is 12 - 24 hours; Specifically, the mutant protein is used in the form of a pure protein, or a crude mutant protein solution obtained by culturing the recombinant host bacterium, or the recombinant host bacterium is used as a biocatalyst for the catalytic reaction.

[0014] In the reaction, a cell lysate of the reductamine dehydrogenase mutant is added. The mass - to - volume ratio of the wet weight of the reductamine dehydrogenase mutant cells to the reaction mixture is 10 g / L; the molar ratio of D - glucose to the ketone is 1.5:1; the amount of nicotinamide adenine dinucleotide phosphate disodium salt NADP+ added in the reaction is 1.0 mmol / L; the amount of glucose dehydrogenase GDH enzyme powder is 1.5 mg / L; the amount of phosphate is 100 mmol / L; the volume of DMSO used is 20% of the total volume of the reaction solution; the pH of the reaction solution is 7.0; the reaction temperature is 30 °C; the reaction time is 20 hours.

[0015] The steps of the reaction include: adding DMSO and sodium phosphate buffer in a volume ratio of 1:4 to the reactor; adding a mixture of amine, D - glucose and NADP+ to the reactor and reacting at 30 °C; the addition amount of benzyl acetone is 100 mmol / L; the molar ratio of the amine to the ketone is 1.2:1; adding the cell lysate of the reductamine dehydrogenase mutant V1 and GDH enzyme powder to the reaction solution for reaction; after reacting for 20 hours, the product is obtained.

[0016] After obtaining the product after reacting for 20 hours, the following steps are further included: quenching the reaction with hydrochloric acid to obtain a solution with a pH of 3.0 - 4.0; loading diatomaceous earth into the bioreactor and stirring for 10 minutes; filtering the mixture and rinsing with water to obtain a filtrate; extracting the filtrate with ethyl acetate successively, alkalizing the aqueous phase to pH = 10.0 with saturated sodium carbonate solution, extracting three times with ethyl acetate and combining the organic phase extracts; concentrating under reduced pressure to obtain the product.

[0017] By semi - rationally designing the active center of the enzyme and carrying out engineering transformation on the basis of previous research, the mutant V1 is obtained in the present invention. The high selectivity, high conversion rate and excellent substrate compatibility are demonstrated by the synthesis of a series of alkylated chiral benzylpropylamines.

[0018] Based on the analysis of the crystal structure of the previous reductive amination enzyme M5, the present invention performs stereoselective modification of M5 through protein engineering techniques such as site-directed mutagenesis and combinatorial mutagenesis, develops a highly efficient mutant for synthesizing the S-enantiomer of alkylated benzylpropylamine, and uses different amine donors such as benzylacetone and benzylamine as substrates to synthesize a series of chiral amines with a selectivity of >99% S, which is of great significance for the industrial green synthesis of such chiral amines. Description of the Drawings

[0019] Figure 1 . Enzymatic synthesis route.

[0020] Figure 2 . Chiral HPLC analysis chromatogram of the reductive amination product 1a catalyzed by M5 and V1.

[0021] Figure 3 . Chiral HPLC analysis chromatogram of the reductive amination product 1b catalyzed by V1.

[0022] Figure 4 . Chiral HPLC analysis chromatogram of the reductive amination product 1c catalyzed by V1.

[0023] Figure 5 . Chiral HPLC analysis chromatogram of the reductive amination product 1e catalyzed by V1.

[0024] Figure 6 . Chiral HPLC analysis chromatogram of the reductive amination product 1f catalyzed by V1.

[0025] Figure 7 . Chiral HPLC analysis chromatogram of the reductive amination product 1g catalyzed by V1.

[0026] Figure 8 . Chiral HPLC analysis chromatogram of the reductive amination product 1j catalyzed by V1.

[0027] Figure 9 . Chiral HPLC analysis chromatogram of the reductive amination product 1k catalyzed by V1.

[0028] Figure 10 . Chiral HPLC analysis chromatogram of the reductive amination product 1l catalyzed by V1.

