Ketoreductase and application thereof in biosynthesis of phenylephrine hydrochloride

Abstract

The invention provides ketoreductase and application thereof in biosynthesis of phenylephrine hydrochloride, and belongs to the technical field of medicines. The invention provides ketoreductase which has an amino acid sequence as shown in SEQ ID NO.1 and can be used for preparing phenylephrine hydrochloride. The invention also provides a method for preparing phenylephrine hydrochloride by using the ketoreductase. The method comprises the steps of alkylation reaction, enzyme catalysis reaction, debenzylation salification and crystallization. The method provided by the invention has the advantages that the raw materials are cheap and easy to obtain, the operation process is simple, the product yield is high, the ee. Value is high, the product quality meets the standard requirements of Chinese pharmacopoeia and European pharmacopoeia, and the like, is green and environment-friendly, is more beneficial to industrial production, and has very important significance on industrial production of phenylephrine hydrochloride.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a ketoreductase and an application thereof in the biosynthesis of phenylephrine hydrochloride. Background Art

[0002] Phenylephrine hydrochloride is an adrenergic receptor agonist, which is mainly used clinically to prevent and treat hypotension caused by spinal anesthesia, general anesthesia, and the use of chlorpromazine, and is also used for supraventricular tachycardia and mydriasis examination. Its chemical structure is as follows: ; As an optically active drug, phenylephrine hydrochloride has two main synthesis methods reported in the literature: chemical synthesis and biocatalysis. The chemical synthesis method for preparing phenylephrine hydrochloride includes: splitting racemic phenylephrine to obtain L-phenylephrine and asymmetric hydrogenation to synthesize L-phenylephrine hydrochloride.

[0003] 1) L-phenylephrine is obtained by splitting racemic phenylephrine: German chemists Bergmann and Sulzbacher reported a method for synthesizing racemic phenylephrine. Using m-hydroxybenzaldehyde as the starting material, racemic phenylephrine was synthesized by the Curtius rearrangement reaction of β-hydroxyacid azide, and then L-phenylephrine was obtained by splitting. The specific process is as follows: ; This process route has complicated steps and involves azidation reaction, which has certain risks and is not suitable for industrial production.

[0004] Teerawutgulrag et al. used m-hydroxybenzaldehyde as the starting material, constructed a double bond through the Wittig reaction, and then aminized the double bond through epoxidation or hydroxyhalogenation to obtain racemic phenylephrine, which was then resolved to obtain L-phenylephrine.

[0005] ; The reaction route has simple steps, high yield in each step, avoids the use of azide compounds and lithium aluminum hydride, has mild reaction conditions, and has high practical value.

[0006] 2) Asymmetric hydrogenation of L-phenylephrine hydrochloride: Boehringer Ingelheim Pharma GmbH & Co. KG uses N-methyl-2-amino-m-hydroxyacetophenone hydrochloride as the starting material and uses a chiral metal catalyst to asymmetric synthesize benzylphenylephrine, and then debenzylates the salt to synthesize acid L-phenylephrine. The specific process is as follows: This process route is simple, but the use of chiral catalysts is relatively expensive, the production cost is high, and it is not suitable for industrial production.

[0007] Compared with the chemical method, the biological method catalyzes the asymmetric synthesis of L-phenylephrine and 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 the Azoarbrio species EBN1 as a biocatalyst and NADP as an electron donor to reduce α-chloro-3-hydroxyacetophenone to a chiral alcohol, and then obtains L-phenylephrine after amination with methylamine. The product has an optical purity of more than 99%, but this process route is relatively simple, but the enzyme-catalyzed substrate concentration is low, only 8.5g / L, and the last step of amination reaction requires a 90°C pressurized reaction, which has a high risk factor and high production cost, and is not suitable for industrial production. The specific reaction route is as follows: ; Patent CN102776251A uses N-methyl-2-amino-m-hydroxyacetophenone hydrochloride as the starting material and ketoreductase KRED 185 as the biocatalyst to catalyze it into chiral alcohol, and then debenzylate and salify to obtain L-phenylephrine hydrochloride. The patent route design is relatively reasonable, but commercial enzymes are used and the specific catalytic enzymes are not disclosed. The specific process is as follows: ; According to the comparison between the chemical method and the enzymatic method, most chemical synthesis methods have the problems of complex process, lengthy steps, splitting, low ee value, low yield, etc. Asymmetric hydrogenation synthesis requires expensive chiral metal catalysts and ligands, which are not suitable for industrial production. The enzymatic process that has been reported so far usually has low enzyme activity, resulting in high cost and cannot reach the industrial level. Summary of the invention

