Omega-transaminase mutants, methods for making and using same

Abstract

This invention discloses an ω-transaminase mutant, its preparation method, and its applications, belonging to the field of biocatalytic organic synthesis technology. Compared with the wild-type ω-transaminase, this ω-transaminase mutant has at least two substitution mutations at the following amino acid sites: positions 57, 86, 153, 180, and 259. The amino acid sequence of the wild-type ω-transaminase is shown in SEQ ID NO:1. The synthesis of (S)-6-methylnicotine using this ω-transaminase mutant exhibits advantages such as high yield, good stereoselectivity (ee>99.5%), and high catalytic efficiency, making it suitable for large-scale production. This provides an efficient preparation route for the supply of (S)-6-methylnicotine and its application in related products.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic organic synthesis technology, and in particular to an ω-transaminase mutant, its preparation method, and its application. Background Technology

[0002] Nicotine and its analogues are important alkaloids with significant value in neuroscience research, drug development (such as smoking cessation therapy), and pesticides. Among them, (S)-6-methylnicotine has attracted attention due to its remarkable pharmacological activity and receptor selectivity. Traditional chemical synthesis of (S)-6-methylnicotine mainly relies on chiral resolution or asymmetric synthesis using stoichiometric chiral auxiliaries. These methods suffer from drawbacks such as lengthy steps, poor atom economy, use of expensive or toxic metal catalysts, generation of large amounts of waste, and unsatisfactory optical purity (ee value).

[0003] Furthermore, biocatalysis is evolving from a traditional chiral synthetic tool into a disruptive synthetic platform, combining the precision of biological systems with the efficiency requirements of industrial production. Its advantages and prospects are significant, making it an important development direction for green chemistry and sustainable manufacturing. There are a few patent reports on the synthesis of (S)-6-methylnicotine using biocatalysis. For example, CN1188552102A provides a method for preparing (S)-demethyl-6-methylnicotine from 6-methylmaxmine using imine reductase catalysis, and then further preparing (S)-6-methylnicotine from (S)-demethyl-6-methylnicotine via methylation. CN118853791A also provides a method for preparing (S)-6-methylnicotine using an aminoketone as a substrate and imine reductase as a catalyst.

[0004] However, the use of imine reductase as a catalyst for the synthesis of chiral amines has problems such as a narrow substrate spectrum, complex coenzyme system, and poor substrate stability. In addition, enzyme resources are limited and often require directed evolution and modification, which limits its industrial applicability.

[0005] Meanwhile, ω-transaminase-catalyzed asymmetric amination reactions provide a green, efficient, and highly selective route for the synthesis of chiral amines. However, currently, no patent reports exist regarding the synthesis of (S)-6-methylnicotine catalyzed by ω-transaminase. Furthermore, natural ω-transaminases typically exhibit extremely low or even no catalytic activity in asymmetric amination reactions, failing to meet the synthetic requirements.

[0006] Therefore, developing novel ω-transaminases capable of efficiently catalyzing the synthesis of (S)-6-methylnicotine is key to achieving its biosynthesis. Summary of the Invention

[0007] To address the aforementioned problem of low catalytic activity of current ω-transaminases, this invention provides an ω-transaminase mutant. Using this ω-transaminase mutant to catalyze the synthesis of (S)-6-methylnicotine offers advantages such as high yield, good stereoselectivity (ee>99.5%), and high catalytic efficiency, making it suitable for large-scale production. This provides an efficient preparation route for the supply of (S)-6-methylnicotine and its application in related products.

[0008] The present invention provides an ω-transaminase mutant, which, compared with wild-type ω-transaminase, has at least two substitution mutations at the following amino acid sites: position 57, position 86, position 153, position 180, and position 259. The amino acid sequence of the wild-type ω-transaminase is shown in SEQ ID NO: 1.

[0009] In some of these embodiments, the substitution mutation at position 57 is A57G / L / A, the substitution mutation at position 86 is N86A / Y, the substitution mutation at position 153 is Y153A / S, the substitution mutation at position 180 is M180A / G, and the substitution mutation at position 259 is D259A / V / I.

[0010] In some embodiments, the ω-transaminase mutant includes the following substitution mutations: A57G and Y153A.

[0011] In some embodiments, the ω-transaminase mutant includes the following substitution mutations: A57G, Y153A, and D259A; or includes the following substitution mutations: A57G, Y153A, M180G, and D259A; or includes the following substitution mutations: A57G, N86Y, Y153A, M180G, and D259A.

