An omega-transaminase mutant and use thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a kind of omega-transaminase mutant and its application.The present application discloses a kind of omega-transaminase mutant and its application in the synthesis of silodosin intermediate, the omega-transaminase mutant is by from Arthrobacter, through homologous modeling, reaction intermediate molecular docking, active site amino acid analysis, homologous sequence analysis, virtual mutation analysis combined with site-directed mutation verification, the single-point mutation of 62th and 223th obtains H62A, S223A mutant.The omega-transaminase mutant of the present application can one-pot catalysis 60mM silodosin intermediate ketone is converted into silodosin intermediate, conversion rate is greater than 92%, compared with wild type, the specific activity of H62A mutant to silodosin intermediate ketone is 1.4 times of wild type, S223A is 1.24 times of wild type, and the optimum reaction temperature is 30 DEG C, and the ee value of product is greater than 99.29%.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an ω-transaminase mutant and its applications. Background Technology

[0002] Biocatalysis, characterized by mild reaction conditions, high efficiency, strong stereo and regio selectivity, and environmental friendliness, has found wide application in the research and development of new drugs. Furthermore, the modification and optimization of biocatalysts through rational design and directed evolution, such as improving catalyst stability and substrate selectivity, will enable their better application in production processes. Transaminases, as biocatalysts, are primarily used in the synthesis of chiral amines.

[0003] Sildosin, chemically named 2,3-dihydro-1-(3-hydroxypropyl)-5-[(2R)-2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylamino]propyl]-1H-indole-7-carboxamide, is an α1A receptor antagonist developed by Kissei Pharmaceutical Co., Ltd. of Japan. It was first launched in Japan in May 2006 and officially launched in the United States in October 2008 after FDA approval, under the brand name Urief. Sildosin is clinically used to treat symptoms associated with benign prostatic hyperplasia (BPH) or enlargement. Its structural formula is as follows:

[0004]

[0005] Currently, there are many methods for preparing optically pure silodosine intermediates, which can be summarized into three categories:

[0006] The first method, as described in patents JP2002265444, WO2013056842, JP2002265444, and CN101759627, involves obtaining chiral intermediates of silodosine through a separation method. However, this method has low yields and high material losses. The second method, as described in patents JP2001199956, CN109305932, and CN101993406A, uses expensive chiral excipients, resulting in intermediates with low optical purity. The method has several drawbacks, including low purity. Thirdly, patents WO2016139773, WO2011030356, CN106045895A, CN103420893A, CN103554003A, KR20150066777, and CN106045895 describe methods for constructing chiral centers by docking with chiral compounds. The optical purity of the intermediates obtained by this method directly derives from the raw materials, but it requires multiple reaction steps and has a low yield. Summary of the Invention

[0007] The purpose of this invention is to provide a highly active enzyme for the asymmetric synthesis of silodoxine intermediates. It offers advantages such as reduced synthetic steps for chiral intermediates, high optical purity, low risk and cost, and environmental friendliness. The raw materials are inexpensive, the operation is simple, and it is more suitable for large-scale industrial production.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The present invention provides an ω-transaminase mutant, which is obtained by single or multiple mutations at position 62 or 223 of the amino acid sequence.

[0010] Preferably, the ω-transaminase mutant is mutated by changing histidine at position 62 to alanine and serine at position 223 to alanine.

[0011] The present invention also provides an expression vector containing the gene encoding the ω-transaminase mutant.

[0012] The present invention also provides recombinant genetically engineered bacteria containing the expression vector.

[0013] The present invention also provides the application of the ω-transaminase mutant in the asymmetric synthesis of silodoxin intermediates from silodoxin intermediate ketones.

[0014] Preferably, the method for synthesizing the silodosine intermediate includes the following steps:

[0015] Step 1: Preparation of recombinant ω-transaminase mutant genetically engineered bacteria

[0016] Point mutations were performed at positions 62 and / or 223, respectively, and the cells were transformed into Escherichia coli DH5α competent cells. Positive transformants were selected and sequenced to identify the recombinant expression vector. The recombinant expression vector was then transformed into Escherichia coli BL21(DE3) strain to obtain a genetically engineered bacterium that can induce the expression of a recombinant ω-transaminase mutant.