[0008] The main purpose of the present invention is to overcome the defects of the prior art and 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 an amino acid sequence as shown in SEQ ID NO.1 and can be used to prepare phenylephrine hydrochloride. The present invention also provides a method for preparing phenylephrine hydrochloride using the ketoreductase, which comprises an alkylation reaction, an enzyme-catalyzed reaction, a debenzylation salt formation and a crystallization step. The method provided by the present invention has the advantages of cheap and readily available raw materials, a simple operation process, a high product yield, oh.The product has the advantages of high value, product quality in line with the requirements of the Chinese Pharmacopoeia and the European Pharmacopoeia, green and environmentally friendly, and more conducive to industrial production. It is of great significance for the industrial production of phenylephrine hydrochloride.

[0009] the term: In the present invention, the term "room temperature" used is 20-30°C.

[0010] In the present invention, the term "DMF" used is N,N-dimethylformamide.

[0011] In the present invention, the term "DCM" used is dichloromethane.

[0012] In the present invention, the term "IPA" used is isopropyl alcohol.

[0013] In the present invention, the term "IPTG" used is isopropylthiogalactoside.

[0014] The technical solution of the present invention: In a first aspect, the present invention provides a ketoreductase having an amino acid sequence as shown in SEQ ID NO.1.

[0015] In a second aspect, the present invention provides a coding gene, which is used to encode the above-mentioned ketoreductase, and the coding gene has a nucleotide sequence as shown in SEQ ID NO.2.

[0016] In a third aspect, the present invention provides a recombinant plasmid comprising the above-mentioned coding gene.

[0017] Specifically, the expression vector of the recombinant plasmid is a pET expression vector.

[0018] Preferably, the pET expression vector is pET24a.

[0019] In a fourth aspect, the present invention provides a ketoreductase strain, wherein the ketoreductase strain comprises the above-mentioned encoding gene.

[0020] Specifically, the host cell of the ketoreductase strain is Escherichia coli.

[0021] Preferably, the Escherichia coli is Escherichia coli BL21 (DE3).

[0022] In a fifth aspect, the present invention provides the use of the above-mentioned ketoreductase, encoding gene or ketoreductase strain in the preparation of phenylephrine hydrochloride.

[0023] In a sixth aspect, the present invention provides a method for preparing phenylephrine hydrochloride, the preparation method comprising: using the above-mentioned ketoreductase, encoding gene or ketoreductase strain.

[0024] Specifically, the preparation method comprises: using one or more of the culture of ketoreductase strains, culture extracts, cell disruptors, bacterial cells, fermentation broth, fermentation broth precipitation, crude enzyme solution, lyophilized powder, and crude enzyme solution lyophilized powder to prepare phenylephrine hydrochloride.

[0025] Preferably, the preparation method comprises: preparing phenylephrine hydrochloride using lyophilized powder of crude enzyme solution of ketoreductase strain.

[0026] Specifically, the preparation method comprises the following steps: S1. In an organic solution containing an acid-binding agent, 2-chloro-3-hydroxyacetophenone is reacted with dichloromethane to generate benzyl deoxyadrenophenone, and a solvent and hydrochloric acid are added to obtain benzyl deoxyadrenophenone hydrochloride; S2, reacting in a mixed solvent containing coenzyme and lyophilized powder of crude ketoreductase enzyme solution to generate benzylphenylephrine; S3. In an organic solution containing a hydrogen donor, benzyl phenylephrine is subjected to a Pd / C reaction to remove the benzyl group, and an alcoholic solution of hydrogen chloride is added to obtain phenylephrine hydrochloride.

