A ketoreductase and its use in the biosynthesis of phenylephrine hydrochloride

Abstract

The present invention provides a ketoreductase and its use in the biosynthesis of phenylephrine hydrochloride, belonging to the field of pharmaceutical technology. The present invention provides a ketoreductase having the amino acid sequence shown in SEQ ID NO.1, which can be used for the preparation of phenylephrine hydrochloride. The present invention also provides a method for preparing phenylephrine hydrochloride using the ketoreductase, which method comprises an alkylation reaction, an enzymatic catalysis reaction, debenzylation and salification, and a crystallization step. The method provided by the present invention has the advantages of cheap and easily available raw materials, simple operation process, high product yield, ee. high value, and the product quality meeting the requirements of the Chinese Pharmacopoeia and the European Pharmacopoeia standards, etc., is green and environmentally friendly, more conducive to industrial production, and is of great significance for the industrial production of phenylephrine hydrochloride.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a ketoreductase and its use in the biosynthesis of phenylephrine hydrochloride. Background Art

[0002] Phenylephrine hydrochloride is an adrenergic receptor agonist drug, which is mainly used clinically for preventing and treating hypotension caused by spinal anesthesia, general anesthesia, application of chlorpromazine, etc., and also for supraventricular tachycardia and mydriasis examination. Its chemical structural formula is as follows:

[0003] ;

[0004] As an optically active drug, the currently reported synthetic methods of phenylephrine hydrochloride mainly include chemical synthesis method and biocatalysis method. The preparation of phenylephrine hydrochloride by chemical synthesis method includes: resolving racemic phenylephrine to obtain L-phenylephrine and asymmetric hydrogenation to synthesize L-phenylephrine hydrochloride.

[0005] 1) Resolving racemic phenylephrine to obtain L-phenylephrine:

[0006] German chemists Bergmann and Sulzbacher reported a method for synthesizing racemic phenylephrine. Using m-hydroxybenzaldehyde as the starting material, racemic phenylephrine was synthesized through the Curtius rearrangement reaction of β-hydroxy acid azide, and then L-phenylephrine was obtained through resolution. The specific process is as follows:

[0007] ;

[0008] This process route has cumbersome steps and also involves azidation reaction in the middle, which has certain risks and is not suitable for industrial production.

[0009] Teerawutgulrag et al. used m-hydroxybenzaldehyde as the starting material, constructed a double bond through the wittig reaction, and converted the double bond to racemic phenylephrine through epoxidation amination or hydroxyhalogenation amination of the double bond, and then L-phenylephrine was obtained through resolution.

[0010] ;

[0011] This reaction route has simple steps, high yields in each step, avoids the use of azide compounds and lithium aluminum hydride, and has mild reaction conditions, with high practical value.

[0012] 2) Asymmetric hydrogenation for synthesizing L-phenylephrine hydrochloride: Boehringer Ingelheim Pharma GmbH & Co. KG uses N-methyl-2-amino-m-hydroxyacetophenone hydrochloride as the starting material, and asymmetrically synthesizes benzylphenylephrine using a chiral metal catalyst, and then synthesizes L-phenylephrine through debenzylation and salification. The specific process is as follows:

[0013] ;

[0014] This process route is simple, but the chiral catalyst used is relatively expensive, resulting in a high production cost and is not suitable for industrial production.

[0015] Compared with the chemical method, the biocatalytic asymmetric synthesis of L-phenylephrine has high selectivity. Patent CN102159719A uses m-hydroxyacetophenone as the starting material, reacts with sulfonyl chloride in the presence of methanol to obtain α-chloro-3-hydroxyacetophenone, and then uses the alcohol dehydrogenase of Azotobacter sp. EBN1 as a biocatalyst and NADP as an electron donor to reduce α-chloro-3-hydroxyacetophenone to chiral alcohol, and then obtains L-phenylephrine after amination with methylamine. The optical purity of the product is greater than 99%. However, this process route is relatively simple, but the enzyme-catalyzed substrate concentration is low, only 8.5 g / L, and the last step of amination reaction requires a pressurized reaction at 90 °C, with a high risk coefficient and high production cost, and is not suitable for industrial production. The specific reaction route is as follows:

[0016] ;