[0029] Figure 11 . Chiral HPLC analysis chromatogram of the reductive amination product 1m catalyzed by V1. Detailed Embodiments

[0030] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods without specific conditions in the following examples are usually carried out under conventional conditions such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0031] Example 1: Semi-rational design of the protein structure based on the IR-G36 mutant M5 The reductase IR-G36 mutant M5, hereinafter referred to as M5, has the DNA sequence described in SEQ ID NO.1 and the amino acid sequence described in SEQ ID NO.2 in the sequence listing. Its co-crystallization result with the cofactor NADP(H) shows that its structure is a classical homodimer. The two monomers are respectively composed of the Rossman domain at the N-terminus and the helical bundle at the C-terminus. Two catalytic active centers are formed at the junctions of the C-terminal and N-terminal domains of the two monomers, and the cofactor NADP(H) binds to the two active pockets.

[0032] The present invention initially selected benzylacetone and benzylamine as the model substrates for the catalytic reductive amination reaction of M5. In order to find potential mutation sites, the imine intermediate was docked into the active site of M5 using Discovery Studio. The docking result shows that the benzylpropyl part faces the side opening of the substrate channel, and the benzyl part is located inside the pocket. Based on the previous research, the residues controlling stereoselectivity are located at the ketone substrate and imine side at the opening of the active pocket. Further analysis found that 6 amino acid residues (W234, F260, I149, Q265, L200, and Y204) at this position have direct interactions with the benzylpropyl and imine parts.

[0033] Subsequently, single-site saturation mutations were carried out at 7 sites respectively. The overall results are shown in Tables 1-4. The mutants of W234, I149, Q265, and Y204 S showed a more obvious improvement in the -configuration product.

[0034] Table 1: Determination results of the ee values of the saturation mutants at the W234 site

[0035] Table 2: Determination results of the ee values of the saturation mutants at the I149 site

[0036] Table 3: Determination results of the ee values of the saturation mutants at the Q265 site

[0037] Table 4. ee value determination results of saturated mutants at Y204 site

[0038] To further improve S- Stereoselectivity, we combined the dominant mutants according to their spatial positions. The mutants and their corresponding ee values ​​are shown in Table 5. We then determined S -Selectivity reached 99% of the conversion rate of the mutants. At a ketone substrate concentration of 100 mmol / L, the double mutant M5-W234A-I149Y had the highest conversion rate, reaching 92.5%, while the conversion rates of the mutants M5-W234A-Q265L and M5-W234C- Q265L were both lower than 70%. Therefore, M5-W234A-I149Y was finally selected as the optimal mutant and abbreviated as V1.

[0039] Table 5. ee value determination results of I149 site saturation mutant

[0040] Example 2 The gene encoding mutant V1 was connected to the pET28a plasmid and transformed into E. coli BL21 (DE3) competent cells for protein expression. Single clones were picked and cultured in LB medium (10 mL) containing 50 µg / mL kanamycin at 37°C and 220 rpm overnight. The above bacterial liquid was inoculated into LB medium containing 50 µg / mL kanamycin at a ratio of 1:100 and cultured at 37°C and 220 rpm until the OD600 value was 0.6-0.8. IPTG (0.2 mM / L) was added to the culture medium to induce gene expression, and the culture was continued at 20°C and 200 rpm for 16 hours. Then, the cells were harvested by centrifugation at 6000 rpm for 10 minutes, resuspended in sodium phosphate buffer (100 mmol / L, pH 7.0), and centrifuged at 6000 rpm for 10 minutes to collect the cells. The above-mentioned cells were washed twice with sodium phosphate buffer, resuspended in sodium phosphate buffer, and disrupted by high-pressure disruptor. The disrupted liquid was centrifuged at 1,2000 rpm, the supernatant was collected, and the protein was purified by Ni+2 affinity chromatography.