[0017] Step 2: Preparation of recombinant ω-transaminase mutants

[0018] The recombinant ω-transaminase mutant genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 12 h to obtain a seed culture. The seed culture was then inoculated into TB liquid medium containing kanamycin resistance at a volume of 1% of the TB liquid medium containing kanamycin resistance. The culture was then incubated at 37°C until the OD600 value reached 2.0. Isopropyl-β-D-galactoside (0.2% concentration) or D-lactose (0.5% concentration) was added, and the culture was continued at 25-28°C for 14-24 h. The bacterial cells were then collected by centrifugation. The collected bacterial cells were washed once with phosphate buffer and resuspended to obtain a cell suspension for later use. The suspension was then sonicated in an ice bath and centrifuged again. The resulting supernatant was the recombinant ω-transaminase, and its protein content was measured for later use.

[0019] Step 3: Preparation of Sildosin Intermediate

[0020] A reaction system was constructed by sequentially adding substrate (final concentration 20–60 mmol / L), cosolvent (final volume percentage concentration 5–50%), cofactor (final concentration 0.1–5 mmol / L), amino donor (final concentration 0.05–0.5 mol / L), and recombinant ω-transaminase mutant cells or recombinant ω-transaminase mutants (final volume percentage concentration 5–25%) to the reaction solution. The reaction system was then reacted at 25–35 °C for 24–26 h. After the reaction, the content of silodoxine intermediate in the reaction solution was measured. The reaction solution was then centrifuged and filtered through a membrane, separated using a semi-preparative liquid phase, and the corresponding components were collected. The mobile phase was removed by rotary evaporation, and the solution was dried to obtain the silodoxine intermediate.

[0021] The substrate is silodoxine intermediate ketone; the reaction solution is a 50-100 mmol / L phosphate buffer with a pH of 6.0-9.0, a glycine-sodium hydroxide buffer with a pH of 6.0-9.0, or a 50-100 mmol / L sodium barbital-hydrochloric acid buffer with a pH of 6.0-9.0; the cosolvent is dimethyl sulfoxide, N,N-dimethylformamide, or methyl tert-butyl ether; the cofactor is pyridoxal phosphate; and the amino donor is R-methylbenzylamine, isopropylamine, or D-alanine.

[0022] Preferably, the specific method of ultrasonic disruption in step two is as follows: the suspension is placed in an ice bath and disrupted by ultrasonic waves (power of 150-200w), with parameters of 2s operation, 3s interval, and 140-160 cycles.

[0023] Preferably, the method for detecting the protein content in the supernatant in step two is the Bradford method.

[0024] Preferably, in step three, the reaction process of the reaction system is sampled and the conversion rate of the silodosin intermediate in the sample is determined qualitatively and quantitatively by thin-layer chromatography and high-performance liquid chromatography in order to monitor the reaction process.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention provides a highly active ω-transaminase using silodosin intermediate ketone as a raw material, which is inexpensive. After the reaction, the generated acetophenone is easily removed and does not interfere with the separation and purification of the product. Only the silodosin intermediate remains in the aqueous phase system, making it easy to separate and helping to improve product purity and yield while reducing separation costs. The required ω-transaminase can be prepared in large quantities by constructing E. coli genetically engineered bacteria and then fermenting it, which is relatively readily available and inexpensive. The reaction is a "one-pot" process, where the substrate and all enzymes are added simultaneously to start the reaction, directly yielding the final product, silodosin intermediate. This process has low cost, a conversion rate of over 92%, and a product yield of over 85%, without the influence of byproducts. This process method is suitable for industrial applications. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the reaction principle for the asymmetric synthesis of the silodoxine intermediate of this invention.

[0029] Figure 2 It is the silodosine structure. Detailed Implementation

[0030] The present invention provides the following technical solution: an ω-transaminase mutant and its application in the synthesis of silodoxine intermediates.

[0031] This invention provides an ω-transaminase mutant, which is obtained by single or multiple mutations at position 62 or 223 of Arthrobacterium. The amino acid sequence of the mutant is shown in SEQ ID NO.1 and SEQ ID NO.2; the original sequence is shown in SEQ ID NO.3.

