Carbonyl reductase mutant and application thereof in synthesis of phenylephrine hydrochloride

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to a carbonyl reductase mutant and application thereof in synthesis of phenylephrine hydrochloride. A method for synthesizing phenylephrine hydrochloride in the field is still far away from industrial production, and a synthesis method which is environmentally friendly, efficient, high in stereoselectivity and more suitable for industrial production is urgently needed to be developed. The mutant with higher enzyme activity is obtained by mutating the LpADH carbonyl reductase, the highest enzyme activity can reach 4720% of that of the unmutated LpADH carbonyl reductase, the activity of the mutant is remarkably improved, the production efficiency can be greatly improved, the production cost can be reduced, industrialization is facilitated, and the method is suitable for industrial production. In addition, the preparation method does not involve the use of various polluting chemicals, so that the risk of environmental pollution is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme catalysis, in particular to a carbonyl reductase mutant and application thereof in synthesizing phenylephrine hydrochloride. Background Art

[0002] Phenylephrine Hydrochloride is a selective α1 adrenergic receptor agonist. It is clinically used to prevent and treat hypotension caused by spinal anesthesia, general anesthesia, and chlorpromazine. It is also used for supraventricular tachycardia and mydriasis examinations. Its chemical structure is as follows:

[0003]

[0004] The carbon atom attached to the hydroxyl group on the side chain of phenylephrine hydrochloride is a chiral carbon atom with an R configuration. The pharmacological activity of phenylephrine hydrochloride is highly dependent on the stereo configuration of the chiral center. Studies have shown that the R-configuration isomer has a much stronger agonist effect on α1 adrenergic receptors than the S-configuration isomer. Therefore, the ability to obtain phenylephrine of acceptable chiral purity is crucial to selecting a synthetic route.

[0005] Phenylephrine hydrochloride is an optically active pharmaceutical. Current literature reports that its industrial synthesis relies primarily on two methods: asymmetric catalytic synthesis and biocatalysis. Asymmetric catalytic synthesis of phenylephrine hydrochloride utilizes heavy metal catalysts. Patent DE 19902229 utilizes a rhodium catalyst for asymmetric reduction to obtain the chiral intermediate (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol. However, these catalysts are expensive, require harsh reaction conditions, have low yields, and pose significant environmental risks. This results in high production costs for phenylephrine hydrochloride, hindering sustainable development. Furthermore, resolution methods are commonly used to synthesize phenylephrine hydrochloride. Patent IN201741038850 utilizes L-tartaric acid to resolve the racemate to obtain phenylephrine. This method, however, offers low yields and is complex. Biocatalytic methods for the production of phenylephrine hydrochloride have promising applications, with advantages including environmental friendliness, high stereoselectivity, and mild reaction conditions. Tokoshima D et al. (J. Journal of Molecular Catalysis B: Enzymatic, 2013, 97:95-99) successfully produced phenylephrine hydrochloride using a whole-cell enzyme to catalyze the reduction of 2-chloro-1-(3-nitrophenyl)ethanone. However, the method's long preparation route and low overall yield make it difficult to achieve large-scale industrial production. Patent CN102159719A uses m-hydroxyacetophenone as a starting material, which reacts with sulfonyl chloride in the presence of methanol to produce α-chloro-3-hydroxyacetophenone. Alcohol dehydrogenase from the Azoarbrio species EBN1 is then used as a biocatalyst, with NADP as an electron donor, to reduce the α-chloro-3-hydroxyacetophenone to a chiral alcohol. The alcohol is then aminized with methylamine to produce L-phenylephrine. The product has an optical purity greater than 99% and a relatively simple process. However, the enzyme-catalyzed substrate concentration is low, only 8.5 g / L, and the final amination reaction requires pressurization at 90°C, which increases the risk factor and production cost, making it unsuitable for industrial production.

[0006] Therefore, there is an urgent need in this field to develop an environmentally friendly, efficient, highly stereoselective method for preparing phenylephrine hydrochloride that is more suitable for industrial production. Summary of the Invention

[0007] In view of the above problems existing in the existing preparation methods, the purpose of the present invention is to provide a novel carbonyl reductase mutant and its application in the synthesis of phenylephrine hydrochloride.

[0008] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a carbonyl reductase mutant;

[0009] The mutant comprises at least one of the following mutations:

[0010] 1) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from Y to G at amino acid position 190;

[0011] 2) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from L to G at position 195;

[0012] 3) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from T to A at amino acid position 152;

[0013] 4) The amino acid at position 197 of the amino acid sequence of the carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from D to A.

[0014] Furthermore, the mutant comprises or consists of the following amino acid sequence:

[0015] The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 190 is mutated from Y to G and the amino acid at position 195 is mutated from L to G, as shown in SEQ ID NO: 1;

[0016] The amino acid sequence of the carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1, wherein the amino acid at position 190 is mutated from Y to G, the amino acid at position 195 is mutated from L to G, and the amino acid at position 197 is mutated from D to A;

[0017] The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1, wherein the amino acid at position 152 is mutated from T to A, the amino acid at position 190 is mutated from Y to G, the amino acid at position 195 is mutated from L to G, and the amino acid at position 197 is mutated from D to A;

[0018] Furthermore, the mutant further comprises at least one of the following mutations based on the above mutations 1) to 4):

[0019] 5) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from A to V at amino acid position 64;

[0020] 6) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1, wherein the amino acid at position 76 is mutated from T to L;

[0021] 7) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 95 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from V to Q;

[0022] 8) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 96 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from S to L;

[0023] 9) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 145 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from E to A;

[0024] 10) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 147 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from F to L;

[0025] 11) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 153 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from L to M;

[0026] 12) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 200 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from E to P;

[0027] 13) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 202 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from A to F;

[0028] 14) The amino acid at position 206 of the carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from M to C.