[0027] Preferably, the organic solution in step S1 comprises: dichloromethane and / or N,N-dimethylformamide.

[0028] Preferably, the acid binding agent in step S1 comprises: triethylamine and / or N,N-diisopropylethylamine.

[0029] Preferably, the reaction conditions in step S1 are 10-40° C. for 6-10 h.

[0030] More preferably, the reaction conditions in step S1 are 10-40° C. for 6-10 h.

[0031] More specifically, step S1 comprises: adding 2-chloro-3-hydroxyacetophenone to an organic solution containing an acid-binding agent to react with dichloromethane, adding water, standing to separate the liquids, concentrating the organic phase under reduced pressure, adding a solvent and hydrochloric acid to the concentrate, crystallizing, filtering the precipitate to dryness, and vacuum drying the solid to obtain benzyl deoxyadrenone hydrochloride.

[0032] More specifically, the mixed solvent in step S2 includes: a mixed solvent of alcohols and water.

[0033] Preferably, the alcohol includes butanol, isopropanol or ethanol.

[0034] Preferably, the alcohol accounts for 20%-50% of the volume of the mixed solution.

[0035] More preferably, the alcohol accounts for 35%-40% of the volume of the mixed solution.

[0036] Preferably, the reaction conditions described in step S2 include: the reaction temperature is 20-40°C.

[0037] More preferably, the reaction temperature is 28-32°C.

[0038] Preferably, the reaction conditions described in step S2 include: the reaction pH value is 5.5-7.5.

[0039] More preferably, the reaction pH value is 6.0-7.0.

[0040] Preferably, based on the volume of the mixed solvent in step S2, the mass volume ratio of benzyl deoxyephedrine hydrochloride in step S2 is 1-100 g / L.

[0041] Further preferably, based on the volume of the mixed solvent described in step S2, the mass volume ratio of benzyl deoxyephedrine hydrochloride described in step S2 is 70-85 g / L.

[0042] Preferably, based on the volume of the mixed solvent in step S2, the mass volume ratio of the lyophilized powder of the crude ketoreductase enzyme solution in step S2 is 1-100 g / L.

[0043] Further preferably, based on the volume of the mixed solvent described in step S2, the mass volume ratio of the ketoreductase crude enzyme solution lyophilized powder described in step S2 is 30-60 g / L.

[0044] Preferably, based on the volume of the mixed solvent in step S2, the mass volume ratio of the coenzyme in step S2 is 0.01-0.12 g / L.

[0045] Further 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 0.05-0.12 g / L.

[0046] More specifically, step S2 comprises: reacting in a mixed solvent containing coenzyme and lyophilized powder of crude ketoreductase enzyme solution, filtering to remove bacteria, concentrating the filtrate, extracting, collecting the organic phase, concentrating and drying to generate benzylphenylephrine Preferably, the hydrogen donor in step S3 includes: formic acid and / or hydrogen; Further preferably, the hydrogen donor in step S3 is formic acid.

[0047] More specifically, the organic solution in step S3 includes alcohols or a mixed solvent of alcohols and water.

[0048] Preferably, the alcohol includes methanol, ethanol or isopropanol.

[0049] More preferably, the alcohol is methanol.

[0050] Preferably, based on the benzylphenylephrine in step S3, the amount of Pd / C in step S3 is 1%-10%.

[0051] More preferably, based on the benzylphenylephrine in step S3, the amount of Pd / C in step S3 is 4%-5%.

[0052] Preferably, the reaction temperature in step S3 is 0-80°C.

[0053] Further preferably, the reaction temperature in step S3 is 20-30°C.

[0054] Preferably, the alcoholic solution of hydrogen chloride in step S3 comprises one or more of: methanolic hydrogen chloride solution, ethanolic hydrogen chloride solution, and isopropanol hydrogen chloride solution.