[0012] In some embodiments, the ω-transaminase mutant has the following substitution mutations: A57G, N86Y, Y153A, M180G, and L259A.

[0013] On the other hand, the present invention also discloses a method for preparing the above-mentioned ω-transaminase mutant, comprising the following steps: Construction and transformation of recombinant vector: Obtain the amino acid sequence of the above-mentioned ω-transaminase mutant, construct a plasmid containing the gene encoding the ω-transaminase mutant, introduce it into host cells, and culture it; Construction and induction of expression vector: Host cells containing the ω-transaminase mutant gene plasmid were induced to express the expression in 2YT medium containing kanamycin resistance.

[0014] In some embodiments, the plasmid is pET-28a; and / or the host cell is Escherichia coli BL21(DE3).

[0015] In some embodiments, the 2YT culture medium contains: 16 g / L yeast extract, 5 g / L sodium chloride, and 10 g / L peptone.

[0016] In some embodiments, the preparation method further includes a purification step, wherein the purification step is as follows: cells containing ω-transaminase mutant are lysed and centrifuged, the supernatant is purified with resin, ultrafiltered, and the filtrate is discarded to obtain the product; preferably, the resin is Ni-NTA resin, and / or the molecular weight cutoff of the ultrafiltration is 10 kDa.

[0017] The present invention also discloses a polynucleotide that encodes the above-mentioned ω-transaminase mutant.

[0018] The present invention also discloses a recombinant expression vector comprising the polynucleotides described above.

[0019] The present invention also discloses a recombinant engineered cell, characterized in that the recombinant engineered cell contains the aforementioned polynucleotides.

[0020] This invention also discloses the application of ω-transaminase as a catalyst in asymmetric amination reaction synthesis.

[0021] In some embodiments, the ω-transaminase is selected from one or more of the ω-transaminases with amino acid sequences as shown in SEQ ID NO: 1 and the ω-transaminase mutants described above.

[0022] In some of these embodiments, the ω-transaminase is selected from one or more of the ω-transaminase mutants.

[0023] This invention also discloses an asymmetric amination reaction, comprising the following steps: Compound II, in the presence of an amino donor and a coenzyme, was contacted with the aforementioned ω-transaminase mutant to yield compound IV:

[0024] in: X is selected from: chlorine, bromine, iodine, methanesulfonic acid group, benzenesulfonic acid group, p-toluenesulfonic acid group, or p-nitrobenzenesulfonic acid group; The amino donor is selected from: primary amines; The coenzyme is selected from pyridoxal 5'-phosphate.

[0025] In some of these implementations, X is chlorine.

[0026] In some embodiments, the amino donor is selected from isopropylamine, propylamine, phenylethylamine; preferably isopropylamine or propylamine; more preferably isopropylamine.

[0027] In some of these embodiments, the coenzyme is selected from 5'-pyridoxal phosphate monohydrate.

[0028] In some embodiments, the reaction is carried out in a buffer salt solution with a pH of 7.0 to 9.5, preferably with a pH of 8.0 to 9.0, more preferably with a pH of 8.5 to 9.0.

[0029] In some embodiments, the reaction is carried out in a buffered salt solution selected from phosphate buffer, Tris-HCl buffer, glycine-NaOH buffer, and borate buffer; preferably, the buffered salt solution is selected from borate buffer or Tris-HCl buffer; more preferably, Tris-HCl buffer.

[0030] In some of these embodiments, the reaction temperature is 35 ± 10 °C, preferably 35 °C.

[0031] In some embodiments, the mass ratio of compound II to the ω-transaminase mutant is 5-10:1, preferably 6.25-7.68:1, and more preferably 6.25:1.

[0032] In some embodiments, the concentration of the amino donor in the reaction system is 1.0-5.0 M, preferably 1.3-1.7 M, for example 1.37, 1.7 M or 2.0 M.

[0033] In some embodiments, the mass ratio of compound II to the coenzyme is 50-100:1, preferably 40-98:1, and more preferably 80:1.

[0034] In some embodiments, compound II generates intermediate compound III, and compound III cyclizes to generate compound IV: .

[0035] In some of these embodiments, compound IV has an optical purity of ≥99%.