[0029] Furthermore, the mutant further comprises at least one of the following mutations based on the above mutations 1) to 4) and mutations 5) to 14):

[0030] 15) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 94 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from A to V;

[0031] 16) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 11 in the amino acid sequence of SEQ ID NO: 1 is mutated from I to L;

[0032] 17) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri, wherein the amino acid at position 99 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from V to L;

[0033] 18) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1, wherein the amino acid at position 148 is mutated from V to I;

[0034] 19) The amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1, wherein the amino acid at position 159 is mutated from S to T;

[0035] 20) The amino acid at position 249 of the amino acid sequence of the carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID NO: 1 is mutated from Y to F.

[0036] Furthermore, the mutant comprises or consists of the following amino acid sequence:

[0037] a) the amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as set forth in SEQ ID NO: 1, in which amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, and amino acid position 206 is mutated from M to C;

[0038] b) the amino acid sequence of carbonyl reductase LpADH from Lentilactobacillus parabuchneri as set forth in SEQ ID NO: 1, in which amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 94 is mutated from A to V, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, and amino acid position 206 is mutated from M to C;

[0039] c) Carbonyl reductase LpADH from Lentilactobacillus parabuchneri as SEQ ID The amino acid sequence of NO:1, in which amino acid position 11 is mutated from I to L, amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 99 is mutated from V to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 148 is mutated from V to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 159 is mutated from S to T, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, amino acid position 206 is mutated from M to C, and amino acid position 249 is mutated from Y to F;

[0040] d) Carbonyl reductase LpADH from Lentilactobacillus parabuchneri as shown in SEQ ID The amino acid sequence of NO:1, in which amino acid position 11 is mutated from I to L, amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 94 is mutated from A to V, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 99 is mutated from V to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 148 is mutated from V to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 159 is mutated from S to T, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, amino acid position 206 is mutated from M to C, and amino acid position 249 is mutated from Y to F;

[0041] e) The carbonyl reductase LpADH from Lentilactobacillus parabuchneri has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid at position 11 is mutated from I to L, the amino acid at position 43 is mutated from I to V, the amino acid at position 54 is mutated from P to T, the amino acid at position 64 is mutated from A to V, the amino acid at position 72 is mutated from E to K, the amino acid at position 76 is mutated from T to I, the amino acid at position 79 is mutated from N to E, the amino acid at position 95 is mutated from V to Q, the amino acid at position 96 is mutated from S to L, the amino acid at position 99 is mutated from V to L, the amino acid at position 101 is mutated from E to D, the amino acid at position 145 is mutated from E to A, and the amino acid at position 147 is mutated from N to E. F mutates to I, the 148th amino acid mutates to I, the 152nd amino acid mutates to A, the 153rd amino acid mutates to M, the 159th amino acid mutates to T, the 190th amino acid mutates to G, the 195th amino acid mutates to G, the 197th amino acid mutates to A, the 200th amino acid mutates to P, the 202nd amino acid mutates to F, the 206th amino acid mutates to C, the 235th amino acid mutates to S and the 249th amino acid mutates to F.

[0042] The amino acid sequence of SEQ ID NO: 1 is specifically:

[0043] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADIGEKAAKS

[0044] IGGPDVIRFVQHDASDEAGWTELFDTTENAFGPVTTVVNNAGIAVSKSVEETT

[0045] TEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPTLGAY

[0046] NASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMS

[0047] QRTKTPMGHIGEPNDIAWICVYLASDEAKFATGAEFVVDGGYTAQ

[0048] In a second aspect, the present invention provides an isolated nucleic acid encoding the carbonyl reductase mutant;

[0049] The nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0050] The nucleotide sequence of SEQ ID NO: 2 is specifically:

[0051] 5'-ATGACAGATAGGCTAAAGGGAAAAGTAGCTATTGTTACCGGCGGTAC -3'

[0052] In a third aspect, the present invention provides an expression vector comprising the nucleic acid, wherein the expression vector includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector and viral vector.

[0053] In a fourth aspect, the present invention provides a recombinant microorganism, wherein the recombinant microorganism is a recombinant microorganism comprising the above-isolated nucleic acid or an expression vector of the nucleic acid;

[0054] Furthermore, the recombinant microorganism is constructed by introducing the above nucleic acid into Escherichia coli via a plasmid, or integrating it into the chromosome of Escherichia coli by genetic engineering means.

[0055] In a fifth aspect, the present invention provides use of the carbonyl reductase mutant, nucleic acid, expression vector or recombinant microorganism in the synthesis of phenylephrine hydrochloride.

[0056] In a sixth aspect, the present invention provides a method for synthesizing phenylephrine, comprising the following steps:

[0057] 2-chloro-3-hydroxyacetophenone is mixed with N-methylbenzylamine to react to generate 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethane-1-one, and concentrated hydrochloric acid is added to obtain 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethane-1-one hydrochloride, 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethane-1-one, also known as benzyl deoxyadrenoside;

[0058] Using 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethan-1-one hydrochloride as a substrate, in the presence of a coenzyme, the above-mentioned carbonyl reductase mutant, nucleic acid, expression vector or recombinant microorganism is used for catalysis to produce a reduction reaction to prepare (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol, (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol, also known as benzylphenylephrine;

[0059] (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol is debenzylated via palladium-carbon reaction in the presence of a hydrogen donor, and concentrated hydrochloric acid is added to obtain phenylephrine hydrochloride.