[0055] Further preferably, the alcoholic solution of hydrogen chloride in step S3 is an isopropanol solution of hydrogen chloride.

[0056] More specifically, step S3 comprises: in an organic solution containing a hydrogen donor, removing the benzyl group from benzyl phenylephrine by Pd / C reaction, filtering to remove palladium carbon, concentrating the filtrate, adding an alcohol solution of hydrogen chloride to the concentrate, filtering, and vacuum drying the filter cake to obtain phenylephrine hydrochloride.

[0057] Beneficial effects of the present invention: The ketoreductase provided by the present invention can be used for biosynthesis of phenylephrine hydrochloride. The biosynthesis method of phenylephrine hydrochloride provided by the present invention has the advantages of cheap and readily available raw materials, simple operation process, low raw material cost, high product yield, high product quality, etc. In particular, the method is more suitable for scaled-up production and is of great significance for the industrial production of phenylephrine hydrochloride. DETAILED DESCRIPTION

[0058] The present invention is further described by way of examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the present invention, and any simple improvements made under the premise of the concept of the present invention should be included in the protection scope of the present invention.

[0059] The culture medium components used in the present invention are as follows: 1) LB medium: each liter contains 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, pH 7.2.

[0060] 2) TB medium: Each liter contains 12 g of peptone, 24 g of yeast extract, 5 g of glycerol, 2.31 g of potassium dihydrogen phosphate, and 16.43 g of potassium dihydrogen phosphate trihydrate, pH 7.2.

[0061] Example 1 Construction of reductase strain and preparation of enzyme The amino acid sequence of the fully gene-synthesized ketoreductase is shown in SEQ ID NO.1: MTDRLKGKVAIVTGGTLGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIALSKSMEDTTTEEWRKLLSVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGIVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVEDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ.

[0062] The nucleotide sequence of the full gene synthesizing ketoreductase is shown in SEQ ID NO.2: .

[0063] The gene sequence of the ketoreductase (SEQ ID NO.2) was synthesized by full gene, and NdeI and BamHI restriction sites were designed at both ends of the gene. The recombinant plasmid was obtained by cloning into the corresponding sites of the expression vector pET24a (purchased from Novagen) using a seamless cloning kit (purchased from MCE, catalog number HY-K1041-50 rxns). The recombinant plasmid was transformed into the Escherichia coli host cell BL21 (DE3) to obtain the ketoreductase strain.

[0064] The constructed ketoreductase strain was inoculated into LB medium containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, and then inoculated into TB medium at 1% inoculum, cultured at 37°C and 200 rpm until OD600 was 5-10, IPTG was added at a final concentration of 0.2 mM, and induced overnight at 25°C and 200 rpm. The fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the obtained bacteria were stored at -20°C for later use.

[0065] Preparation of crude ketoreductase enzyme solution: The bacterial cells obtained by centrifugation of the fermentation broth are resuspended in 4 times the volume of water, and then crushed with an ultrasonic cell crusher to obtain a crude ketoreductase enzyme solution.

[0066] Preparation of ketoreductase crude enzyme solution freeze-dried powder: The ketoreductase crude enzyme solution was centrifuged at 12000 rpm for 20 min, and the supernatant was freeze-dried to obtain the ketoreductase crude enzyme solution freeze-dried powder.

[0067] Example 2 Preparation of benzyl deoxyepinephrine hydrochloride

[0068] Method 1: Add 210 g of 2-chloro-3-hydroxyacetophenone and 630 mL of dichloromethane into a 2L four-necked bottle, add 150 g of triethylamine, stir, add dropwise 165 g of N-methylbenzylamine, react at 20-30°C for 8 h, add 630 mL of tap water, stand for separation, concentrate the organic phase under reduced pressure, add 210 mL of tap water to the concentrate, add dropwise 155 mL of concentrated hydrochloric acid while stirring, stir, solid precipitates, filter, wash with tap water, drain, and dry the solid at 60°C in vacuum to obtain 288 g of benzyl deoxyadrenone hydrochloride (molar yield 80%).