[0041] Example 3 The purified mutant protein V1 was used to determine the reductive amination reaction between benzylacetone and benzylamine. In a 5 ml reaction mixture, 1 mmol / L of NADP+ was added, 5 mg / mL of purified enzyme was added, 100 mmol / L of benzylacetone was added, the amount of benzylamine added was in a molar ratio of 1.2:1 to the ketone, the amount of D-glucose added was in a molar ratio of 1.5:1 to the ketone, 0.4 mg / mL of GDH enzyme powder was added, DMSO with a volume ratio of 20% was added, and the concentration of the sodium phosphate buffer was 100 mmol / L (pH 7.0). All reactions were carried out at 30 °C and 220 rpm for 20 hours, and then quenched by adding 5 mL of acetonitrile containing 1 mol / L acetic acid. Centrifugation was carried out at 12000 rpm for 10 minutes, and the supernatant was analyzed by HPLC to determine the conversion rate.

[0042] Example 4

[0043] In a 5 mL reaction system, benzylacetone (100 mmol / L, 1 equivalent), amine (120 mmol / L, 1.2 equivalents), the cell lysate of Escherichia coli expressing mutant V1 (the wet cell weight accounted for 10 g / L of the total reaction volume), D-glucose (1.5 times the equivalent of the ketone), NADP + with a concentration of 1 mmol / L), GDH enzyme powder (0.4 mg / mL), sodium phosphate buffer with a concentration of 100 mmol / L (pH 7.0) and 20% (by volume of the total reaction solution) of DMSO were added. The solution was adjusted to pH 7.0 with 1 mol / L hydrochloric acid, and the mixture was reacted at 30 °C and 220 rpm. After 20 hours, the reaction was quenched by adjusting the pH value to 3.0 with acetic acid, and diatomaceous earth was added. The mixture was filtered and washed with water. The filtrate was extracted with ethyl acetate (15 mL × 3) to remove neutral substances. The aqueous phase was alkalized to pH 10.0 with saturated sodium carbonate and then extracted with ethyl acetate (15 mL × 3). The solvent of the combined organic extracts was removed under reduced pressure to obtain the corresponding chiral amine product. The reaction system, conversion rate and ee value are shown in Table 6 (where no chiral column for separating 1d and 1h was found).

[0044] Table 5 5 mL reaction system catalyzed by mutant V1 with different amines

[0045] Example 5: ( S )-α-Methyl-N-(benzyl)benzylamine ( S -1a) Enzymatic synthesis

[0046] According to the methods of Examples 3 and 4, using benzylacetone and benzylamine as raw materials and mutant V1 as the catalyst, a colorless oil (>99%, S ) was obtained with a conversion rate of 92.5%.

[0047] 1 H NMR δ H (400 MHz, Chloroform- d ) δ 7.39 (dd, J = 7.1, 2.4 Hz, 2H),7.34-7.25 (m, 5H), 7.24 (s, 1H), 7.19 (t, J = 7.3 Hz, 1H), 7.15-7.09 (m, 2H),3.96-3.83 (m, 2H), 2.95 (ddd, J = 10.1, 6.7, 3.9 Hz, 1H), 2.71 (ddd, J =14.6, 9.6, 5.4 Hz, 1H), 2.52 (ddd, J = 14.0, 9.4, 6.9 Hz, 1H), 2.13 (dddd, J = 13.5, 10.2, 6.8, 3.8 Hz, 1H), 1.84 (ddd, J = 13.8, 9.4, 5.2 Hz, 1H), 1.30(d, J = 6.5 Hz, 3H). 13 C NMR δ C (100 MHz, Chloroform- d ) δ 140.4, 131.8, 130.0,128.9, 128.6, 128.3, 126.3, 51.7, 47.7, 34.4, 31.7, 16.2。

[0048] Example 6: Enzymatic synthesis of ( S )-α-methyl-N-(methyl)amphetamine ( S -1b)

[0049] According to the methods of Examples 3 and 4, using benzylacetone and methylamine as raw materials and mutant V1 as the catalyst, a colorless oil (>99%, S ) was obtained with a conversion rate of 70.4%.