[0060] Furthermore, the coenzyme is the oxidative coenzyme NAD + , NADP + or reduced coenzyme, the concentration of coenzyme is 0.05-0.2g / L;

[0061] In the present invention, "coenzyme" refers to a coenzyme that can realize electron transfer in redox reaction. Typically, the coenzyme of the present invention is selected from the reducing coenzyme NADH, NADPH or the oxidizing coenzyme NADPH. + , NADP + Since the cost of reducing coenzymes is high, the oxidative coenzyme NAD is preferred. + , NADP + ;

[0062] Furthermore, the concentration of 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethan-1-one hydrochloride in the above reduction reaction is 1-200 g / L;

[0063] Furthermore, in the above reduction reaction, the reduction reaction system further comprises a co-substrate, the co-substrate being selected from one of isopropanol, amine formate or glucose, and the reduction reaction system is a phosphate buffer system;

[0064] Furthermore, a cosolvent is added to the above reduction reaction, and the cosolvent is selected from any one of dimethyl sulfoxide, DMF, isopropanol, acetonitrile or dimethylacetamide;

[0065] Further, the cosolvent is dimethyl sulfoxide;

[0066] Furthermore, in the above reduction reaction, the carbonyl reductase mutant is added using the cell lysate of the above recombinant microorganism, and the added amount is an amount equivalent to 3-10g of wet cells per 1L of reaction system;

[0067] Furthermore, the reduction reaction temperature is 20-40°C and the pH is 6.5-7.5;

[0068] Furthermore, in the above palladium-carbon reaction, the hydrogen donor is selected from one of hydrogen, formic acid or ammonium formate, and the amount of the hydrogen donor is 1-5eq of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol;

[0069] Furthermore, when isopropyl alcohol is selected as the hydrogen donor, the amount of isopropyl alcohol used is greater than that of other hydrogen donors because it serves as both a cosolvent and a hydrogen donor.

[0070] Furthermore, the temperature of the palladium-carbon reaction is 20-60°C;

[0071] Furthermore, in the above palladium-carbon reaction, the mass ratio of palladium-carbon to (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol added is 1:10-100;

[0072] Furthermore, in the above palladium-carbon reaction, the amount of hydrochloric acid used is 1-1.5 eq of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol.

[0073] Furthermore, the synthesis method of the above-mentioned phenylephrine hydrochloride comprises the following steps:

[0074] 2-chloro-3-hydroxyacetophenone was dissolved in acetone and heated to 60°C. Sodium carbonate and N-methylbenzylamine were added, and the mixture was mixed to react. The reaction was monitored by liquid chromatography. After the reaction was completed, the inorganic salt was filtered out. The filtrate was cooled to 5°C, concentrated hydrochloric acid was slowly added dropwise, and the precipitated solid was filtered. The filter cake was dried to obtain a white solid, i.e., 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethane-1-one hydrochloride.

[0075] Dissolve 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethane-1-one hydrochloride in isopropanol, add phosphate buffer, heat to 20-40°C, adjust the pH to 6.5-7.5, add coenzyme, add the above carbonyl reductase mutant, react on a shaker, and monitor the reaction conversion rate by HPLC; after the reaction is completed, adjust the pH of the reaction solution to 2.0-3.0 and heat to 80°C, stir, add diatomaceous earth and filter, and adjust the pH of the filtrate to The product precipitates after 9-10 min, and is filtered to obtain a white solid, namely (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol. The prepared (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol is dissolved in methanol and stirred to dissolve clearly. Then, wet palladium carbon (10% by weight of the substrate) is added, and the product is replaced by a hydrogen donor. Concentrated hydrochloric acid is added dropwise, and the palladium carbon is recovered by filtration. The filtrate is concentrated to obtain a light yellow solid, namely, phenylephrine hydrochloride.

[0076] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0077] The mutation site Y190G causes the activity of carbonyl reductase LpADH after mutation to reach 209% of the pre-mutation level, the mutation site T152A / Y190G / L195G / D197A causes the activity of carbonyl reductase LpADH after mutation to reach 912% of the pre-mutation level, and the mutation site I11L / I43V / P54T / A64V / E72K / T76I / N79E / V95Q / S96L / V99L / E101D / E145A / F147I / / V148I / T152A / L153M / S159T / Y190G / L195G / D197A / E200P / A202F / M206C / A235S / Y249F causes the activity of carbonyl reductase LpADH after mutation to reach 4720% of the pre-mutation level. The mutant carbonyl reductase provided herein can very efficiently prepare (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol with a stereochemical conformation (ee value ≥ 99.9%), which can be used to synthesize phenylephrine hydrochloride. Compared with chemical synthesis methods, this method significantly reduces the risk of environmental pollution. Furthermore, by increasing enzyme activity, the method of the present application greatly improves production efficiency and reduces production costs, which is conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The chiral spectrum of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol prepared in Example 4 was obtained;

[0079] Figure 2 The HPLC spectrum of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol prepared in Example 4 was obtained;

[0080] Figure 3 HPLC spectrum of the crude phenylephrine hydrochloride prepared in Example 9;

[0081] Figure 4 The HPLC spectrum of recrystallized phenylephrine hydrochloride prepared in Example 12 was obtained. DETAILED DESCRIPTION

[0082] Example 1 Construction and screening of carbonyl reductase

[0083] The culture medium components involved are as follows:

[0084] LB medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L;

[0085] Fermentation medium: yeast extract 24 g / L, soy peptone 24 g / L, sodium chloride 3 g / L, glycerol 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L.