[0017] Patent CN102776251A uses N-methyl-2-amino-m-hydroxyacetophenone hydrochloride as the starting material, and ketoreductase KRED 185 as a biocatalyst to catalyze it into chiral alcohol, and then obtains L-phenylephrine hydrochloride through debenzylation and salification. The route design of this patent is relatively reasonable, but it uses a commercial enzyme and does not disclose the specific enzyme used for catalysis. The specific process is as follows:

[0018] ;

[0019] From the comparison of the above chemical and enzymatic processes, it can be seen that most chemical synthesis methods have problems such as complex processes, long steps, the need for resolution, low ee values, and low yields. Asymmetric hydrogenation synthesis also requires expensive chiral metal catalysts and ligands and is not suitable for industrial production. Currently reported enzymatic processes usually have low enzyme activity, resulting in high costs and unable to reach the industrial level. Summary of the Invention

[0020] The object of the present invention is mainly to overcome the defects existing in the prior art, and to provide a method for preparing phenylephrine hydrochloride by an enzymatic process suitable for industrial production. The present invention provides a ketoreductase and its use in the biosynthesis of phenylephrine hydrochloride. The ketoreductase has the amino acid sequence shown in SEQ ID NO.1 and can be used for the preparation of phenylephrine hydrochloride. The present invention also provides a method for preparing phenylephrine hydrochloride using the ketoreductase, which method comprises an alkylation reaction, an enzymatic catalysis reaction, debenzylation and salification, and crystallization steps. The method provided by the present invention has the advantages of cheap and easily available raw materials, simple operation process, high product yield, ee. high optical purity, the product quality meeting the requirements of the Chinese Pharmacopoeia and the European Pharmacopoeia standards, etc., is green and environmentally friendly, more conducive to industrial production, and has very important significance for the industrial production of phenylephrine hydrochloride.

[0021] Terms:

[0022] In the present invention, the term "room temperature" used is 20 - 30 °C.

[0023] In the present invention, the term "DMF" used is N,N-dimethylformamide.

[0024] In the present invention, the term "DCM" used is dichloromethane.

[0025] In the present invention, the term "IPA" used is isopropyl alcohol.

[0026] In the present invention, the term "IPTG" used is isopropyl β-D-thiogalactopyranoside.

[0027] Technical solutions of the present invention:

[0028] In the first aspect, the present invention provides a ketoreductase, and the ketoreductase has the amino acid sequence shown in SEQ ID NO.1.

[0029] In the second aspect, the present invention provides a coding gene, and the coding gene is used for encoding the above-mentioned ketoreductase, and the coding gene has the nucleotide sequence shown in SEQ ID NO.2.

[0030] In the third aspect, the present invention provides a recombinant plasmid, and the recombinant plasmid contains the above-mentioned coding gene.

[0031] Specifically, the expression vector of the recombinant plasmid is a pET expression vector.

[0032] Preferably, the pET expression vector is pET24a.

[0033] In the fourth aspect, the present invention provides a ketoreductase strain, and the ketoreductase strain contains the above-mentioned coding gene.

[0034] Specifically, the host cell of the ketoreductase strain is Escherichia coli.

[0035] Preferably, the Escherichia coli is Escherichia coli BL21(DE3).

[0036] In a fifth aspect, the present invention provides the use of the above-mentioned ketoreductase, coding gene or ketoreductase strain in the preparation of phenylephrine hydrochloride.

[0037] In a sixth aspect, the present invention provides a method for preparing phenylephrine hydrochloride, which includes: using the above-mentioned ketoreductase, coding gene or ketoreductase strain.

[0038] Specifically, the preparation method includes: using one or more of the culture of the ketoreductase strain, culture extract, cell lysate, bacterial cells, fermentation broth, fermentation broth precipitate, crude enzyme solution, lyophilized powder, and freeze-dried powder of crude enzyme solution to prepare phenylephrine hydrochloride.

[0039] Preferably, the preparation method includes: using the freeze-dried powder of crude enzyme solution of the ketoreductase strain to prepare phenylephrine hydrochloride.