[0069] Method 2: Add 210 g of 2-chloro-3-hydroxyacetophenone and 420 mL of DMF into a 2 L four-necked bottle, add 150 g of triethylamine, stir, add dropwise 165 g of N-methylbenzylamine, react at 20-30°C for 2 h, add 1050 mL of tap water, add dropwise 186 mL of concentrated hydrochloric acid, stir to precipitate solid, filter, wash with 420 mL of tap water, drain, and dry the solid at 60°C in a vacuum oven to obtain 216 g of benzyl deoxyadrenone hydrochloride (molar yield 60%).

[0070] Example 3 Preparation of Benzylphenylephrine

[0071] Method 1: In a 5000mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8g of benzyl deoxyepinephrine hydrochloride, 1.5L of isopropanol, 1.5L of tap water, 140g of the lyophilized powder of the crude ketoreductase prepared in Example 1, 0.4g of NADP, 500mL of tap water, and react at a temperature of 28-32°C and a pH of 6.0-7.0. When the substrate conversion is complete, filter to remove the bacteria, concentrate the filtrate at 60°C under reduced pressure, add 1000mL of ethyl acetate to extract twice, combine the organic phases, and concentrate and dry the organic phases at 45°C under reduced pressure to obtain 240g of benzyl epinephrine (molar yield 93.4%) with an ee value of 99.9%.

[0072] Method 2: In a 5000mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8g of benzyl deoxyepinephrine hydrochloride, 2.0L of isopropanol, 1.0L of tap water, 200g of the lyophilized powder of the crude ketoreductase prepared in Example 1, 0.4g of NADP, 500mL of tap water, and react at a temperature of 28-32°C and a pH of 6.0-6.5. When the substrate conversion is complete, filter out the bacteria, concentrate the filtrate at 60°C under reduced pressure, add 1000mL of ethyl acetate to extract twice, combine the organic phases, and concentrate and dry the organic phases at 45°C under reduced pressure to obtain 231g of benzyl epinephrine (molar yield 89.9%) with an ee value of 99.9%.

[0073] Method 3: In a 5000mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 291.8g of benzyl deoxyepinephrine hydrochloride, 2.0L of isopropanol, 1.5L of tap water, 200g of the lyophilized powder of the crude ketoreductase prepared in Example 1, 0.4g of NADP, 500mL of tap water, and react at a temperature of 30-35°C and a pH of 5.5-6.0. When the substrate conversion is complete, filter out the bacteria, concentrate the filtrate at 60°C under reduced pressure, add 1000mL of ethyl acetate to extract twice, combine the organic phases, and concentrate and dry the organic phases at 45°C under reduced pressure to obtain 227g of benzyl epinephrine (molar yield 88.3%) with an ee value of 99.9%.

[0074] Method 4: In a 5000mL four-necked round-bottom flask equipped with a stirrer and a thermometer, 291.8g of benzyl deoxyepinephrine hydrochloride, 1.5L of isopropanol, 2.0L of tap water, 120g of the lyophilized powder of the crude ketoreductase prepared in Example 1, and 0.2g of NAD+ were added, and the temperature was controlled at 25-30°C and pH 7.0-7.5 for reaction. When the substrate conversion was complete, the bacteria were filtered out, the filtrate was concentrated under reduced pressure at 60°C, 1000mL of ethyl acetate was added for extraction twice, the organic phases were combined, and the organic phases were concentrated under reduced pressure at 45°C to obtain 235g of benzyl epinephrine (molar yield 91.4%), with an ee value of 99.9%.

[0075] Example 4 Preparation of Phenylephrine Hydrochloride

[0076] Method 1: In a 500mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 25.7g of benzyl epinephrine, 127mL of isopropanol, 1.50g of palladium carbon, and 2.0g of formic acid, and react at 50°C. After the substrate reaction is complete as detected by HPLC, the palladium carbon is filtered out, and the filtrate is concentrated under reduced pressure at 50°C until no fraction is distilled out. 50mL of isopropanol is added to the concentrate, and 50mL of 4M hydrogen chloride isopropanol solution is slowly added. Stir at room temperature for 2h, filter, rinse the filter cake with 25mL of isopropanol, drain, and vacuum dry the filter cake at 50°C to obtain 14.82g of phenylephrine hydrochloride (yield 73.0%), with a purity of 99.9% and an ee value of 99.9%.