[0050] 1 1H NMR δ H (400 MHz, Chloroform- d ) δ 7.21 (dt, J J = 38.0, 7.4 Hz, 5H),2.98 (s, 1H), 2.75 (d, J J = 9.1 Hz, 1H), 2.59 (d, J J = 10.4 Hz, 1H), 2.50 (s,3H), 2.13 (s, 1H), 1.83 (d, J J = 11.6 Hz, 1H), 1.33 (d, J J = 5.7 Hz, 3H). 13C NMR δ C (100 MHz, Chloroform- d ) δ 140.3, 128.6, 128.3, 126.3, 54.17, 34.3,31.6, 29.6, 15.7。

[0051] Example 7: S (-)-α-Methyl-N-(propargyl)amphetamine ( S -1c) Enzymatic Synthesis

[0052] According to the methods of Examples 3 and 4, using benzylacetone and propargylamine as raw materials and mutant V1 as the catalyst, a pale yellow oil (>99%, S ) was obtained with a conversion rate of 84.1%.

[0053] 1 1H NMR δ H (400 MHz, Chloroform- d ) δ 7.31 (dd, J J = 8.4, 6.5 Hz, 2H),7.22 (t, J J = 7.8 Hz, 3H), 3.79-3.62 (m, 2H), 3.35-3.22 (m, 1H), 2.78 (ddd, J J = 15.0, 10.0, 5.5 Hz, 1H), 2.66 (ddd, J= 13.9, 9.9, 6.4 Hz, 1H), 2.35 (d, J = 2.2 Hz, 1H), 2.16 - 2.02 (m, 1H), 1.93 - 1.79 (m, 1H), 1.33 (d, J = 6.4 Hz, 3H). 13 C NMR δ C (100 MHz, Chloroform - d ) δ 140.70, 128.59, 128.37, 126.25, 76.00, 75.39, 51.61, 35.40, 33.80, 31.77, 16.85。

[0054] Example 8: ( S ) - α - Methyl - N - (n - propyl) phenylpropylamine ( S - 1d) Enzymatic Catalytic Synthesis

[0055] According to the methods of Examples 3 and 4, using benzylacetone and n - propylamine as raw materials and mutant V1 as the catalyst, a colorless oil (>99%, S ) with a conversion rate of 68.2% was obtained.

[0056] 1 H NMR δ H (400 MHz, Chloroform - d ) δ 7.27 (t, J = 7.3 Hz, 2H), 7.22 - 7.12 (m, 3H), 3.04 (tt, J = 10.1, 6.5 Hz, 1H), 2.74 (ddt, J = 18.1, 14.1, 5.6Hz, 3H), 2.55 (ddd, J = 13.8, 9.6, 6.9 Hz, 1H), 2.16 (dddd, J = 13.5, 10.3, 6.8, 3.6 Hz, 1H), 1.84 (dtd, J = 14.8, 9.8, 5.2 Hz, 1H), 1.68 (p, J = 7.6 Hz, 2H), 1.34 (d, J= 6.5 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H). 13 C NMR δ C (101 MHz, Chloroform- d ) δ 140.3, 128.6, 128.3, 126.3, 53.1, 46.1, 34.3, 31.8, 19.6, 15.9, 11.3。

[0057] Example 9: ( S )-α-Methyl-N-(n-pentyl)benzylamine ( S -1e) Enzymatic Synthesis

[0058] According to the methods of Examples 3 and 4, using benzylacetone and n-pentylamine as raw materials and mutant V1 as the catalyst, a colorless oil (98%, S ) with a conversion rate of 63.8% was obtained.