[0032] Preferably, the transaminase mutant is an amino acid sequence mutation of one of the following: (1) histidine at position 62 is mutated to alanine H62A; (2) tryptophan at position 223 is mutated to alanine S223A. Due to the special nature of amino acid sequences, any fragment of the polypeptide or its variants, such as its conserved variants, bioactive fragments, or derivatives, as long as the fragment or variant of the polypeptide has more than 95% homology with the aforementioned amino acid sequence, falls within the scope of protection of this invention. The changes may include the deletion, insertion, or substitution of amino acids in the amino acid sequence; for conserved changes in variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved changes, such as replacing alanine with tryptophan.

[0033] The present invention also relates to an expression vector containing the ω-transaminase mutant encoding gene, and a recombinant genetically engineered bacterium containing the expression vector, wherein the expression vector is based on plasmid pET28a(+) or a vector capable of expressing the enzyme; the recombinant genetically engineered bacterium is obtained by transforming a recombinant plasmid into a host bacterial cell, wherein the host bacterial cell includes E. coli BL21(DE3) cells or a host cell capable of expressing the enzyme.

[0034] The present invention also provides the application of the ω-transaminase mutant in the asymmetric synthesis of silodoxin intermediates from silodoxin intermediate ketones.

[0035] In this invention, the method for synthesizing the silodosine intermediate includes the following steps:

[0036] 1. Design of mutant primers

[0037] To obtain a highly active ω-transaminase for the synthesis of silodoxine intermediates, the original enzyme was modeled using EasyModeller 4.0, and the intermediates were molecularly docked using AutoDock. It was found that amino acids at positions 62 and 223 exhibited significant steric hindrance, severely hindering the substrate from reaching the catalytic site. H62A and S223A were found to reduce this steric hindrance, and site-directed mutagenesis was performed using SnapGene (Table 1, underlined bases are mutants).

[0038] Table 1. Mutation sites and primer sequences

[0039] H62A-f <![CDATA[5’-GATCAAGGCTATCTG GCA AGCGATGTGA-3’]]> H62A-r <![CDATA[5’- TGC CAGATAGCCTTGATCAAAAATGCTAATGC-3’]]> S223A-f <![CDATA[5’-CTGCTGGCGGAAGGC GCA GGCTTTAACGT-3’]]> S223A-r <![CDATA[5’- TGC GCCTTCCGCCAGCAGGCCATCGCCATCCAGTAA-3’]]>

[0040] As shown in SEQ ID NO.4~7.

[0041] 2. Recombinant genetically engineered mutant strains

[0042] Using the primers in Table 1 (synthesized from Qingke Biotechnology, Nanjing, China), with the original enzyme as a template, and employing the Fast site-directed mutagenesis kit (purchased from TransGen, Beijing, China), site-directed mutagenesis was performed using PCR technology to obtain H62A and S223A mutants. The PCR reaction system (total volume 50 μL) is as follows: 25 μL of 10× DNA Polymerase Buffer, 2 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP), 1 μL each of forward and reverse primers (50 μM each), 1 μL of template DNA, 2 μL of DNA Polymerase, and 18 μL of ddH2O.

[0043] A BioRad PCR instrument was used. The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 6 min, for a total of 30 cycles, and a final extension at 72℃ for 10 min. The PCR products were then digested with DMT enzyme at 37℃ for 1 hour to completely consume the template.

[0044] Take 5 μL of PCR product and add it to 50 μL of DMT competent cell suspension in an ice bath. Incubate on ice for 30 min. Heat shock the transformation product at 42℃ for 120 s, then quickly cool it on ice for 2 min. Add 500 μL of LB liquid medium to the tube and incubate at 37℃ and 200 rpm for 60 min. Take 100 μL of the above bacterial solution and spread it evenly on LB solid medium containing a final concentration of 50 μg / mL kanamycin. After the bacterial solution is completely absorbed by the medium, incubate upside down at 37℃ for 12 h. After picking a single colony to verify it as a positive clone, use a plasmid miniprep kit (purchased from TransGen, Beijing, China) to extract the plasmid and sequence it for verification (Qingke Biotechnology, Nanjing, China). Take 5 μL of plasmid and add it to 50 μL of BL21(DE3) competent cell suspension in an ice bath. Incubate on ice for 30 min. Heat shock the transformation product at 42℃ for 120 s, then quickly cool on ice for 2 min. Add 500 μL of LB liquid medium to the tube and incubate at 37℃ and 200 rpm for 60 min. Take 100 μL of the above bacterial solution and spread it evenly on LB solid medium containing a final concentration of 50 μg / mL kanamycin. After the bacterial solution is completely absorbed by the medium, incubate upside down at 37℃ for 12 h. After picking a single colony to verify a positive clone, inoculate it into LB test tube medium containing 50 μg / mL kanamycin and incubate at 37℃ and 200 rpm for 12 h to obtain recombinant genetically engineered mutant strains H62A and S223A.