[0036] On the other hand, the present invention also discloses a method for synthesizing (S)-6-methylnicotine, which is synthesized according to the following route:

[0037] in: R is selected from: C1-C4 alkyl, preferably methyl, ethyl, isopropyl or tert-butyl, more preferably ethyl; X is selected from: chlorine, bromine, iodine, methanesulfonic acid group, benzenesulfonic acid group, p-toluenesulfonic acid group or p-nitrobenzenesulfonic acid group, preferably chlorine; Step S1: Under alkaline conditions, 6-methylnicotinic acid ester reacts with γ-butyrolactone ester by condensation to give compound I; Step S2: Compound I is heated under acidic conditions, which causes ring-opening to form compound II; Step S3: Proceed according to the asymmetric amination reaction steps described above; Step S4: Compound IV undergoes the Eschweiler-Clarke reaction under acidic conditions to produce (S)-6-methylnicotine.

[0038] In some embodiments, in step S1, the alkaline condition is the presence of sodium ethoxide, preferably, the concentration of sodium ethoxide in the reaction system is 1-1.1M.

[0039] In some embodiments, in step S1, the reaction is carried out in a solvent, preferably toluene.

[0040] In some embodiments, in step S1, the reaction is carried out under the protection of an inert gas, preferably nitrogen.

[0041] In some of these implementations, the reaction temperature in step S1 is 80-85°C.

[0042] In some embodiments, in step S1, the molar ratio of 6-methylnicotinic acid ester to γ-butyrolactone is 1:1.2.

[0043] In some embodiments, in step S2, the acidic condition is the presence of concentrated hydrochloric acid or hydrobromic acid, preferably, the reaction equivalent of the concentrated hydrochloric acid or hydrobromic acid is 10-12 eq of compound I.

[0044] In some embodiments, the reaction in step S2 is carried out at 110±10°C.

[0045] In some of these embodiments, the molar ratio of compound IV to formaldehyde is selected to be 1:3-4.

[0046] In some embodiments, in step S4, the acidic condition is the presence of formic acid, preferably, the reaction equivalent of the formic acid is 4-6 eq of compound IV.

[0047] In some implementations, the reaction in step S4 is carried out at 40-45°C.

[0048] definition: The aforementioned "substitution mutation" refers to a mutation in which an amino acid residue at a specific position in an amino acid sequence is replaced by another different amino acid residue, while the total length of the sequence remains unchanged. For example, N86A / Y indicates that the asparagine (Asn, N) at position 86 has undergone a substitution mutation, changing to Ala alanine or Tyr tyrosine. In other words, the symbol " / " represents "or".

[0049] The aforementioned "recombinant expression vector" refers to a DNA molecule constructed by inserting the target gene (i.e., the aforementioned polynucleotide) into a vector DNA molecule using DNA recombination technology. This molecule is capable of being introduced into host cells and expressing the target gene. For example, common prokaryotic expression vector systems can be used.

[0050] The aforementioned “recombinant engineered cells” refer to host cells that have been introduced into the host cell through genetic engineering techniques (including transformation, transfection, transduction, etc.) to carry the foreign gene in their genome or plasmid, thereby being endowed with specific genetic traits and capable of stable replication and heterologous expression of the target protein / enzyme.

[0051] The aforementioned "asymmetric amination reaction" refers to the chemical reaction process in which an amino (-NH2) or amine group is introduced into a prochiral substrate (such as a ketone, aldehyde, olefin, or imine) under the action of the ω-transaminase of this invention as a chiral catalyst, and a single enantiomer (R or S configuration) chiral amine is generated with high selectivity by controlling the stereochemical environment. This invention provides for the highly selective generation of S-configuration chiral amines.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows: The ω-transaminase mutant of the present invention can efficiently catalyze the synthesis of (S)-6-methylnicotine with advantages of high yield, good stereoselectivity (ee>99.5%) and high catalytic efficiency, making it suitable for large-scale production and providing an efficient preparation route for the supply of (S)-6-methylnicotine and its application in related products. Attached Figure Description

[0055] Figure 1 The image shows the 1H-NMR spectrum of (S)-6-methylnicotine prepared in Example 1. Detailed Implementation

[0056] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0057] The optical purity and chemical purity involved in the following examples were determined by the following HPLC methods: ①HPLC conditions for determining the chemical purity of compound IV: Stationary phase: Waters XTERRA RP18 5μm 4.6×150mm.