[0059] 1 H NMR δ H (400 MHz, Chloroform- d ) δ 7.28 (t, J = 7.4 Hz, 2H), 7.24 - 7.14 (m, 3H), 3.05 (dqd, J = 10.2, 6.5, 3.6 Hz, 1H), 2.78 (dddd, J = 27.3, 14.7, 8.1, 4.8 Hz, 3H), 2.56 (ddd, J = 14.0, 9.5, 7.0 Hz, 1H), 2.18 (dddd, J = 13.4, 10.2, 7.0, 3.6 Hz, 1H), 1.85 (dtd, J = 14.6, 9.7, 5.2 Hz, 1H), 1.67 (q, J = 7.4 Hz, 2H), 1.35 (d, J = 6.5 Hz, 3H), 1.32 - 1.20 (m, 4H), 0.90 - 0.82 (m, 3H). 13 C NMR δ C(100 MHz, Chloroform- d ) δ 140.3, 128.6, 128.3, 126.3, 52.9, 44.5, 34.3, 31.8, 28.9, 25.7, 22.1, 15.9, 13.9。

[0060] Example 10: S )-α-Methyl-N-(cyclopropyl)amphetamine ( S -1f) Enzymatic Synthesis

[0061] According to the methods of Examples 3 and 4, using benzylacetone and cyclopropylamine as raw materials and mutant V1 as the catalyst, a colorless oil (93%, S ) with a conversion rate of 91.5% was obtained.

[0062] 1 H NMR δ H (400 MHz, Chloroform- d ) δ 7.23 (dd, J = 8.3, 6.6 Hz, 2H), 7.18 - 7.09 (m, 3H), 3.13 (ddd, J = 10.1, 6.7, 3.9 Hz, 1H), 2.70 (ddd, J = 14.8, 10.2, 5.2 Hz, 1H), 2.53 (ddd, J = 13.9, 9.9, 6.7 Hz, 1H), 2.36 (tt, J = 7.5, 3.9 Hz, 1H), 2.18 (dddd, J = 13.8, 10.5, 6.7, 3.8 Hz, 1H), 1.82 (dtd, J = 14.6, 9.7, 5.2 Hz, 1H), 1.34 (d, J = 6.5 Hz, 3H), 0.93 (dt, J = 6.1, 3.1 Hz, 2H), 0.63 (d, J = 7.4 Hz, 2H). 13 C NMR δ C (100 MHz, Chloroform- d) δ 140.5, 128.6, 128.3, 126.2, 54.5, 34.4, 31.7, 27.5, 16.2, 3.6, 3.4。

[0063] Example 11:( S )-α-methyl-N-(cyclobutyl)amphetamine ( S -1g) Enzymatic synthesis

[0064] According to the methods of Examples 3 and 4, using benzylacetone and cyclobutylamine as raw materials and mutant V1 as the catalyst, a colorless oil (96%, S ) with a conversion rate of 41.2% was obtained.

[0065] 1 H NMR δ H (400 MHz, Chloroform- d ) δ 7.36 (t, J = 7.4 Hz, 2H), 7.27(dd, J = 14.5, 7.0 Hz, 3H), 3.71 (p, J = 8.2 Hz, 1H), 3.08 (ddt, J = 13.3,10.4, 5.0 Hz, 1H), 2.83 (ddt, J = 17.1, 12.4, 5.9 Hz, 1H), 2.64 (ddd, J =14.2, 9.5, 6.8 Hz, 1H), 2.41 (pd, J = 10.0, 3.5 Hz, 2H), 2.30 - 2.13 (m, 3H),1.98 - 1.74 (m, 3H), 1.40 (d, J = 6.6 Hz, 3H). 13 C NMR δ C (100 MHz, Chloroform- d ) δ 140.5, 128.7, 128.4, 126.4, 51.7, 49.5, 34.6, 31.7, 27.3, 27.2, 16.4,15.6。

[0066] Example 12:( S )-α-methyl-N-(cyclopentyl)amphetamine (S Enzymatic synthesis of -1h

[0067] According to the methods of Examples 3 and 4, using benzylacetone and cyclopentylamine as raw materials and mutant V1 as the catalyst, a colorless oil (>99%, S ) was obtained, with a conversion rate of 8.3%.