[0040] Specifically, the preparation method includes the following steps:

[0041] S1. In an organic solution containing an acid-binding agent, react 2-chloro-3-hydroxyacetophenone with dichloromethane to form benzyl deoxyadrenone, add a solvent and hydrochloric acid to obtain benzyl deoxyadrenone hydrochloride;

[0042] S2. In a mixed solvent containing a coenzyme and the freeze-dried powder of crude enzyme solution of ketoreductase, react to form benzyl phenylephrine;

[0043] S3. In an organic solution containing a hydrogen donor, benzyl phenylephrine is debenzylated by reaction with Pd / C, and an alcoholic solution of hydrogen chloride is added to obtain phenylephrine hydrochloride.

[0044] Preferably, the organic solution in step S1 includes: dichloromethane and / or N,N-dimethylformamide.

[0045] Preferably, the acid-binding agent in step S1 includes: triethylamine and / or N,N-diisopropylethylamine.

[0046] Preferably, the reaction conditions in step S1 are: reacting at 10-40 °C for 6-10 h.

[0047] More preferably, the reaction conditions in step S1 are: reacting at 10-40 °C for 6-10 h.

[0048] More specifically, step S1 includes: adding 2-chloro-3-hydroxyacetophenone to an organic solution containing an acid-binding agent to react with dichloromethane, adding water, standing for liquid separation, concentrating the organic phase under reduced pressure, adding a solvent and hydrochloric acid to the concentrate, crystallizing out, filtering to obtain the precipitate and drying it by suction, and drying the solid under vacuum to obtain benzyl deoxyephedrine hydrochloride.

[0049] More specifically, the mixed solvent described in step S2 includes: a mixed solvent of an alcohol and water.

[0050] Preferably, the alcohol includes butanol, isopropanol or ethanol.

[0051] Preferably, the percentage of the alcohol in the volume of the mixed solution is 20%-50%.

[0052] More preferably, the percentage of the alcohol in the volume of the mixed solution is 35%-40%.

[0053] Preferably, the reaction conditions described in step S2 include: the reaction temperature is 20-40°C.

[0054] More preferably, the reaction temperature is 28-32°C.

[0055] Preferably, the reaction conditions described in step S2 include: the reaction pH value is 5.5-7.5.

[0056] More preferably, the reaction pH value is 6.0-7.0.

[0057] Preferably, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the benzyl deoxyephedrine hydrochloride described in step S2 is 1-100 g / L.

[0058] More preferably, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the benzyl deoxyephedrine hydrochloride described in step S2 is 70-85 g / L.

[0059] Preferably, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the freeze-dried powder of the crude ketoreductase solution described in step S2 is 1-100 g / L.

[0060] More preferably, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the freeze-dried powder of the crude ketoreductase solution described in step S2 is 30-60 g / L.

[0061] Preferably, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the coenzyme described in step S2 is 0.01-0.12 g / L.

[0062] Further preferably, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the coenzyme in step S2 is 0.05 - 0.12 g / L.

[0063] More specifically, step S2 includes: reacting in a mixed solvent containing a coenzyme and a freeze-dried powder of crude ketoreductase solution, filtering to remove the thallus, concentrating the filtrate, extracting, collecting the organic phase, concentrating and drying to produce benzyl deoxyepinephrine.

[0064] Preferably, the hydrogen donor described in step S3 includes: formic acid and / or hydrogen;

[0065] Further preferably, the hydrogen donor in step S3 is formic acid.

[0066] More specifically, the organic solution described in step S3 includes: alcohols or a mixed solvent of alcohols and water.

[0067] Preferably, the alcohols include methanol, ethanol or isopropanol.

[0068] Further preferably, the alcohol is methanol.

[0069] Preferably, based on the benzyl deoxyepinephrine described in step S3, the dosage of Pd / C in step S3 is 1% - 10%.

[0070] Further preferably, based on the benzyl deoxyepinephrine described in step S3, the dosage of Pd / C in step S3 is 4% - 5%.

[0071] Preferably, the temperature of the reaction described in step S3 is 0 - 80 °C.

[0072] Further preferably, the temperature of the reaction described in step S3 is 20 - 30 °C.

[0073] Preferably, the alcoholic solution of hydrogen chloride described in step S3 includes: one or more of hydrogen chloride methanol solution, hydrogen chloride ethanol solution, and hydrogen chloride isopropanol solution.