[0045] 3. Preparation of recombinant ω-transaminase mutants

[0046] The recombinant ω-transaminase mutant genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 12 h to obtain a seed culture. The seed culture was then inoculated into TB liquid medium containing kanamycin resistance at a volume of 1% of the TB liquid medium containing kanamycin resistance. The culture was then incubated at 37°C until the OD600 value reached 1.8-2.2. Isopropyl-β-D-galactoside or D-lactose at a final concentration of 0.2% or 0.5% was added, and the culture was continued at 25-28°C for 14-24 h. The bacterial cells were then collected by centrifugation. The collected bacterial cells were washed once with phosphate buffer and resuspended. The suspension was then sonicated in an ice bath and centrifuged again. The resulting supernatant was the recombinant ω-transaminase mutant. The protein content was measured and used for later use.

[0047] The specific method of ultrasonic disruption is as follows: the suspension is placed in an ice bath and ultrasonic disruption is performed with parameters of 2 seconds of operation, 3 seconds of interval, and 150 cycles.

[0048] 4. Mutant enzyme activity assay:

[0049] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of the product silodoxine intermediate per hour at 30°C.

[0050] Assay method: 5 mL reaction system, 40 mM substrate (10.3 g / L), 50 mM amino donor R-methylbenzylamine, 10% co-solvent DMSO or DMF, 5% cell addition, 1 mM PLP dissolved in 100 mM, pH 7.0 phosphate buffer. React at 30 °C, 180 rpm for 1 h, and detect product formation using liquid chromatography.

[0051] Chromatographic conditions: The liquid chromatography column was an Alphasil VC-C18 column (4.6 mm × 25 cm) from Dalian Huapu Technology Co., Ltd.; column temperature was 30℃; detection wavelength was 254 nm; flow rate was 1.0 ml / min; injection volume was 20 μL; mobile phase: A: 100% acetonitrile; B: pure water (+0.1% trifluoroacetic acid).

[0052] Table 2. Liquid Chromatography Conditions

[0053]

[0054] By combining the standard curve of the product standard, the product yield is calculated, and the ee value is determined using a chiral column.

[0055] Table 3. Mutant enzyme activity and selectivity

[0056]

[0057] 5. Preparation of Sildosin Intermediates

[0058] A reaction system was constructed by sequentially adding substrate (final concentration 20–60 mmol / L), cosolvent (volume concentration 5–50%), cofactor (final concentration 0.1–5 mmol / L), amino donor (final concentration 0.05–0.5 mol / L), and recombinant ω-transaminase genetically engineered bacterial cell suspension or recombinant ω-transaminase (final volume percentage concentration 5–25%) to the reaction solution. The reaction system was then reacted at 25–30 °C for 24–26 h. After the reaction, the content of silodoxin intermediate in the reaction solution was measured. The reaction solution was then centrifuged and filtered through a membrane, separated using a semi-preparative liquid phase, and the product was collected. The mobile phase was removed by rotary evaporation, and the product was dried to obtain the silodoxin intermediate.

[0059] Samples were taken and the optical purity (ee value) and conversion rate of the silodosine intermediate were determined by high performance liquid chromatography to monitor the reaction process.

[0060] The substrate is silodoxine intermediate ketone; the reaction solution is a 50-100 mmol / L phosphate buffer with a pH of 6.0-9.0, a glycine-sodium hydroxide buffer with a pH of 6.0-9.0, or a 50-100 mmol / L sodium barbital-hydrochloric acid buffer with a pH of 6.0-9.0; the cosolvent is dimethyl sulfoxide, N,N-dimethylformamide, or methyl tert-butyl ether; the cofactor is pyridoxal phosphate; and the amino donor is R-methylbenzylamine, isopropylamine, or D-alanine.