[0058] Mobile phase: (i) 0.15% acetic acid-water solution, (ii) acetonitrile solution with pH adjusted to 10.0 by ammonia; gradient elution program: initial ratio of (i):(ii) 100:0 for 0-3 min, (i):(ii) 74:26 for 3.01-28.00 min, (i):(ii) 60:40 for 28.01-35.00 min, (i):(ii) 100:0 for 35.01-40 min.

[0059] The flow rate was 1.0 ml / min, the column temperature was 30 °C, and the injection volume was 10 μl. The detector conditions were UV absorption at a wavelength of 254 nm.

[0060] ②HPLC conditions for determining the optical purity of compound IV: Stationary phase: CHIRALCEL® IC, 4.6 × 250 mm, 5 μm column, eluted with a mobile phase of isopropanol:n-hexane:diethylamine = 5:95:0.1, flow rate 1.0 mL / min, column temperature 30 °C, injection volume 5 μL. Detector conditions: UV absorption at 261 nm.

[0061] Example 1

[0062] Preparation of ω-transaminase and its mutants.

[0063] 1. Construction and transformation of recombinant vectors

[0064] The following amino acid sequence of wild-type ω-transaminase was obtained (SEQ ID NO: 1). Plasmids encoding wild-type ω-transaminase and its variants were constructed using methods known in the art, such as the Gibson assembly method (see: Ji Zhicheng et al., Construction of Plant Expression Vectors Using Gibson Assembly Method, Journal of South China Agricultural University, 2014, 35(5): 112-116). The specific vector plasmid was pET-28a. Using *Escherichia coli* BL21(DE3) as the host cell, the recombinant plasmid containing wild-type ω-transaminase and its variants was introduced into the host cell and cultured to obtain host cells BL21(DE3) containing the recombinant plasmid.

[0065] Specifically, the amino acid sequence of the above-mentioned wild-type ω-transaminase is as follows: MQKQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFFKTTHPAVVELSSLLAEVTPAGFDRVF YTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGA GGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVOR VKDDIGPYMOKRWRETFSRFEHVDDVRGVGMVOAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRACGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEOTLKARGLA (SEQ ID NO: 1).

[0066] ω-transaminase mutant A has the following two substitution mutations: A57G and Y153A.

[0067] ω-transaminase mutant B has the following three substitution mutations: A57G, Y153A and D259A.

[0068] ω-transaminase mutant C has the following four substitution mutations: A57G, Y153A, M180G and D259A.

[0069] The ω-transaminase mutant D has the following five substitution mutations: A57G, N86Y, Y153A, M180G, and D259A.

[0070] 2. Construction and Induction of Expression Vectors

[0071] The host cells BL21(DE3) containing the recombinant plasmids of ω-transaminase and its variant genes obtained in step 1 were directly plated onto solid LB agar plates containing 25 μg / mL kanamycin resistance. They were cultured at 30°C for 12-16 h to obtain single colonies. Single colonies of BL21(DE3) containing the recombinant vector were picked from the LB agar plates and inoculated into 100 mL of liquid 2YT medium containing 25 μg / mL kanamycin resistance. The culture was incubated at 30°C with shaking for 12 h. IPTG (purchased from Shanghai Maclean Biotechnology Co., Ltd.) was added for induced expression for 20 h. The supernatant was then removed by centrifugation to obtain bacterial sludge.

[0072] The liquid 2×YT medium was prepared by sterilizing at 121°C for 20 minutes with 16 g / L yeast extract, 5 g / L sodium chloride, and 10 g / L peptone.

[0073] 3. Enzyme purification

[0074] The bacterial sludge obtained in step 2 was resuspended and lysed. The buffer solution consisted of 50 mM PB pH 7, 300 mM NaCl, 5 mM imidazole and 10% (v / v) glycerol. The cells were lysed using a high-pressure homogenizer at 12000xg, 4℃ for 20-40 min, and the soluble supernatant was collected.

[0075] The supernatant was purified with Ni-NTA resin, centrifuged using an ultrafiltration tube (Millipore, 10 kDa) to obtain a concentrated solution, and then resuspended in phosphate buffer at pH 7 to obtain the final product.

[0076] Example 2

[0077] Preparation of (S)-6-methylnicotine.