[0068] 1 H NMR δ H (400 MHz, Chloroform-d) δ 7.24 (t, J = 7.3 Hz, 2H), 7.19-7.10 (m, 3H), 3.38 (q, J = 7.5 Hz, 1H), 3.02 (ddd, J = 10.2, 6.7, 3.6 Hz,1H), 2.72 (ddd, J = 14.5, 9.9, 5.1 Hz, 1H), 2.51 (ddd, J = 13.9, 9.6, 6.8 Hz,1H), 2.18 (dddd, J = 13.5, 10.3, 7.0, 3.4 Hz, 1H), 1.84 (ddt, J = 17.7, 9.0,5.7 Hz, 3H), 1.67 (p, J = 5.9 Hz, 4H), 1.45 (h, J = 8.5, 6.9 Hz, 2H), 1.33(d, J = 6.4 Hz, 3H). 13 C NMR δ C (100 MHz, Chloroform- d ) δ 140.4, 128.5, 128.3,126.2, 55.6, 52.0, 34.4, 31.8, 29.5, 29.2, 23.8, 16.0。

[0069] Example 13: ( S )-α-Methyl-N-(cyclohexyl)benzylamine ( S Enzymatic synthesis of -1i

[0070] According to the methods of Examples 3 and 4, using benzylacetone and cyclohexylamine as raw materials and mutant V1 as the catalyst, the conversion rate was 0.5%, and the ee value was not determined.

[0071] 1 H NMR δH (400 MHz, Chloroform- d ) δ 7.29 (t, J = 7.4 Hz, 2H), 7.18 (t, J = 5.4 Hz, 3H), 3.17 (ddt, J = 9.9, 6.8, 3.5 Hz, 1H), 2.93 (tt, J = 11.7,3.9 Hz, 1H), 2.76 (ddd, J = 14.6, 9.8, 5.2 Hz, 1H), 2.54 (ddd, J = 13.8, 9.5,6.9 Hz, 1H), 2.18 (dddd, J = 13.3, 10.2, 6.9, 3.3 Hz, 1H), 1.98 (d, J = 12.4Hz, 1H), 1.89 (ddd, J = 13.7, 9.8, 4.6 Hz, 2H), 1.73 (d, J = 10.9 Hz, 1H),1.63 (q, J = 10.1 Hz, 2H), 1.45-1.32 (m, 5H), 1.22-1.10 (m, 3H). 13 C NMR δ C (100 MHz, Chloroform- d ) δ 140.5, 128.6, 128.4, 126.3, 53.4, 49.3, 34.3, 32.0,29.3, 28.8, 25.0, 24.7, 15.9。

[0072] Example 14: S )-α-Methyl-N-(thiophenylethyl)amphetamine ( S -1j) Enzymatic Synthesis

[0073] According to the methods of Examples 3 and 4, using benzylacetone and thiophenethylamine as raw materials and mutant V1 as the catalyst, a pale yellow solid (>99%, S ) was obtained with a conversion rate of 85.5%.

[0074] 1 H NMRδ H (400 MHz, Chloroform- d ) δ 7.34 - 7.10 (m, 6H), 6.96 - 6.79 (m, 2H), 3.25 (d, J = 8.1 Hz, 2H), 3.20 - 3.07 (m, 3H), 2.76 (td, J = 9.4, 4.9 Hz, 1H), 2.64 - 2.52 (m, 1H), 2.20 (d, J = 7.1 Hz, 1H), 1.89 (dd, J = 9.1, 4.7 Hz, 1H), 1.38 (d, J = 6.5 Hz, 3H). 13 C NMR δ C (100MHz, Chloroform- d ) δ 140.2, 138.9, 128.7, 128.3, 127.2, 126.4, 126.0, 124.4, 53.3, 45.9, 34.5, 31.7, 26.7, 16.1。

[0075] Example 15: S )-α-Methyl-N-(phenethyl)benzylamine ( S -1k) Enzymatic Synthesis

[0076] According to the methods of Examples 3 and 4, using benzyl acetone and phenethylamine as raw materials and mutant V1 as the catalyst, a colorless oil (98%, S ) was obtained with a conversion rate of 82.3%.