[0086] The following genes were synthesized after codon optimization:

[0087] Carbonyl reductase LpADH from Lentilactobacillus parabuchneri (accession number: WP_057909612.1);

[0088] Carbonyl reductase EbADH from Empedobacter brevis ZJUY-1401 (accession number: ALZ42979.1);

[0089] Carbonyl reductase LKADH from Lactobacillus kefir (accession number: AY267012.1);

[0090] Carbonyl reductase EA from Exiguobacterium sp. F42 (accession number: BAD32703.1);

[0091] Carbonyl reductase LsADH from Leifsonia sp. S749 (Accession No.: AB213459);

[0092] The above genes were codon-optimized and synthesized and cloned into the pET28a(+) vector, and then introduced into the host Escherichia coli BL21(DE3) competent cells, cultured on kanamycin-resistant plates, and single colonies were picked and cultured in LB medium to finally obtain recombinant genetically engineered bacteria expressing carbonyl reductases LpADH, EbADH, LKADH, EA and LsADH.

[0093] The above-mentioned recombinant genetically engineered bacteria were inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm to obtain seed liquid. 10 μL of seed liquid was transferred to a shake flask containing 50 mL of fermentation medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 220 rpm until the OD600 value was >0.8. Isopropylthiogalactoside (IPTG) was used at a final concentration of 0.1 mM and the induction temperature was adjusted to 25°C to induce the expression of carbonyl reductase, and then cultured overnight. After fermentation, the cells were centrifuged at 12,000 g for 10 minutes to obtain wet E. coli cells.

[0094] Five carbonyl reductases were screened. The reaction conditions for LpADH, EbADH, LKADH, and LsADH were as follows: a reaction solution containing 10 g / L 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethanone (substrate I), 10% isopropanol (v / v), 5 g / L wet bacterial cells, 0.1 g / L NAD(P), and 100 mM phosphate buffer (pH 7.0) was prepared and reacted in a 2 mL reaction system at 25°C and 220 rpm for 24 h. The reaction conditions for EA were as follows: a reaction solution containing 5 g / L 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethanone (substrate I), 10 g / L wet bacterial cells, 10 g / L glucose, 0.1 g / L NADP, and 100 mM phosphate buffer (pH 7.0) was prepared and reacted in a 2 mL reaction system at 25°C and 220 rpm for 24 h. The reaction was terminated by adding anhydrous ethanol, and the supernatant was collected by centrifugation and detected by HPLC.

[0095] The chromatographic conditions for HPLC detection of reaction conversion were as follows: chromatographic column: Waters SPHERISORB 5μm ODS1 column (4.6mm×250mm); buffer: 2.35g sodium octanesulfonate monohydrate dissolved in 1L water, pH adjusted to 2.8 with phosphoric acid; mobile phase A: buffer and acetonitrile (volume ratio: 90:10), mobile phase B: buffer and acetonitrile (volume ratio: 10:90), gradient elution (0-3min, A 93%; 3-13min, A 93%-70%; 13-20min, A 70%; 20-21min, A 70%-93%; 21-30min, A 93%); detection wavelength: 215nm; column temperature: 45°C; flow rate: 1mL / min; injection volume: 5μL.

[0096] The above reaction formula is shown below, but is not limited to the specific reaction formula:

[0097]

[0098] The screening results are shown in Table 1:

[0099] Table 1 Results of catalytic reduction of 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethanone by different carbonyl reductases

[0100]

[0101] The results in the table show that carbonyl reductase LpADH has the best activity and selectivity, with a conversion rate of 66%, an ee value of 98%, and the product is the desired R configuration. Therefore, LpADH was selected for further modification to improve its selectivity and activity.

[0102] Example 2 Construction and screening of carbonyl reductase LpADH mutant enzymes

[0103] The wild-type carbonyl reductase gene, LpADH, was mutated through directed evolution to generate a plasmid library containing evolved carbonyl reductase genes. The constructed mutant plasmid library was transformed into Escherichia coli BL21(DE3) cells, plated onto LB solid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C in a 37°C incubator. Single colonies were picked and transferred to a 96-well plate containing 400 μL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C, 200 rpm to obtain a mutant seed solution of CsADH. 10 μL of the mutant seed solution was transferred to a 96-well plate containing 400 μL of fermentation medium containing 50 μg / mL kanamycin and cultured at 37°C, 200 rpm to an OD600 value >0.8. Expression of the mutants was induced with isopropylthiogalactoside (IPTG) at a final concentration of 1 mM at 25°C, followed by further incubation for 20 hours. The 96-well plate was placed in a centrifuge and centrifuged at 4000g for 30 minutes to collect the cells. The cells were then resuspended in 200μL of lysis buffer (0.1M phosphate buffer, pH 7.0, containing 1000U of lysozyme) and lysed at 30°C for 1 hour. The cells were then centrifuged at 4000g for 30 minutes at 4°C. The clarified supernatant was aspirated to measure the activity of the mutants. 190μL of the reaction solution (containing 0.4mM substrate and 1mM NADPH) was added to a new 96-well plate, and the change in NADPH was measured at 340nm. The NADPH consumption reflects the activity of the mutants. The carbonyl reductase mutants shown in Table 2 were screened.