[0078] The synthetic route for preparing (S)-6-methylnicotine is as follows:

[0079]

[0080] in: R is methyl, ethyl, isopropyl, or tert-butyl; X can be chlorine, bromine, iodine, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or p-nitrobenzenesulfonic acid.

[0081] In this embodiment, (S)-6-methylnicotine was prepared using ethyl 6-methylpyridinenicotinate as a raw material.

[0082] 1. Synthesis of Compound I

[0083] Compound I

[0084] Under nitrogen protection and stirring, toluene (300 mL), sodium ethoxide (26.8 g), and γ-butyrolactone (31.3 g) were added sequentially to a 2 L four-necked reaction flask. After the addition was complete, the mixture was heated to 85 °C, and 6-methylpyridine nicotinic acid ethyl ester (50.0 g) was added dropwise. The mixture was kept at 80-85 °C for 6 hours. After confirming the reaction was successful by TLC, the mixture was cooled to ambient temperature to obtain a solution of compound I. This solution did not require further purification and was used directly in the next reaction.

[0085] 2. Synthesis of Compound II

[0086] (1) Synthesis of compound IIa

[0087] 300 mL of concentrated hydrochloric acid was added dropwise to the reaction solution of compound I. After the addition was complete, the temperature was raised to 110 °C and the reaction was stirred for 5 hours. After confirming the reaction was qualified by TLC, the temperature was lowered to ambient temperature. Under ice-water bath conditions, 30 wt% sodium hydroxide aqueous solution was added to adjust the pH to 9. After extraction with dichloromethane, the organic phases were combined, concentrated and dried to obtain crude compound IIa (60.3 g, purity 92.3%, yield: 93%).

[0088] (2) Synthesis of compound IIb

[0089] 300 mL of hydrobromic acid was added dropwise to the reaction solution of compound I. After the addition was complete, the temperature was raised to 110 °C and the reaction was stirred for 5 hours. After confirming the reaction was qualified by TLC, the temperature was lowered to ambient temperature. The pH was adjusted to 9 by adding 30% sodium hydroxide aqueous solution under ice-water bath conditions. After extraction with dichloromethane, the organic phases were combined, concentrated and dried to obtain crude compound IIb (71.4 g, purity 93.1%, yield: 90.6%).

[0090] 3. Synthesis of Compound IV

[0091] (1) Compound IIa is the reactant, and ω-transaminase mutant A catalyzes the reaction.

[0092] Under stirring conditions, 195 mL of prepared borate buffer solution (pH 8.5–9.0), 54.2 g of compound IIa (purity 92.3%), 25 mL of dimethyl sulfoxide, 625 mg of pyridoxal 5'-phosphate monohydrate, 35 mL of isopropylamine, and 19.1 g of ω-transaminase mutant A (solid content 42.1%, purified to 8 g) were added to a 500 mL reaction flask. The volume was adjusted to 300 mL with purified water, and the mixture was heated to 35 °C. During the reaction, 30 wt% liquid alkali (sodium hydroxide) was added to maintain the pH of the reaction system at 8.5–9.0. Sampling and testing were started after 12 hours of reaction, with sampling intervals of 2 hours, until the conversion rate of IIa was greater than 99.0%.

[0093] The pH of the reaction solution was adjusted to >12 by adding 30 wt% liquid alkali dropwise. The solution was extracted three times with 300 mL of dichloromethane. The organic phases were combined, and a small amount of anhydrous sodium sulfate was added to the combined organic phase. The mixture was dried for 15 minutes and then transferred to a round-bottom flask. The solvent was removed by rotary evaporation under reduced pressure in a 35°C water bath to obtain the crude product. The crude product was then distilled under reduced pressure to give 37.82 g of compound IV as a colorless liquid. The yield of compound IV was 92.1%, the chemical purity was 98.1%, and the optical purity was 97.3%.

[0094] (2) Compound IIa is the reactant, and ω-transaminase mutant B catalyzes the reaction.

[0095] Under stirring conditions, 195 mL of prepared Tris-HCl buffer solution (pH 8.5-9.0), 54.5 g of compound IIa (purity 92.3%), 25 mL of dimethyl sulfoxide, 1.25 g of pyridoxal 5'-phosphate monohydrate, 35 mL of propylamine, and 17.5 g of ω-transaminase mutant B (solid content 45.7%, purified to 8 g) were added to a 500 mL reaction flask. The volume was adjusted to 250 mL with purified water, and the mixture was heated to 35 °C. During the reaction, 30 wt% liquid alkali (sodium hydroxide) was added to maintain the pH of the reaction system at 8.5-9.0. Sampling and testing were started after 12 hours of reaction, with sampling intervals of 2 hours, until the conversion rate of IIa was greater than 99.0%.