[0074] Further preferably, the alcoholic solution of hydrogen chloride in step S3 is hydrogen chloride isopropanol solution.

[0075] More specifically, step S3 includes: in an organic solution containing a hydrogen donor, benzyl deoxyepinephrine reacts with Pd / C to remove the benzyl group, filtering to remove the palladium carbon, concentrating the filtrate, adding an alcoholic solution of hydrogen chloride to the concentrate, filtering, and vacuum drying the filter cake to obtain deoxyepinephrine hydrochloride.

[0076] Advantages of the present invention:

[0077] The ketoreductase provided by the present invention can be used for the biosynthesis of phenylephrine hydrochloride. The biosynthesis method of phenylephrine hydrochloride provided by the present invention has the advantages of cheap and easily available raw materials, simple operation process, low raw material cost, high product yield, high product quality, etc. In particular, this method is more suitable for large-scale production and is of great significance for the industrial production of phenylephrine hydrochloride. Detailed implementation manners

[0078] The present invention will be further illustrated by the following examples. It should be understood that the following examples are only used to illustrate the present invention and not to limit the present invention. Any simple improvements made under the premise of the concept of the present invention should be covered within the protection scope of the present invention.

[0079] The culture medium components used in the present invention are as follows:

[0080] 1) LB medium: containing 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride per liter, pH 7.2.

[0081] 2) TB medium: containing 12 g of peptone, 24 g of yeast extract, 5 g of glycerol, 2.31 g of potassium dihydrogen phosphate, 16.43 g of dipotassium hydrogen phosphate trihydrate per liter, pH 7.2.

[0082] Example 1 Construction of reductase strain and preparation of enzyme

[0083] The amino acid sequence of the ketoreductase synthesized by total gene synthesis is shown in SEQ ID NO.1:

[0084] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIALSKSMEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGIVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVEDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ.

[0085] The nucleotide sequence of the ketoreductase synthesized by total gene synthesis is shown in SEQ ID NO.2:

[0086] ATGACCGACCGTCTGAAAGGTAAAGTTGCTATCGTTACCGGTGGTACCCTGGGTATCGGTCTGGCTATCGCTGACAAATTCGTTGAAGAAGGTGCTAAAGTTGTTATCACCGGTCGTCACGCTGACGTTGGTGAAAAAGCTGCTAAATCTATCGGTGGTACCGACGTTATCCGTTTCGTTCAGCACGACGCTTCTGACGAAGCTGGTTGGACCAAACTGTTCGACACCACCGAAGAAGCTTTCGGTCCGGTTACCACCGTTGTTAACAACGCTGGTATCGCTCTGTCTAAATCTATGGAAGACACCACCACCGAAGAATGGCGTAAACTGCTGTCTGTTAACCTGGACGGTGTTTTCTTCGGTACCCGTCTGGGTATCCAGCGTATGAAAAACAAAGGTCTGGGTGCTTCTATCATCAACATGTCTTCTATCGAAGGTATCGTTGGTGACCCGTCTCTGGGTGCTTACAACGCTTCTAAAGGTGCTGTTCGTATCATGTCTAAATCTGCTGCTCTGGACTGCGCTCTGAAAGACTACGACGTTCGTGTTAACACCGTTCACCCGGGTTACATCAAAACCCCGCTGGTTGAAGACCTGGAAGGTGCTGAAGAAATGATGTCTCAGCGTACCAAAACCCCGATGGGTCACATCGGTGAACCGAACGACATCGCGTGGATCTGCGTCTACCTGGCGTCTGACGAATCTAAATTCGCTACCGGTGCTGAATTCGTTGTTGACGGTGGTTACACCGCTCAGTAA。

[0087] The gene sequence of the ketoreductase was synthesized by total gene synthesis (SEQ ID NO.2), and NdeI and BamHI restriction sites were designed at both ends of the gene. Using a seamless cloning kit (purchased from MCE, product number HY-K1041-50 rxns), it was cloned into the corresponding site of the expression vector pET24a (purchased from Novagen) to obtain the recombinant plasmid. The recombinant plasmid was transformed into the Escherichia coli host cell BL21(DE3) to obtain the ketoreductase strain.