[0104] Table 2 Carbonyl reductase LpADH mutants and their relative activities

[0105]

[0106] Note: The activity of wild-type carbonyl reductase LpADH was set as 100%.

[0107] As shown in Table 2, the mutant with sequence number M12 has 25 mutation sites and the relative activity is as high as 4720%.

[0108] Example 3

[0109] Synthesis of 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethan-1-one hydrochloride (I)

[0110]

[0111] 2-Chloro-3-hydroxyacetophenone (100 g, 586.2 mmol) and acetone (500 mL) were added to the reaction flask, stirred and dissolved, and then heated to 60°C. Sodium carbonate (124.3 g, 1172.4 mmol) was added, and N-methylbenzylamine (63.9 g, 527.6 mmol) was added. The reaction was followed by liquid chromatography. After completion of the reaction, the inorganic salts were filtered out, the filtrate was cooled to 5°C, and concentrated hydrochloric acid (52.8 ml) was slowly added dropwise. The precipitated solid was filtered, and the filter cake was dried to obtain 154.8 g of a white solid with a yield of 90.5% and an HPLC purity of 99.5%.

[0112] The results of nuclear magnetic resonance and mass spectrometry of 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethan-1-one hydrochloride prepared in Example 3 are as follows:

[0113] ESI-MS (m / z): 256.3 [M+H] +.

[0114] 1H NMR (400MHz, DMSO-d6) δ: 10.75 (s, 1H), 10.33 (s, 1H), 7.65 (dd, J = 6.6, 3.0Hz, 2H), 7.49~7.42 (m, 3H), 13C NMR (101MHz, DMSO-d6) δ: 191.43, 158.03, 135.04, 131.47, 130.02, 129.64, 128.83, 121.89, 118.84, 114.23, 59.59, 59.05.

[0115] Example 4

[0116] Biosynthesis of (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol(II)

[0117]

[0118] Substrate I (5 g), isopropanol (10 mL), and 0.1 M phosphate buffer (pH 7.0, 40 mL) were added to the reaction flask. After heating to 35°C, 30% sodium hydroxide solution was added dropwise to adjust the pH to 7.0. M11 wet bacteria (0.4 g) and NADP+ (0.01 g) were added. The reaction was tracked by liquid chromatography to monitor the reaction conversion rate. The reaction was terminated when the reaction conversion rate was >99.5%.

[0119] After the reaction was completed, the pH of the reaction solution was adjusted to 2.0-3.0 and the temperature was raised to 80°C, stirred for 3 h, and filtered through diatomaceous earth. The pH of the filtrate was adjusted to 9.3 to precipitate the product, and 4.6 g of a white solid was obtained by filtration, with a yield of 92%, a HPLC purity of 99.8%, and an ee value of 99.9%.

[0120] like Figure 1 As shown, the chiral spectrum of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl) indicates that (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol was successfully prepared;

[0121] like Figure 2 As shown, the HPLC retention time is 19.261 min, indicating that (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol was successfully prepared.

[0122] Example 5

[0123] Biosynthesis of (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol(II)

[0124]

[0125] Substrate I (6 g), isopropanol (10 mL), and 0.1 M phosphate buffer (pH 7, 40 mL) were added to the reaction flask. After heating to 35°C, 30% sodium hydroxide solution was added dropwise to adjust the pH to 7.0. M12 wet bacteria (0.3 g) and NADP+ (0.01 g) were added. The reaction was tracked by liquid chromatography to monitor the reaction conversion rate. The reaction was terminated when the reaction conversion rate was >99.5%.

[0126] After the reaction was completed, the pH of the reaction solution was adjusted to 2.0-3.0 and the temperature was raised to 80°C and stirred for 3 h. Celite was added for filtration, and the pH of the filtrate was adjusted to 9.3 to precipitate the product. 5.5 g of white solid was obtained by filtration, with a yield of 91.6%, a HPLC purity of 99.8%, and an ee value of 99.9%.

[0127] Example 6

[0128] Biosynthesis of (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol(II)

[0129]

[0130] Substrate I (100.0 g, 342.7 mmol), isopropanol (123.6 g, 2056.4 mmol, 157 mL), dimethyl sulfoxide (83 mL), and 0.1 M phosphate buffer (pH 7, 600 mL) were added to the reaction flask. After heating to 35°C, 30% sodium hydroxide solution was added dropwise to adjust the pH to 7.0. M9 wet bacteria (3.0 g) and NADP+ (0.08 g) were added and the reaction was allowed to react for 20 h. The reaction was tracked by liquid chromatography to monitor the reaction conversion rate. The reaction was terminated when the reaction conversion rate was >99.5%.

[0131] After the reaction was completed, the pH of the reaction solution was adjusted to 2.0-3.0 and the temperature was raised to 80°C and stirred for 3 h. Celite was added for filtration, and the pH of the filtrate was adjusted to 9.3 to precipitate the product. 85.1 g of a white solid was obtained by filtration, with a yield of 85.1%, a HPLC purity of 99.8%, and an ee value of 99.9%.

[0132] HPLC detection of compound II chromatographic conditions: chromatographic column Chiralcel OJ H column (4.6 mm × 250 mm); mobile phase n-hexane and isopropanol (volume ratio 80:20); detection wavelength 215 nm; column temperature 25 ° C; flow rate 1 mL / min; injection volume 5 μL.

[0133] The results of nuclear magnetic resonance and mass spectrometry of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol prepared in Example 6 are as follows:

[0134] EMI-MS (m / z): 258.14 [M+H] +.