[0061] In this invention, the specific method of ultrasonic fragmentation is as follows: the ultrasonic power is preferably 175W, the operation time is 2s, the interval is 3s, and the number of cycles is 150.

[0062] In this invention, the substrate is silodosine intermediate ketone;

[0063] In this invention, the reaction solution is preferably a 50-100 mmol / L phosphate buffer with a pH of 6.0-9.0; more preferably a 75 mmol / L phosphate buffer with a pH of 7.5.

[0064] In this invention, the cosolvent is preferably dimethyl sulfoxide.

[0065] In this invention, the amino donor is preferably R-methylbenzylamine.

[0066] In this invention, the final concentration of the substrate is preferably 30-50 mmol / L; more preferably 40 mmol / L.

[0067] In this invention, the final volume percentage concentration of the co-solvent is preferably 10-40%; more preferably 20-30%; and even more preferably 25%.

[0068] In this invention, the final concentration of the cofactor is preferably 1 to 4 mmol / L; more preferably 2 to 3 mmol / L; and even more preferably 3 mmol / L.

[0069] In this invention, the final concentration of the amino donor is preferably 0.1 to 0.4 mol / L; more preferably 0.2 to 0.3 mol / L; and even more preferably 0.3 mol / L.

[0070] In this invention, the final volume percentage concentration of the recombinant ω-transaminase is 5-25%; more preferably; even more preferably.

[0071] In this invention, the reaction temperature is preferably 27–33°C; more preferably 29–31°C; and even more preferably 30°C.

[0072] In this invention, the reaction time is preferably 25 hours.

[0073] In this invention, the volume percentage concentration of the final recombinant ω-transaminase genetically engineered bacterial cell suspension or recombinant ω-transaminase is preferably 5-15%; more preferably 10%.

[0074] In this invention, Figure 1 R is a hydrogen or other hydroxyl protecting group, such as: p-nitrobenzoate, benzoate, benzyl ether, MOM, TBDPS, tert-butyl ester.

[0075] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0076] The reagents described in this invention are all commonly used reagents and can be purchased from the market; the Escherichia coli BL21(DE3) is a commonly used bacterial strain and can be purchased from the market.

[0077] The composition of the LB liquid culture medium is as follows: by weight percentage, the LB liquid culture medium contains 1% tryptone, 0.5% yeast extract, 1% sodium chloride, and the balance is water.

[0078] The composition of the TB culture medium is as follows: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 g / L, potassium dihydrogen phosphate 17 mmol / L, dipotassium hydrogen phosphate 72 mmol / L, and the remainder is water.

[0079] Example 1

[0080] An asymmetric synthesis method for silodosine intermediates, comprising the following steps:

[0081] Step 1: Preparation of recombinant ω-transaminase mutant S223A

[0082] The recombinant ω-transaminase mutant S223A genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 12 h to obtain a seed culture. The seed culture was then inoculated into TB liquid medium containing kanamycin resistance at a volume of 1% of the TB liquid medium containing kanamycin resistance. The culture was then incubated at 37°C until the OD600 value reached 2.0. Isopropyl-β-D-galactoside or D-lactose at a final concentration of 0.2% or 0.5% was added, and the culture was continued at 25°C for 16 h. The bacterial cells were then collected by centrifugation. The collected bacterial cells were washed once with phosphate buffer and resuspended. The suspension was then sonicated in an ice bath and centrifuged again. The resulting supernatant was the recombinant ω-transaminase S223A mutant. The protein content was measured and used for later use.

[0083] The specific method of ultrasonic disruption is as follows: the suspension is placed in an ice bath and ultrasonic disruption is performed with parameters of 2 seconds of operation, 3 seconds of interval, and 150 cycles.