[0088] The constructed ketoreductase strain was inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37 °C with shaking at 200 rpm. Then, it was inoculated into TB medium at an inoculation amount of 1% and cultured at 37 °C with shaking at 200 rpm until the OD600 reached 5 - 10. IPTG with a final concentration of 0.2 mM was added, and induction was carried out overnight at 25 °C with shaking at 200 rpm. The fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the obtained cells were stored at -20 °C for later use.

[0089] Preparation of crude ketoreductase solution: The cells obtained by centrifuging the fermentation broth were resuspended with 4 volumes of water and then disrupted using an ultrasonic cell disruptor to obtain the crude ketoreductase solution.

[0090] Preparation of freeze-dried powder of crude ketoreductase solution: The crude ketoreductase solution was centrifuged at 12000 rpm for 20 min, and the supernatant was taken for freeze-drying to obtain the freeze-dried powder of the crude ketoreductase solution.

[0091] Example 2 Preparation of benzyl deoxyadrenalone hydrochloride

[0092]

[0093] Method 1: 210 g of 2-chloro-3-hydroxyacetophenone and 630 mL of dichloromethane were added into a 2 L four-necked flask, 150 g of triethylamine was added, and the mixture was stirred. 165 g of N-methylbenzylamine was added dropwise, and the reaction was carried out at 20 - 30 °C for 8 h. 630 mL of tap water was added, and the mixture was allowed to stand for liquid separation. The organic phase was concentrated under reduced pressure. 210 mL of tap water was added to the concentrate, and 155 mL of concentrated hydrochloric acid was added dropwise with stirring. Solids precipitated out, and after filtration, washing with tap water, and suction drying, 288 g of benzyl deoxyadrenalone hydrochloride (molar yield 80%) was obtained by vacuum drying at 60 °C.

[0094] Method 2: 210 g of 2-chloro-3-hydroxyacetophenone and 420 mL of DMF were added into a 2 L four-necked flask, 150 g of triethylamine was added, and the mixture was stirred. 165 g of N-methylbenzylamine was added dropwise, and the reaction was carried out at 20 - 30 °C for 2 h. 1050 mL of tap water was added, and 186 mL of concentrated hydrochloric acid was added dropwise with stirring. Solids precipitated out, and after filtration, washing with 420 mL of tap water, and suction drying, 216 g of benzyl deoxyadrenalone hydrochloride (molar yield 60%) was obtained by vacuum drying at 60 °C.

[0095] Example 3 Preparation of benzyl deoxyadrenaline

[0096]

[0097] Method 1: In a 5000 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8 g of benzyl deoxyadrenalone hydrochloride, 1.5 L of isopropanol, 1.5 L of tap water, 140 g of the freeze-dried powder of the crude ketoreductase solution prepared in Example 1, 0.4 g of NADP, and 500 mL of tap water. Control the temperature at 28 - 32 °C and react at pH 6.0 - 7.0. When the substrate conversion is complete, filter to remove the cells. Concentrate the filtrate under reduced pressure at 60 °C, extract twice with 1000 mL of ethyl acetate, combine the organic phases, and concentrate and dry the organic phases under reduced pressure at 45 °C to obtain 240 g of benzyl adrenaline (molar yield 93.4%), with an ee value of 99.9%.

[0098] Method 2: In a 5000 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8 g of benzyl deoxyadrenalone hydrochloride, 2.0 L of isopropanol, 1.0 L of tap water, 200 g of the freeze-dried powder of the crude ketoreductase solution prepared in Example 1, 0.4 g of NADP, and 500 mL of tap water. Control the temperature at 28 - 32 °C and react at pH 6.0 - 6.5. When the substrate conversion is complete, filter to remove the cells. Concentrate the filtrate under reduced pressure at 60 °C, extract twice with 1000 mL of ethyl acetate, combine the organic phases, and concentrate and dry the organic phases under reduced pressure at 45 °C to obtain 231 g of benzyl adrenaline (molar yield 89.9%), with an ee value of 99.9%.