[0135] 1H NMR(400MHz, DMSO-d6)δ:9.25(s,1H),7.32~7.17(m,5H),7.08(t,J=7.8Hz,1H),6.77(t,J=2.0Hz,1H),6.72(dt,J=7.6,1.3Hz,1H),6.63(m,J =8.1,2.6,1.0Hz,1H),4.93(s,1H),4.65(dd,J=8.0,4.7Hz,1H),2.53(dd,J=12.6,8.1Hz,1H),2.43(dd,J=12.7,4.7Hz,1H),2.20(s,3H); 13C NMR(101MHz,DMSO d6)δ:157.11,146.19,139.00,128.77,128.74,128.05,126.78,116.73,113.68,112.96,70.25,65.21,61.80,42.33.

[0136] Example 7

[0137] Biosynthesis of (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol(II)

[0138]

[0139] Substrate I (1.0 g, 3.427 mmol), isopropanol (123.6 g, 2056.4 mmol, 157 mL), dimethyl sulfoxide (83 mL), and 0.1 M phosphate buffer (pH 7, 760 mL) were added to the reaction flask. After heating to 40°C, 30% sodium hydroxide solution was added dropwise to adjust the pH to 6.5. M7 wet bacteria (3.0 g) and NAD+ (0.2 g) were added and reacted for 20 h. The reaction was tracked by liquid chromatography to monitor the reaction conversion rate. The reaction was terminated when the reaction conversion rate was >99.5%.

[0140] After the reaction was completed, the pH of the reaction solution was adjusted to 2.0-3.0 and the temperature was raised to 80°C and stirred for 3 h. Celite was added for filtration, and the pH of the filtrate was adjusted to 9.3 to precipitate the product. 0.82 g of a white solid was obtained by filtration, with a yield of 82.0%, a HPLC purity of 99.8%, and an ee value of 99.9%.

[0141] Example 8

[0142] Biosynthesis of (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol(II)

[0143]

[0144] Substrate I (200.0 g, 685.4 mmol), isopropanol (123.6 g, 2056.4 mmol, 157 mL), dimethyl sulfoxide (83 mL), and 0.1 M phosphate buffer (pH 7, 760 mL) were added to the reaction flask. After heating to 20°C, 30% sodium hydroxide solution was added dropwise to adjust the pH to 7.5. M8 wet bacteria (10.0 g) and NADPH (0.05 g) were added and reacted for 20 h. The reaction was tracked by liquid chromatography to monitor the reaction conversion rate. The reaction was terminated when the reaction conversion rate was >99.5%.

[0145] After the reaction was completed, the pH of the reaction solution was adjusted to 2.0-3.0 and the temperature was raised to 80°C and stirred for 3 h. Celite was added for filtration, and the pH of the filtrate was adjusted to 9.3 to precipitate the product. 171.2 g of a white solid was obtained by filtration, with a yield of 85.6%, a HPLC purity of 99.8%, and an ee value of 99.9%.

[0146] Example 9

[0147] Preparation of crude phenylephrine (III) hydrochloride

[0148]

[0149] II (100.0 g, 388.6 mmol) and methanol (600 mL) were added to the reaction flask, and after stirring to dissolve, 10% wet palladium carbon (10 g) was added. After hydrogen replacement, the reaction was carried out at 30°C for 5 h, and 1.0 eq of concentrated hydrochloric acid was added dropwise. The palladium carbon was recovered by filtration, and the filtrate was concentrated to obtain a pale yellow solid crude product of III.

[0150] The crude product of III and isopropanol (500 mL) were added to a 1 L three-necked flask, heated to 60°C to dissolve, cooled naturally to room temperature, and then placed in an ice bath to maintain the internal temperature at 0-5°C for crystallization for 3 h. The mixture was filtered and the filter cake was dried to obtain 60.9 g of a light yellow solid with a yield of 76.9%, HPLC purity of 99.56%, and ee value of 99.9%.

[0151] The chromatographic conditions for HPLC detection of compound III are as follows: chromatographic column Chiralcel ADH column (4.6 mm×250 mm); mobile phase consisting of n-hexane, isopropanol, and diethylamine (volume ratio 880:120:1); detection wavelength 215 nm; column temperature 25°C; flow rate 1 mL / min; injection volume 5 μL.

[0152] like Figure 3 As shown, the HPLC retention time is 11.469 min, indicating that phenylephrine hydrochloride is successfully prepared.

[0153] Example 10

[0154] Preparation of crude phenylephrine (III) hydrochloride

[0155]

[0156] II (100.0 g, 388.6 mmol) and methanol (600 mL) were added to the reaction flask. After stirring to dissolve, 10% wet palladium carbon (1 g) was added, followed by 1 eq formic acid. After reacting at 20°C for 5 h, 1.5 eq concentrated hydrochloric acid was added dropwise. The palladium carbon was recovered by filtration, and the filtrate was concentrated to obtain a pale yellow solid crude product III.

[0157] The crude product of III and isopropanol (500 mL) were added to a 1 L three-necked flask, heated to 60°C to dissolve, cooled naturally to room temperature, and then placed in an ice bath to maintain the internal temperature at 0-5°C for crystallization for 3 h. The mixture was filtered and the filter cake was dried to obtain 67.7 g of a light yellow solid with a yield of 85.5%, HPLC purity of 99.56%, and ee value of 99.9%.