[0077] Method 2: In a 500mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 25.7g of benzyl epinephrine, 200mL of isopropanol, 1.0g of palladium carbon, and 1.5g of formic acid, and react at 35°C. After the substrate reaction is complete as detected by HPLC, the palladium carbon is filtered out, and the filtrate is concentrated under reduced pressure at 50°C until no fraction is distilled out. 50mL of isopropanol is added to the concentrate, and 50mL of 4M hydrogen chloride isopropanol solution is slowly added. Stir at room temperature for 2h, filter, rinse the filter cake with 25mL of isopropanol, drain, and vacuum dry the filter cake at 50°C to obtain 15.22g of phenylephrine hydrochloride (molar yield 75.0%), with a purity of 99.9% and an ee value of 99.9%.

[0078] Example 5 Preparation process of phenylephrine hydrochloride S1. Add 420 g of 2-chloro-3-hydroxyacetophenone and 1260 mL of dichloromethane into a 5 L four-necked bottle, add 300 g of triethylamine, stir, add dropwise 330 g of N-methylbenzylamine, react at 20-30 ° C for 8 h, add 1260 mL of tap water, stand for separation, concentrate the organic phase under reduced pressure, add 400 mL of tap water to the concentrate, add dropwise 300 mL of concentrated hydrochloric acid under stirring, stir, solid precipitates, filter, wash with tap water, drain, and dry the solid at 60 ° C in vacuum to obtain 580 g of benzyl deoxyadrenone hydrochloride.

[0079] S2, in a 5L four-necked round-bottom flask equipped with a stirrer and a thermometer, add 350g of benzyl deoxyepinephrine hydrochloride, add 2.2L of isopropanol, add 1.1L of tap water, add 200g of the ketoreductase crude enzyme solution freeze-dried powder prepared in Example 1, 0.4g of NADP, add 500mL of tap water, control the temperature at 28-32°C, pH 6.0-6.5, and react. When the substrate conversion is complete, filter out the bacteria, concentrate the filtrate at 60°C under reduced pressure, add 1500mL of ethyl acetate to extract twice, combine the organic phases, and concentrate and dry the organic phase at 45°C under reduced pressure to obtain 270g of benzyl epinephrine.

[0080] S3. In a 5000mL four-necked round-bottom flask equipped with a stirrer and a thermometer, add 257g of benzyl epinephrine, 2000mL of isopropanol, 10g of palladium carbon, and 15g of formic acid, and react at 35°C. After the substrate reaction is complete as detected by HPLC, the palladium carbon is filtered out, and the filtrate is concentrated under reduced pressure at 50°C until no fraction is evaporated. 500mL of isopropanol is added to the concentrate, and 500mL of 4M hydrogen chloride isopropanol solution is slowly added. Stir at room temperature for 2h, filter, rinse the filter cake with 250mL of isopropanol, drain, and vacuum dry the filter cake at 50°C to obtain 152g of phenylephrine hydrochloride (total molar yield 56.4%), with a purity of 99.9% and an ee value of 99.9%. Comparative Example 1 The difference between the comparative example 1 and the method 1 of the embodiment 3 is that the lyophilized powder of the crude ketoreductase enzyme solution used in step S2 is different. The preparation method of the lyophilized powder of the crude ketoreductase enzyme solution of the comparative example 1 is as follows: The gene sequence of the ketoreductase (SEQ ID NO.3) was synthesized by full gene, and NdeI and BamHI restriction sites were designed at both ends of the gene. The recombinant plasmid was obtained by cloning into the corresponding sites of the expression vector pET24a (purchased from Novagen) using a seamless cloning kit (purchased from MCE, catalog number HY-K1041-50 rxns). The recombinant plasmid was transformed into the Escherichia coli host cell BL21 (DE3) to obtain the ketoreductase strain.