[0077] 1 H NMR δ H(400 MHz, Chloroform-d) δ 7.32-7.22 (m, 5H), 7.18 (q, J =6.8 Hz, 5H), 3.14-3.00 (m, 5H), 2.77 (ddd, J = 14.6, 9.8, 5.3 Hz, 1H), 2.65-2.53 (m, 1H), 2.22 (dddd, J = 13.5, 10.3, 6.9, 3.8 Hz, 1H), 1.91 (dtd, J =14.6, 9.6, 5.3 Hz, 1H), 1.40 (d, J = 6.5 Hz, 3H). 13 C NMR δ C (100 MHz,Chloroform- d ) δ 140.2, 137.2, 128.8, 128.7, 128.6, 128.3, 127.0, 126.4, 53.2,46.1, 34.5, 32.6, 31.8, 16.2。

[0078] Example 16: S )-α-methyl-N-(phenylpropyl)benzylamine ( S -1l) Enzymatic synthesis

[0079] According to the methods of Examples 3 and 4, using benzylacetone and phenylpropylamine as raw materials and mutant V1 as the catalyst, a colorless oil (97%, S ) was obtained with a conversion rate of 72.8,.

[0080] 1 H NMR δ H(400 MHz, Chloroform-d) δ 7.25 (ddd, J = 23.3, 7.2, 3.9 Hz,6H), 7.14 (t, J = 7.5 Hz, 4H), 3.02 (ddd, J = 10.1, 6.7, 3.8 Hz, 1H), 2.83(q, J = 7.5 Hz, 2H), 2.70 (ddd, J = 14.5, 9.7, 5.3 Hz, 1H), 2.62-2.46 (m,3H), 2.14 (dddd, J = 13.4, 10.2, 7.0, 3.6 Hz, 1H), 2.02 (q, J = 7.7 Hz, 2H),1.82 (dtd, J = 14.5, 9.5, 5.2 Hz, 1H), 1.31 (d, J = 6.5 Hz, 3H). 13 C NMR δ C (100 MHz, Chloroform-d) δ 140.2, 140.2, 128.6, 128.6, 128.3, 126.4, 126.3,53.2, 44.0, 34.3, 32.8, 31.7, 27.4, 15.9。

[0081] Example 17:( S )-α-Methyl-N-(2-(indol-3-yl)ethyl)benzylamine ( S -1m) Enzymatic synthesis

[0082] According to the methods of Examples 3 and 4, using benzyl acetone and 2-(indol-3-yl)ethylamine as raw materials and mutant V1 as the catalyst, a colorless solid (>99%, S ) was obtained, and the conversion rate was 47.8%.

[0083] 1 H NMR δ H (400 MHz, Methanol- d 4 ) δ 7.58 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 7.4 Hz, 2H), 7.23-7.11 (m, 5H), 7.07 (t,J = 7.4 Hz, 1H), 3.30 - 3.09 (m, 5H), 2.72 (ddd, J = 14.8, 10.2, 5.2 Hz, 1H), 2.57(ddd, J = 13.8, 9.8, 6.8 Hz, 1H), 2.07 (dddd, J = 13.8, 10.4, 6.8, 3.9 Hz, 1H), 1.80 (dtd, J = 14.8, 9.7, 5.2 Hz, 1H), 1.34 (d, J = 6.5 Hz, 3H). 13 C NMR δ C (100 MHz, Methanol - d4) δ 140.4, 136.9, 128.3, 128.0, 126.8, 126.1, 123.0, 121.5, 118.8, 117.6, 111.3, 109.0, 53.6, 47.5, 47.3, 47.1, 44.8, 34.5, 31.1, 22.1, 14.9。

Claims

1. A mutant protein of carbonyl reductase, characterized in that The mutant protein has one or more substitution mutations at positions 85, 87, 128, 134, 162, 197, 219, 234, 235 based on the amino acid sequence shown in SEQ ID NO:

1.