[0158] Example 11

[0159] Preparation of crude phenylephrine (III) hydrochloride

[0160]

[0161] II (100.0 g, 388.6 mmol) and methanol (600 mL) were added to the reaction flask. After stirring to dissolve, 10% wet palladium carbon (5 g) was added, followed by 5 eq of ammonium formate. The mixture was reacted at 60°C for 5 h, and then 1.2 eq of concentrated hydrochloric acid was added dropwise. The palladium carbon was recovered by filtration, and the filtrate was concentrated to obtain a pale yellow solid crude product of III.

[0162] The crude product of III and isopropanol (500 mL) were added to a 1 L three-necked flask, heated to 60°C to dissolve, cooled naturally to room temperature, and then placed in an ice bath to maintain the internal temperature at 0-5°C for crystallization for 3 h. The mixture was filtered and the filter cake was dried to obtain 70.9 g of a light yellow solid with a yield of 89.5%, HPLC purity of 99.56%, and ee value of 99.9%.

[0163] Example 12

[0164] Recrystallization of crude phenylephrine (III) hydrochloride

[0165]

[0166] 100.0 g of the crude product III was added to a 1 L four-necked flask, and isopropanol (800 mL) was added. The temperature was raised to 60 ° C. and stirred to dissolve. Activated carbon (5.0 g) was added and stirring was continued for 0.5 h. The filtrate was filtered while hot and transferred to another 1 L four-necked flask. After stirring at 60 ° C. for 20 min, heating was stopped, and the temperature was naturally cooled to room temperature. After adding an ice bath, the internal temperature was maintained at 0-5 ° C. and crystallization was carried out for 3 h. The filter cake was rinsed with cold isopropanol and dried to obtain 90.5 g of a white solid with a yield of 90.5% and an HPLC purity of 99.9%.

[0167] The results of nuclear magnetic resonance and mass spectrometry of the recrystallized phenylephrine hydrochloride prepared in Example 12 are as follows:

[0168] EMI-MS (m / z): 168.09 [M+H] +.

[0169] 1H NMR(400MHz, DMSO-d6)δ:9.59(s,1H),7.13(t,J=7.8Hz,1H),6.82(t,J=2.1Hz,1H),6.76(dd,J=7.7,1.4Hz,1H),6.74~6.69(m,1H) 13C NMR (101MHz, DMSO-d6) δ: 157.57, 143.28, 129.34, 116.31, 114.70, 112.81, 68.06, 54.98, 32.72.

[0170] like Figure 4 As shown, the HPLC retention time is 12.442 min, indicating that the recrystallized final product of phenylephrine hydrochloride is successfully prepared.

Claims

1. A carbonyl reductase mutant, characterized in that The amino acid sequence of the carbonyl reductase mutant is an amino acid sequence in which the amino acid sequence encoded by SEQ ID NO: 1 is mutated, and the mutation comprises at least one of the following mutations: As shown in SEQ ID NO: 1, amino acid position 190 is mutated from Y to G; As shown in SEQ ID NO: 1, amino acid 195 is mutated from L to G; As shown in SEQ ID NO: 1, amino acid 152 is mutated from T to A; As shown in SEQ ID NO: 1, the 197th amino acid in the amino acid sequence is mutated from D to A.

2. The carbonyl reductase mutant according to claim 1, characterized in that The mutation further comprises at least one of the following mutations: As shown in SEQ ID NO: 1, amino acid position 64 is mutated from A to V; As shown in SEQ ID NO: 1, amino acid position 76 is mutated from T to L; As shown in SEQ ID NO: 1, amino acid position 95 is mutated from V to Q; As shown in SEQ ID NO: 1, the amino acid at position 96 is mutated from S to L; As shown in SEQ ID NO: 1, amino acid 145 is mutated from E to A; As shown in SEQ ID NO: 1, amino acid 147 is mutated from F to L; As shown in SEQ ID NO: 1, amino acid 153 is mutated from L to M; As shown in SEQ ID NO: 1, the amino acid at position 200 is mutated from E to P; As shown in SEQ ID NO: 1, amino acid 202 is mutated from A to F; Or the amino acid at position 206 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from M to C.

3. The carbonyl reductase mutant according to claim 1 or 2, characterized in that The mutation further comprises at least one of the following mutations: As shown in SEQ ID NO: 1, amino acid 94 is mutated from A to V; For example, the 11th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is mutated from I to L; As shown in SEQ ID NO: 1, amino acid position 99 is mutated from V to L; As shown in SEQ ID NO: 1, amino acid position 148 is mutated from V to I; As shown in SEQ ID NO: 1, amino acid 159 is mutated from S to T; Or the amino acid at position 249 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from Y to F.