[0084] Step 2: Preparation of Sildosin Intermediate

[0085] A reaction system was constructed by sequentially adding substrate (final concentration 20 mmol / L), cosolvent (final volume percentage concentration 10%), cofactor (final concentration 1 mmol / L), amino donor (final concentration 0.5 mol / L), and recombinant ω-transaminase S223A (final volume percentage concentration 10%) to the reaction solution. The reaction system was then reacted at 30°C for 24 h. During the reaction process, samples were taken and analyzed by high-performance liquid chromatography (HPLC) to determine the optical purity (ee value) of the silodoxine intermediate, which was 99% and the conversion rate was 94%. The reaction solution was then centrifuged and filtered through a membrane, separated using a semi-preparative HPLC system, and the corresponding components were collected. The mobile phase was removed by rotary evaporation, and the solution was dried to obtain the silodoxine intermediate with a yield of 89%.

[0086] The substrate is silodoxine intermediate ketone; the solution is a 100 mmol / L sodium barbital-hydrochloric acid buffer solution with a pH of 8.0; the solubilizer is methyl tert-butyl ether; the cofactor is pyridoxal phosphate; and the amino donor is D-alanine.

[0087] Example 2

[0088] A method for asymmetric synthesis of silodosine, comprising the following steps:

[0089] Step 1: Preparation of recombinant ω-transaminase mutant H62A

[0090] The recombinant ω-transaminase mutant genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 16 h to obtain a seed culture. The seed culture was then inoculated into TB liquid medium containing kanamycin resistance at a volume of 1% of the TB liquid medium containing kanamycin resistance. The culture was then incubated at 37°C until the OD600 value reached 2.0. Isopropyl-β-D-galactoside or D-lactose at a final concentration of 0.01 mmol / L or 0.5% by mass was added, and the culture was continued at 28°C for 24 h. The bacterial cells were then collected by centrifugation. The collected bacterial cells were washed once with phosphate buffer and resuspended. The suspension was ultrasonically disrupted in an ice bath and then centrifuged again. The resulting supernatant was the recombinant ω-transaminase H62A. The protein content was measured and used for later use.

[0091] The specific method of ultrasonic disruption is as follows: the suspension is placed in an ice bath and ultrasonic disruption is performed with parameters of 2 seconds of operation, 3 seconds of interval, and 150 cycles.

[0092] Step 2: Preparation of Sildosin Intermediate

[0093] A reaction system was constructed by sequentially adding substrate (final concentration 60 mmol / L), cosolvent (final mass percentage concentration 20%), cofactor (final concentration 1 mmol / L), amino donor (final concentration 0.1 mol / L), and recombinant ω-transaminase H62A (final volume percentage concentration 20%) to the reaction solution. The reaction system was then reacted at 35 °C for 26 h. During the reaction process, samples were taken and analyzed using high-performance liquid chromatography (HPLC). The optical purity (ee value) of the silodoxine intermediate was 99%, and the conversion rate was 95%. The reaction solution was then centrifuged and filtered through a membrane. Semi-preparative HPLC was used for separation, and the corresponding components were collected. The mobile phase was removed by rotary evaporation, and the solution was dried to obtain the silodoxine intermediate with a yield of 87%.

[0094] The substrate is silodoxine intermediate ketone; the reaction solution is 80 mmol / L glycine sodium hydroxide buffer solution with pH 7.0; the cosolvent is dimethyl sulfoxide; the cofactor is pyridoxal phosphate; and the amino donor is R-methylbenzylamine.

[0095] Example 3

[0096] A biological preparation method for silodosine intermediate, comprising the following steps:

[0097] Step 1: Preparation of recombinant ω-transaminase mutant H62A

[0098] The recombinant ω-transaminase mutant H62A genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 16 h to obtain a seed culture. The seed culture was then inoculated into TB liquid medium containing kanamycin resistance at a volume of 1% of the TB liquid medium containing kanamycin resistance. The culture was then incubated at 37°C until the OD600 value reached 2.0. Isopropyl-β-D-galactoside or D-lactose at a final concentration of 0.2% or 0.5% was added, and the culture was continued at 26°C for 20 h. The bacterial cells were then collected by centrifugation. The collected bacterial cells were washed once with phosphate buffer and resuspended to obtain the mutant H62A cell suspension for later use.