[0099] Method 3: In a 5000 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8 g of benzyl deoxyadrenalone hydrochloride, 2.0 L of isopropanol, 1.5 L of tap water, 200 g of the freeze-dried powder of the crude ketoreductase solution prepared in Example 1, 0.4 g of NADP, and 500 mL of tap water. Control the temperature at 30 - 35 °C and react at pH 5.5 - 6.0. When the substrate conversion is complete, filter to remove the cells. Concentrate the filtrate under reduced pressure at 60 °C, extract twice with 1000 mL of ethyl acetate, combine the organic phases, and concentrate and dry the organic phases under reduced pressure at 45 °C to obtain 227 g of benzyl adrenaline (molar yield 88.3%), with an ee value of 99.9%.

[0100] Method 4: In a 5000 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8 g of benzyl deoxyadrenalone hydrochloride, 1.5 L of isopropanol, 2.0 L of tap water, 120 g of the freeze-dried powder of the crude ketoreductase solution prepared in Example 1, and 0.2 g of NAD+. Control the temperature at 25 - 30 °C and react at pH 7.0 - 7.5. When the substrate conversion is complete, filter to remove the cells. Concentrate the filtrate under reduced pressure at 60 °C, extract twice with 1000 mL of ethyl acetate, combine the organic phases, and concentrate and dry the organic phases under reduced pressure at 45 °C to obtain 235 g of benzyl adrenaline (molar yield 91.4%), with an ee value of 99.9%.

[0101] Example 4 Preparation of Phenylephrine Hydrochloride

[0102]

[0103] Method 1: In a 500 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 25.7 g of benzyladrenaline, 127 mL of isopropanol, 1.50 g of palladium-carbon, and dropwise add 2.0 g of formic acid, and react at 50 °C. After HPLC detection shows that the substrate reaction is complete, filter off the palladium-carbon, and concentrate the filtrate under reduced pressure at 50 °C until no distillate is distilled out. Add 50 mL of isopropanol to the concentrate, slowly add 50 mL of 4M hydrogen chloride isopropanol solution, stir at room temperature for 2 h, filter, wash the filter cake with 25 mL of isopropanol, drain, and vacuum dry the filter cake at 50 °C to obtain 14.82 g of phenylephrine hydrochloride (yield 73.0%), purity 99.9%, and ee value 99.9%.

[0104] Method 2: In a 500 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 25.7 g of benzyladrenaline, 200 mL of isopropanol, 1.0 g of palladium-carbon, and dropwise add 1.5 g of formic acid, and react at 35 °C. After HPLC detection shows that the substrate reaction is complete, filter off the palladium-carbon, and concentrate the filtrate under reduced pressure at 50 °C until no distillate is distilled out. Add 50 mL of isopropanol to the concentrate, slowly add 50 mL of 4M hydrogen chloride isopropanol solution, stir at room temperature for 2 h, filter, wash the filter cake with 25 mL of isopropanol, drain, and vacuum dry the filter cake at 50 °C to obtain 15.22 g of phenylephrine hydrochloride (molar yield 75.0%), purity 99.9%, and ee value 99.9%.

[0105] Example 5 Whole Process of Preparation of Phenylephrine Hydrochloride

[0106] S1. Add 420 g of 2-chloro-3-hydroxyacetophenone and 1260 mL of dichloromethane into a 5 L four-necked flask, add 300 g of triethylamine, stir, and dropwise add 330 g of N-methylbenzylamine, and react at 20 - 30 °C for 8 h. Add 1260 mL of tap water, let it stand for liquid separation, concentrate the organic phase under reduced pressure, add 400 mL of tap water to the concentrate, stir and dropwise add 300 mL of concentrated hydrochloric acid, stir, and a solid precipitates. Filter, wash with tap water, drain, and vacuum dry the solid at 60 °C to obtain 580 g of benzyl deoxyadrenalone hydrochloride.

[0107] S2. In a 5L four-necked round-bottom flask equipped with a stirrer and a thermometer, add 350 g of benzyl deoxyadrenalone hydrochloride, 2.2 L of isopropanol, 1.1 L of tap water, 200 g of the freeze-dried crude ketoreductase solution prepared in Example 1, 0.4 g of NADP, and 500 mL of tap water. Control the temperature at 28 - 32 °C and react at pH 6.0 - 6.5. When the substrate conversion is complete, filter off the cells. Concentrate the filtrate under reduced pressure at 60 °C, extract twice with 1500 mL of ethyl acetate, combine the organic phases, and concentrate and dry the organic phase under reduced pressure at 45 °C to obtain 270 g of benzyl adrenaline.