2. The mutant protein according to claim 1, characterized in that Based on the amino acid sequence shown in SEQ ID NO: 1, there is a mutation at any of the following sites: the phenylalanine at position 85 mutates to cysteine ​​or threonine; the phenylalanine at position 87 mutates to tryptophan; the tyrosine at position 128 mutates to alanine or glycine; the phenylalanine at position 134 mutates to valine, isoleucine or leucine; the histidine at position 162 mutates to alanine; the valine at position 197 mutates to threonine or serine; the tyrosine at position 219 mutates to isoleucine or phenylalanine; the proline at position 234 mutates to arginine; and the valine at position 235 mutates to isoleucine.

3. The mutant protein according to claim 2, characterized in that Based on the amino acid sequence shown in SEQ ID NO: 1, there is a mutation at any of the following positions: a combined mutation at positions 85 and 87; a combined mutation at positions 85, 87 and 128; a combined mutation at positions 85, 87 and 134; a combined mutation at positions 85, 87, 134 and 197; a combined mutation at positions 85, 87, 134, 197 and 219.

4. A nucleic acid encoding the mutant protein according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid according to claim 4.

6. A recombinant host bacteria containing the nucleic acid according to claim 4 or the expression vector according to claim 5.

7. Use of the mutant protein according to any one of claims 1 to 3 in the synthesis of S-bosone.

8. A method for reducing 1-C-(β-D-xylopyranosyl)-acetone, characterized in that: Includes steps: The mutant protein according to any one of claims 1 to 3 is contacted with a reaction substrate 1-C-(β-D-xylopyranosyl)-acetone, and a coenzyme circulation system is added to carry out a catalytic reaction, thereby obtaining the S - configuration Bose; after the reaction is completed, the reduced pressure is concentrated, and the S - Boserain.

9. The method according to claim 8, characterized in that The pH of the catalytic reaction system is 6.0-8.0; the substrate concentration is 80-240 g / L; and the reaction time is 10-48 hours; Preferably, the pH of the catalytic reaction system is pH 6.0-7.0; the substrate concentration is 120-200 g / L; the reaction time is 12-24 hours; Specifically, the mutant protein is in the form of pure protein, or a mutant crude protein solution is obtained by culturing the recombinant host bacteria as claimed in claim 6, or a catalytic reaction is carried out using the recombinant host bacteria as claimed in claim 6 as a biocatalyst.

10. The method according to claim 9, characterized in that , The cell lysate of the reductive aminase mutant is added to the reaction, the mass volume ratio of the wet weight of the reductive aminase mutant cells to the reaction mixture is 10 g / L; the molar ratio of D-glucose to ketone is 1.5:1; the amount of nicotinamide adenine dinucleotide phosphate disodium salt NADP+ added to the reaction is 1.0 mmol / L; the amount of glucose dehydrogenase GDH enzyme powder is 1.5 mg / L; the amount of phosphate is 100 mmol / L; the volume of DMSO is 20% of the total volume of the reaction solution; the pH of the reaction solution is 7.0; the reaction temperature is 30°C; and the reaction time is 20 hours; The reaction steps include: adding DMSO and sodium phosphate buffer in a volume ratio of 1:4 into a reactor; adding a mixed solution of amine, D-glucose and NADP+ into the reactor and reacting at 30°C; the amount of benzyl acetone added is 100 mmol / L; the molar ratio of amine to ketone is 1.2:1; adding cell lysate of reductive aminase mutant V1 and GDH enzyme powder into the reaction solution for reaction; and obtaining a product after reacting for 20 hours; After the product is obtained after reacting for 20 hours, the method further includes the following steps: quenching the reaction with hydrochloric acid to obtain a solution with a pH of 3.0-4.0; loading diatomaceous earth into a bioreactor and stirring for 10 minutes; filtering the mixture and washing with water to obtain a filtrate; extracting the filtrate with ethyl acetate in sequence, alkalizing the aqueous phase with a saturated sodium carbonate solution to a pH of 10.0, extracting with ethyl acetate three times and combining the organic phase extracts; and concentrating under reduced pressure to obtain the product.

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