[0099] Step 2: Preparation of Sildodoxine intermediate amine

[0100] A reaction system was constructed by sequentially adding substrate (final concentration 40 mmol / L), cosolvent (final mass percentage concentration 10%), cofactor (final concentration 5.0 mmol / L), amino donor (final concentration 0.5 mol / L), and recombinant ω-transaminase mutant H62A cell suspension (final volume percentage concentration 25%) to the reaction solution. The reaction system was then incubated at 30°C for 26 h. During the reaction process, samples were taken and analyzed using high-performance liquid chromatography (HPLC) to determine the optical purity (ee value) of the silodoxine intermediate, which was 99% and the conversion rate was 92%. The reaction solution was then centrifuged and filtered through a membrane, separated using a semi-preparative HPLC system, and the corresponding components were collected. The mobile phase was removed by rotary evaporation, and the solution was dried to obtain the silodoxine intermediate with a yield of 85%.

[0101] The substrate is silodoxine intermediate ketone; the reaction solution is 100 mmol / L phosphate buffer at pH 7.0; the cosolvent is N,N-dimethylformamide; the cofactor is pyridoxal phosphate; and the amino donor is isopropylamine.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An omega-transaminase mutant, characterized in that, The amino acid sequence of the omega-transaminase mutant is the amino acid sequence obtained by mutating the histidine at position 62 or the serine at position 223 of the amino acid sequence shown in SEQ ID NO. 3 to alanine.

2. An expression vector containing the coding gene of the omega-transaminase mutant of claim 1.

3. A recombinant genetically engineered bacterium containing the expression vector of claim 2.

4. Use of the omega-transaminase mutant of claim 1 in catalyzing the asymmetric synthesis of silodosin intermediate ketone, which comprises sequentially adding silodosin intermediate ketone as shown in formula 1, a cosolvent, a cofactor, an amino donor and the omega-transaminase mutant of claim 1 into a reaction solution to form a reaction system, and then allowing the reaction to proceed to obtain silodosin intermediate as shown in formula 2. Formula 1 Formula 2.

5. Use according to claim 4, characterized in that: The synthesis method of the silodosin intermediate comprises the following steps: (1) constructing a genetically engineered bacterium expressing the omega-transaminase mutant of claim 1; (2) after the genetically engineered bacterium obtained in step (1) is cultured, the bacterial cells are collected by centrifugation, washed once and then resuspended to obtain a cell suspension; the cell suspension is subjected to ultrasonic disruption in an ice bath and then centrifuged, and the obtained supernatant is the recombinant omega-transaminase mutant; (3) a reaction system is formed by sequentially adding silodosin intermediate ketone as shown in formula 1, a cosolvent, a cofactor, an amino donor and the recombinant omega-transaminase mutant obtained in step (2) into a reaction solution; after the reaction is completed, the reaction solution is centrifuged and membrane-filtered, semi-preparative liquid chromatography is used for separation, the mobile phase is removed by rotary evaporation, and drying is performed to obtain silodosin intermediate as shown in formula 2. Formula 1 Formula 2.

6. The use according to claim 5, characterized in that: In step (2), the ultrasonic disruption is performed at a power of 150-200 W, with a working time of 2 s, an intermittent time of 3 s, and a cycle number of 140-160.

7. The use according to claim 5, characterized in that: In step (3), the reaction solution is a 50-100 mmol / L phosphate buffer having a pH value of 6.0-9.0, a glycine-sodium hydroxide buffer having a pH value of 6.0-9.0, or a 50-100 mmol / L barbital sodium-hydrochloric acid buffer having a pH value of 6.0-9.

0. The cosolvent is dimethyl sulfoxide, N,N-dimethylformamide or methyl tert-butyl ether; the cofactor is pyridoxal phosphate; and the amino donor is R-methylbenzylamine, isopropylamine or D-alanine.

8. Use according to claim 7, wherein: In step (3), the final concentration of silodosin intermediate ketone is 20-60 mmol / L, the final volume percentage concentration of the cosolvent is 5-50%, the final concentration of the cofactor is 0.1-5 mmol / L, the final concentration of the amino donor is 0.05-0.5 mol / L, and the final volume percentage concentration of the recombinant omega-transaminase mutant is 5-25%.

9. The use according to claim 5, characterized in that: In step (3), the reaction temperature is 25-35℃, and the reaction time is 24-26 h.

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