[0096] The pH of the reaction solution was adjusted to >12 by adding 30wt% liquid alkali dropwise. The solution was extracted three times with 300 mL of dichloromethane. The organic phases were combined, and a small amount of anhydrous sodium sulfate was added to the combined organic phase. The mixture was dried for 15 minutes. The dried organic phase was transferred to a round-bottom flask and the solvent was removed by rotary evaporation under reduced pressure in a 35°C water bath to obtain the crude product. The crude product was then distilled under reduced pressure to give 39.01 g of compound IV as a colorless liquid with a yield of 94.5%, a chemical purity of 97.8%, and an optical purity of 97.2%.

[0097] (3) Compound IIb is the reactant, and the ω-transaminase mutant C catalyzes the reaction.

[0098] Under stirring conditions, 195 mL of prepared Tris-HCl buffer solution (pH 8.5–9.0), 66.0 g of compound IIb (purity 93.1%), 25 mL of dimethyl sulfoxide, 625 mg of pyridoxal 5'-phosphate monohydrate, 35 mL of isopropylamine, and 15.9 g of ω-transaminase mutant C (solid content 50.3%, purified to 8 g) were added to a 500 mL reaction flask. The volume was adjusted to 300 mL with purified water, and the mixture was heated to 35 °C. During the reaction, 30 wt% liquid alkali (sodium hydroxide) was added to maintain the pH of the reaction system at 8.5–9.0. Sampling and testing were started after 12 hours of reaction, with sampling intervals of 2 hours, until the conversion rate of 2b was greater than 99.0%.

[0099] 30wt% liquid alkali was added dropwise to the reaction solution to adjust the pH to >12. The mixture was extracted three times with 300 mL of dichloromethane. The organic phases were combined, and a small amount of anhydrous sodium sulfate was added to the combined organic phase. The mixture was dried for 15 minutes. The dried organic phase was transferred to a round-bottom flask, and the solvent was removed by rotary evaporation under reduced pressure in a 35°C water bath to obtain the crude product. The crude product was then distilled under reduced pressure to give 39.44 g of compound IV as a colorless liquid, with a yield of 95.8%, a chemical purity of 98.5%, and an optical purity of 97.9%.

[0100] (4) Compound IIa is the reactant, and ω-transaminase mutant D catalyzes the reaction.

[0101] Under stirring conditions, 19.5 L of prepared Tris-HCl buffer solution (pH 8.5-9.0), 53.4 g of compound IIa (purity 92.3%), 25 mL of dimethyl sulfoxide, 625 mg of pyridoxal 5'-phosphate monohydrate, 35 mL of isopropylamine, and 17.9 g of ω-transaminase mutant D (solid content 44.6%, 8 g pure) were added to a 50 mL reactor. The mixture was then brought to a final volume of 300 mL with purified water and heated to 35 °C. During the reaction, 30 wt% liquid alkali was added to maintain the pH of the reaction system at 8.5-9.0. Sampling and testing were started after 12 hours of reaction, with sampling intervals of 2 hours, until the conversion rate of IIa was greater than 99.0%.

[0102] 30wt% liquid alkali was added dropwise to the reaction solution to adjust the pH to >12. The solution was extracted three times with 300mL of dichloromethane. The organic phases were combined and the solvent was removed under reduced pressure in a 35°C water bath to obtain the crude product. The crude product was then distilled under reduced pressure to give 38.92g of compound IV as a colorless liquid, with a yield of 96.2%, a chemical purity of 99.1%, and an optical purity of 98.2%.