[0108] S3. In a 5000 mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 257 g of benzyl adrenaline, 2000 mL of isopropanol, 10 g of palladium carbon, and dropwise add 15 g of formic acid. React at 35 °C. After HPLC detects that the substrate reaction is complete, filter off the palladium carbon. Concentrate the filtrate under reduced pressure at 50 °C until no distillate is distilled out. Add 500 mL of isopropanol to the concentrate, slowly add 500 mL of 4M hydrogen chloride isopropanol solution, stir at room temperature for 2 h, filter, wash the filter cake with 250 mL of isopropanol, drain, and vacuum dry the filter cake at 50 °C to obtain 152 g of deoxyadrenaline hydrochloride (total molar yield 56.4%), purity 99.9%, and ee value 99.9%

[0109] Comparative Example 1

[0110] The difference between Comparative Example 1 and Method 1 of Example 3 lies in that the freeze-dried powder of the crude ketoreductase solution used in Step S2 is different. The preparation method of the freeze-dried powder of the crude reductase solution in Comparative Example 1 is as follows:

[0111] Fully synthesize the gene sequence of ketoreductase (SEQ ID NO.3), design NdeI and BamHI restriction enzyme sites at both ends of the gene, and use a seamless cloning kit (purchased from MCE, product number HY-K1041-50 rxns) to clone it to the corresponding site of the expression vector pET24a (purchased from Novagen) to obtain the recombinant plasmid. Transform the recombinant plasmid into the Escherichia coli host cell BL21(DE3) to obtain the ketoreductase strain.

[0112] Inoculate the constructed ketoreductase strain into LB medium containing 50 μg / mL kanamycin, culture overnight at 37 °C and 200 rpm, then inoculate it into TB medium at an inoculation amount of 1%, culture at 37 °C and 200 rpm until the OD600 is 5 - 10, add IPTG with a final concentration of 0.2 mM, and induce overnight at 25 °C and 200 rpm. Centrifuge the fermentation broth at 8000 rpm for 10 min, remove the supernatant, and store the obtained cells at -20 °C for later use.

[0113] Preparation of crude ketoreductase enzyme solution: The cells obtained by centrifuging the fermentation broth were resuspended in 4 volumes of water and then disrupted using an ultrasonic cell disruptor to obtain the crude ketoreductase enzyme solution.

[0114] Preparation of freeze-dried powder of crude ketoreductase enzyme solution: The crude ketoreductase enzyme solution was centrifuged at 12,000 rpm for 20 min, and the supernatant was taken and freeze-dried to obtain the freeze-dried powder of the crude ketoreductase enzyme solution.

[0115] SEQ ID NO.3:

[0116] ATGACCGACCGTCTGAAAGGTAAAGTTGCTATCGTTACCGGTGGTACCCTGGGTATCGGTCTGGCTATCGCTGACAAATTCGTTGAAGAAGGTGCTAAAGTTGTTATCACCGGTCGTCACGCTGACGTTGGTGAAAAAGCTGCTAAATCTATCGGTGGTACCGACGTTATCCGTTTCGTTCAGCACGACGGTTCTGACGAAGCTGGTTGGACCAAACTGTTCGACACCACCGAAGAAGCTTTCGGTCCGGTTACCACCGTTGTTAACAACGCTGGTATCGCTGTTACCAAATCTGTTGAAGACACCACCACCGAAGAATGGCGTAAACTGCTGTCTGTTAACCTGGACGGTGTTTTCTTCGGTACCCGTCTGGGTATCCAGCGTATGAAAAACAAAGGTCTGGGTGCTTCTATCATCAACATGTCTTCTATCGACGGTTTCGTTGGTGACCCGACCCTGGGTGCTTACAACGCTTCTAAAGGTGCTGTTCGTATCATGTCTAAATCTGCTGCTCTGGACTGCGCTCTGAAAGACTACGACGTTCGTGTTAATACCGTTCACCCGGGCTTCATCAAAACCCCACTGGTTGACGACCTGGAAGGTGCTGAAGAAATGGTTTCTCAGCGTACCAAAACCCCGATGGGTCACATCGGTGAACCGAACGACATCGCGTGGATCTGCGTCTACCTGGCGTCTGACGAATCTAAATTCGCTACCGGTGCTGAATTCGTTGTTGACGGTGGTTACACCGCTCAGTAA.