4. The carbonyl reductase mutant according to any one of claims 1 to 3, characterized in that The carbonyl reductase mutant comprises or consists of the following amino acid sequence: a) an amino acid sequence as set forth in SEQ ID NO: 1, in which the amino acid at position 64 is mutated from A to V, the amino acid at position 76 is mutated from T to I, the amino acid at position 95 is mutated from V to Q, the amino acid at position 96 is mutated from S to L, the amino acid at position 145 is mutated from E to A, the amino acid at position 147 is mutated from F to I, the amino acid at position 152 is mutated from T to A, the amino acid at position 153 is mutated from L to M, the amino acid at position 190 is mutated from Y to G, the amino acid at position 195 is mutated from L to G, the amino acid at position 197 is mutated from D to A, the amino acid at position 200 is mutated from E to P, the amino acid at position 202 is mutated from A to F, and the amino acid at position 206 is mutated from M to C; b) an amino acid sequence as set forth in SEQ ID NO: 1, in which amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 94 is mutated from A to V, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, and amino acid position 206 is mutated from M to C; c) an amino acid sequence as set forth in SEQ ID NO: 1, in which amino acid position 11 is mutated from I to L, amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 99 is mutated from V to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 148 is mutated from V to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 159 is mutated from S to T, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, amino acid position 206 is mutated from M to C, and amino acid position 249 is mutated from Y to F; d) SEQ ID The amino acid sequence of NO:1, in which amino acid position 11 is mutated from I to L, amino acid position 64 is mutated from A to V, amino acid position 76 is mutated from T to I, amino acid position 94 is mutated from A to V, amino acid position 95 is mutated from V to Q, amino acid position 96 is mutated from S to L, amino acid position 99 is mutated from V to L, amino acid position 145 is mutated from E to A, amino acid position 147 is mutated from F to I, amino acid position 148 is mutated from V to I, amino acid position 152 is mutated from T to A, amino acid position 153 is mutated from L to M, amino acid position 159 is mutated from S to T, amino acid position 190 is mutated from Y to G, amino acid position 195 is mutated from L to G, amino acid position 197 is mutated from D to A, amino acid position 200 is mutated from E to P, amino acid position 202 is mutated from A to F, amino acid position 206 is mutated from M to C, and amino acid position 249 is mutated from Y to F; e) in the amino acid sequence shown in SEQ ID NO: 1, the 11th amino acid mutates from I to L, the 43rd amino acid mutates from I to V, the 54th amino acid mutates from P to T, the 64th amino acid mutates from A to V, the 72nd amino acid mutates from E to K, the 76th amino acid mutates from T to I, the 79th amino acid mutates from N to E, the 95th amino acid mutates from V to Q, the 96th amino acid mutates from S to L, the 99th amino acid mutates from V to L, the 101st amino acid mutates from E to D, the 145th amino acid mutates from E to A, the 147th amino acid mutates from N to E, F mutates to I, the 148th amino acid mutates to I, the 152nd amino acid mutates to A, the 153rd amino acid mutates to M, the 159th amino acid mutates to T, the 190th amino acid mutates to G, the 195th amino acid mutates to G, the 197th amino acid mutates to A, the 200th amino acid mutates to P, the 202nd amino acid mutates to F, the 206th amino acid mutates to C, the 235th amino acid mutates to S and the 249th amino acid mutates to F. A nucleic acid encoding the carbonyl reductase mutant according to any one of claims 1 to 4. An expression vector comprising the nucleic acid of claim 5 .

7. The expression vector according to claim 6, wherein The expression vector is recombinant DNA, expression cassette, transposon, plasmid vector, or viral vector.

8. A recombinant microorganism comprising the nucleic acid according to claim 5 or the expression vector according to claim 6 or 7.

9. Use of the carbonyl reductase mutant according to any one of claims 1 to 4, the nucleic acid according to claim 5, the expression vector according to claim 6 or 7, or the recombinant microorganism according to claim 8 in the synthesis of phenylephrine hydrochloride.

10. A method for synthesizing phenylephrine hydrochloride, characterized in that: The steps include: Using 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethan-1-one hydrochloride as a substrate, in the presence of a coenzyme, the carbonyl reductase mutant according to any one of claims 1 to 4, the nucleic acid according to claim 5, the expression vector according to claim 6 or 7, or the recombinant microorganism according to claim 8 is used for catalysis to produce (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol; (R)-3-(2-(Benzyl(methyl)amino)-1-hydroxyethyl)phenol is debenzylated via palladium-carbon reaction in the presence of a hydrogen donor, and concentrated hydrochloric acid is added to obtain phenylephrine hydrochloride.

11. The synthesis method according to claim 10, characterized in that The coenzyme is the oxidative coenzyme NAD + , NADP + Or reducing coenzymes NADH, NADPH.

12. The synthesis method according to claim 10, characterized in that The concentration of the 1-(3-hydroxyphenyl)-2-[methyl(phenylmethyl)amino]ethan-1-one hydrochloride is 1-200 g / L.

13. The synthesis method according to claim 10, characterized in that In the reduction reaction, the concentration of the coenzyme is 0.05-0.2 g / L.

14. The synthesis method according to claim 10, characterized in that The reduction reaction system further includes a co-substrate, which is selected from one of isopropanol, glucose or ammonium formate, and the reduction reaction system is a phosphate buffer system.

15. The synthesis method according to claim 10, characterized in that The carbonyl reductase mutant is added using the cell lysate of the recombinant microorganism according to claim 8, and the added amount is an amount equivalent to 3-10 g of wet cells per 1 L of reaction system.

16. The synthesis method according to claim 10, characterized in that The reduction reaction temperature is 20-40° C., and the pH is 6.5-7.

5.

17. The synthesis method according to claim 10, characterized in that The hydrogen donor is selected from one of hydrogen, formic acid or ammonium formate, and the amount of the hydrogen donor is 1-5eq of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol.

18. The synthesis method according to claim 10, characterized in that The temperature of the palladium carbon reaction is 20-60°C.

19. The synthesis method according to claim 10, characterized in that In the palladium-carbon reaction, the mass ratio of palladium-carbon to (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol is 1:10-100.

20. The synthesis method according to claim 10, characterized in that In the palladium-carbon reaction, the amount of hydrochloric acid used is 1-1.5 eq of (R)-3-(2-(benzyl(methyl)amino)-1-hydroxyethyl)phenol.

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