[0103] 4. Synthesis of (S)-6-methylnicotine

[0104] Add 200g of compound IV (catalyzed by ω-transaminase mutant D) and 300mL of 37wt% formaldehyde aqueous solution to a reaction flask. Heat to 40-45℃, and after the addition is complete, maintain the temperature and stir for 1 hour. After TLC detection shows complete conversion of compound IV to the intermediate state, add 240mL of formic acid dropwise to the reaction solution. Continue stirring for 3 hours after the addition is complete. TLC detection shows complete conversion of the intermediate state to the target product. Cool to ambient temperature, and adjust the pH of the reaction solution to > 12 with 30wt% liquid alkali (sodium hydroxide). Then, use 300 mL of 300 mL of formic acid solution to further adjust the pH. Extracted three times with dichloromethane, the organic phases were combined, and an appropriate amount of anhydrous sodium sulfate was added to the combined organic phases for drying for 30 minutes. The dried organic phase was transferred to a round-bottom flask, and the solvent was removed under reduced pressure at 35°C using a rotary evaporator. Further reduced pressure distillation and fractional distillation yielded 206.2 g of (S)-6-methylnicotine, a colorless oily liquid, with a yield of 94.9%, chemical purity of 99.95%, and optical purity of 98.5%. The 1H NMR spectrum is attached. Figure 1 As shown.

[0105] Example 2

[0106] Compound IV was prepared according to the method described in item 3, “Synthesis of Compound IV” in Example 1, “(4) Compound IIa as reactant, ω-transaminase mutant D as catalytic reaction”. Following the same method, (S)-6-methylnicotine was synthesized using wild-type ω-transaminases with amino acid sequences as shown in SEQ ID NO: 1, and NCBI accession numbers XP_007730450, 5FR9_A, WP_040602310, and XP_748821 as catalytic enzymes.

[0107] The catalytic abilities of different transaminases were compared, and the results are shown in the table below.

[0108] Table 1. Catalytic results of each ω-transaminase

[0109] The “dosage” mentioned above refers to the enzyme mass / substrate molar amount.

[0110] The results show that, under the same enzyme dosage (32 g / mol), the ω-transaminase mutant D can achieve a substrate conversion rate of over 99% in just 18 hours, with an optical purity of 98.2%, a chemical purity of 99.1%, and a yield of 96.2%, ranking first in all four indicators: reaction rate, stereoselectivity, product purity, and yield.

[0111] In comparison, although wild-type ω-transaminase has slightly inferior performance, its catalytic efficiency, optical purity, and yield are superior to XP_007730450, 5FR9_A, WP_040602310, and XP_748821.

[0112] In summary, site-directed modification of ω-transaminase mutants not only significantly improved catalytic efficiency (reducing reaction time by more than 20%), but also greatly improved stereoselectivity and product quality.

Claims

1. An omega-transaminase mutant, characterized in that, Compared with the wild-type ω-transaminase, the ω-transaminase mutant has at least two substitution mutations at the following amino acid sites: positions 57, 86, 153, 180, and 259. The amino acid sequence of the wild-type ω-transaminase is shown in SEQ ID NO:

1.

2. The ω-transaminase mutant as described in claim 1, characterized in that, The substitution mutation at position 57 is A57G / L / A, the substitution mutation at position 86 is N86A / Y, the substitution mutation at position 153 is Y153A / S, the substitution mutation at position 180 is M180A / G, and the substitution mutation at position 259 is D259A / V / I. Preferably, the ω-transaminase mutant includes the following substitution mutations: A57G and Y153A; for example, the ω-transaminase mutant includes the following substitution mutations: A57G, Y153A, and D259A; or includes the following substitution mutations: A57G, Y153A, M180G, and D259A; or includes the following substitution mutations: A57G, N86Y, Y153A, M180G, and D259A; More preferably, the ω-transaminase mutant has the following substitution mutations: A57G, N86Y, Y153A, M180G, and D259A.

3. The method for preparing the ω-transaminase mutant according to any one of claims 1-2, characterized in that, Includes the following steps: Construction and transformation of recombinant vector: Obtain the amino acid sequence of the ω-transaminase mutant according to any one of claims 1-2, construct a plasmid containing the gene encoding the ω-transaminase mutant, introduce it into host cells, and culture it; Construction and induction of expression vector: Host cells containing the ω-transaminase mutant gene plasmid were induced to express the expression in 2YT medium containing kanamycin resistance. The preparation method meets one or more of the following conditions: (1) The plasmid is pET-28a; and / or the host cell is Escherichia coli BL21(DE3); (2) The 2YT culture medium contains: 16 g / L yeast extract, 5 g / L sodium chloride and 10 g / L peptone; (3) It also includes a purification step, wherein the purification step is as follows: the cells containing the ω-transaminase mutant are lysed and centrifuged, the supernatant is purified with resin, ultrafiltered, and the filtrate is discarded to obtain the product; preferably, the resin is Ni-NTA resin, and / or the molecular weight cutoff of the ultrafiltration is 10 kDa.