[0117] The results showed that no benzyl epinephrine product was formed when the freeze-dried powder of the crude ketoreductase solution fermented and prepared with the ketoreductase sequence disclosed in the above-mentioned CN118165946A was used to reduce benzyl deoxyadrenone hydrochloride.

[0118] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A ketoreductase, characterized in that, The amino acid sequence of the ketoreductase described above is shown in SEQ ID NO.

1.

2. A coding gene, characterized in that, The encoding gene described above is used to encode the ketoreductase according to claim 1, and the encoding gene has a nucleotide sequence shown in SEQ ID NO.

2.

3. A ketoreductase strain, characterized in that, The ketoreductase strain described above contains the encoding gene according to claim 2.

4. Use of the ketoreductase according to claim 1, the encoding gene according to claim 2, or the ketoreductase strain according to claim 3 in the preparation of phenylephrine hydrochloride.

5. A preparation method of phenylephrine hydrochloride, characterized in that, The preparation method described above includes: using the ketoreductase according to claim 1, the encoding gene according to claim 2, or the ketoreductase strain according to claim 3 to prepare phenylephrine hydrochloride.

6. The preparation method according to claim 5, characterized in that, The preparation method described above includes: using one or more of the culture, cell lysate, cell body, fermentation broth, crude enzyme solution, freeze-dried powder, and freeze-dried powder of crude enzyme solution of the ketoreductase strain to prepare phenylephrine hydrochloride.

7. The preparation method according to claim 6, characterized in that The preparation method described above includes the following steps: S1. In an organic solution containing an acid-binding agent, react 2-chloro-3-hydroxyacetophenone with dichloromethane to generate benzyl deoxyadrenalone, add a solvent and hydrochloric acid to obtain benzyl deoxyadrenalone hydrochloride; S2. React in a mixed solvent containing a coenzyme and freeze-dried powder of crude ketoreductase solution to generate benzyl deoxyephedrine; S3. In an organic solution containing a hydrogen donor, benzyl deoxyephedrine reacts with Pd / C to remove the benzyl group, add an alcoholic solution of hydrogen chloride to obtain phenylephrine hydrochloride.

8. The preparation method according to claim 7, characterized in that, The organic solution described in step S1 includes: dichloromethane and / or N,N-dimethylformamide; And / or, the acid-binding agent described in step S1 includes: triethylamine and / or N,N-diisopropylethylamine.

9. The preparation method according to claim 7, wherein The coenzyme described in step S2 includes: nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate; And / or, the mixed solvent described in step S2 includes: a mixed solvent of alcohol and water, and the percentage of the alcohol in the mixed solution by volume is 20%-50%; And / or, the reaction conditions described in step S2 include: the reaction temperature is 20-40°C, and the reaction pH value is 5.5-7.5; And / or, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the benzyl deoxyadrenalone hydrochloride described in step S2 is 1-100 g / L; And / or, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the freeze-dried powder of crude ketoreductase solution described in step S2 is 1-100 g / L; And / or, based on the volume of the mixed solvent described in step S2, the mass-volume ratio of the coenzyme described in step S2 is 0.01-0.12 g / L.

10. The preparation method according to claim 7, wherein, The hydrogen donor described in step S3 includes: formic acid and / or hydrogen; And / or, the organic solution described in step S3 includes: alcohol or a mixed solvent of alcohol and water; the alcohol includes methanol, ethanol or isopropanol; And / or, based on the benzyl deoxyephedrine described in step S3, the dosage of Pd / C described in step S3 is 1%-10%; And / or, the reaction temperature described in step S3 is 0-80°C; And / or, the alcoholic solution of hydrogen chloride described in step S3 includes one or more of: methanol solution of hydrogen chloride, ethanol solution of hydrogen chloride, and isopropanol solution of hydrogen chloride.

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