[0081] The constructed ketoreductase strain was inoculated into LB medium containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, and then inoculated into TB medium at 1% inoculum, cultured at 37°C and 200 rpm until OD600 was 5-10, IPTG was added at a final concentration of 0.2 mM, and induced overnight at 25°C and 200 rpm. The fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the obtained bacteria were stored at -20°C for later use.

[0082] Preparation of crude ketoreductase enzyme solution: The bacterial cells obtained by centrifugation of the fermentation broth are resuspended in 4 times the volume of water, and then crushed with an ultrasonic cell crusher to obtain a crude ketoreductase enzyme solution.

[0083] Preparation of ketoreductase crude enzyme solution freeze-dried powder: The ketoreductase crude enzyme solution was centrifuged at 12000 rpm for 20 min, and the supernatant was freeze-dried to obtain the ketoreductase crude enzyme solution freeze-dried powder.

[0084] SEQ ID NO.3: .

[0085] The results showed that when the lyophilized powder of crude ketoreductase prepared by fermentation with the ketoreductase sequence disclosed in CN118165946A was used to reduce benzylphenylephrine hydrochloride, no benzylephrine product was generated.

[0086] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A ketoreductase, characterized in that The ketoreductase has an amino acid sequence as shown in SEQ ID NO.

1.

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

2.

3. A ketoreductase strain, characterized in that The ketoreductase strain comprises 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 method for preparing phenylephrine hydrochloride, characterized in that: The preparation method comprises: 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 comprises: preparing phenylephrine hydrochloride using one or more of the culture of ketoreductase strains, culture extracts, cell disruptors, bacterial cells, fermentation broth, fermentation broth precipitation, crude enzyme solution, lyophilized powder, and crude enzyme solution lyophilized powder.

7. The preparation method according to claim 6, characterized in that: The preparation method comprises the following steps: S1. In an organic solution containing an acid-binding agent, 2-chloro-3-hydroxyacetophenone is reacted with dichloromethane to generate benzyl deoxyadrenophenone, and a solvent and hydrochloric acid are added to obtain benzyl deoxyadrenophenone hydrochloride; S2, reacting in a mixed solvent containing coenzyme and lyophilized powder of crude ketoreductase enzyme solution to generate benzylphenylephrine; S3. In an organic solution containing a hydrogen donor, benzyl phenylephrine is subjected to a Pd / C reaction to remove the benzyl group, and an alcoholic solution of hydrogen chloride is added to obtain phenylephrine hydrochloride.

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

9. The preparation method according to claim 7, characterized in that: The coenzyme described in step S2 includes: nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate; And / or, the mixed solvent in step S2 comprises: a mixed solvent of alcohol and water, wherein the alcohol accounts for 20%-50% of the volume of the mixed solution; And / or, the reaction conditions described in step S2 include: reaction temperature of 20-40° C., reaction pH of 5.5-7.5; and / or, based on the volume of the mixed solvent in step S2, the mass volume ratio of benzyl deoxyadrenone hydrochloride in step S2 is 1-100 g / L; and / or, based on the volume of the mixed solvent in step S2, the mass volume ratio of the ketoreductase crude enzyme solution and the lyophilized powder 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, characterized in that: The hydrogen donor in step S3 includes: formic acid and / or hydrogen; And / or, the organic solution in step S3 includes: alcohols or a mixed solvent of alcohols and water; the alcohols include methanol, ethanol or isopropanol; and / or, based on the benzylphenylephrine described in step S3, the amount of Pd / C described in step S3 is 1%-10%; and / or, the temperature of the reaction in step S3 is 0-80°C; And / or, the alcoholic solution of hydrogen chloride described in step S3 includes: one or more of: methanolic hydrogen chloride solution, ethanolic hydrogen chloride solution, and isopropanol hydrogen chloride solution.

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