4. A polynucleotide, characterized in that, The polynucleotide encodes the ω-transaminase mutant according to any one of claims 1-2.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide as described in claim 4.

6. A recombinant engineered cell, characterized in that, The recombinant engineered cells comprise the polynucleotides as described in claim 4.

7. The application of ω-transaminase as a catalyst in asymmetric amination synthesis, characterized in that, The ω-transaminase is selected from one or more of the ω-transaminase with the amino acid sequence shown in SEQ ID NO: 1 and the ω-transaminase mutant according to any one of claims 1-2; preferably, the ω-transaminase is selected from one or more of the ω-transaminase mutants.

8. An asymmetric amination reaction, characterized in that, Includes the following steps: Compound II, in the presence of an amino donor and a coenzyme, is contacted with the ω-transaminase mutant according to any one of claims 1-2 to yield compound IV: ； in: X is selected from: chlorine, bromine, iodine, methanesulfonic acid group, benzenesulfonic acid group, p-toluenesulfonic acid group, or p-nitrobenzenesulfonic acid group; The amino donor is selected from: primary amines; The coenzyme is selected from pyridoxal 5'-phosphate.

9. The asymmetric amination reaction as described in claim 8, characterized in that, Meets one or more of the following conditions: (1) X is chlorine; (2) The amino donor is selected from: isopropylamine, propylamine, phenylethylamine; preferably isopropylamine or propylamine; more preferably isopropylamine; (3) The coenzyme is selected from: pyridoxal 5'-phosphate monohydrate; (4) The reaction is carried out in a buffer salt solution with a pH of 7.0 to 9.

5. Preferably, the pH of the buffer salt solution is 8.0 to 9.0, and more preferably, it is 8.5 to 9.

0. (5) The reaction is carried out in a buffer salt solution, wherein the buffer salt solution is selected from one of the following: phosphate buffer, Tris-HCl buffer, glycine-NaOH buffer, and borate buffer; preferably, the buffer salt solution is selected from borate buffer or Tris-HCl buffer; more preferably, Tris-HCl buffer. (6) The reaction temperature is 35±10℃, preferably 35℃; (7) The mass ratio of compound II to the ω-transaminase mutant is 5-10:1, preferably 6.25-7.68:1, more preferably 6.25:1; (8) The concentration of the amino donor in the reaction system is 1.0-5.0 M, preferably 1.3-1.7 M, for example 1.37, 1.7 M or 2.0 M; (9) The mass ratio of compound II to the coenzyme is 50-100:1, preferably 40-98:1, and more preferably 80:1; (10) Compound II forms intermediate compound III, and compound III undergoes cyclization to form compound IV: ； (11) The optical purity of compound IV is ≥99%.

10. A method for synthesizing (S)-6-methylnicotine, characterized in that, Synthesize according to the following route: ； in: R is selected from: C1-C4 alkyl, preferably methyl, ethyl, isopropyl or tert-butyl, more preferably ethyl; X is selected from: chlorine, bromine, iodine, methanesulfonic acid group, benzenesulfonic acid group, p-toluenesulfonic acid group or p-nitrobenzenesulfonic acid group, preferably chlorine; Step S1: Under alkaline conditions, 6-methylnicotinic acid ester reacts with γ-butyrolactone ester by condensation to give compound I; Step S2: Compound I is heated under acidic conditions, which causes ring-opening to form compound II; Step S3: The asymmetric amination reaction step as described in claim 7 or 8 is carried out; Step S4: Compound IV undergoes the Eschweiler-Clarke reaction under acidic conditions to produce (S)-6-methylnicotine; Preferably, the synthesis method meets one or more of the following conditions: (1) In step S1, the alkaline condition is the presence of sodium ethoxide, preferably, the concentration of sodium ethoxide in the reaction system is 1-1.1M; (2) In step S1, the reaction is carried out in a solvent, preferably toluene; (3) In step S1, the reaction is carried out under the protection of an inert gas, preferably nitrogen. (4) In step S1, the reaction temperature is 80~85℃; (5) In step S1, the molar ratio of 6-methylnicotinic acid ester to γ-butyrolactone is 1:1.2; (6) In step S2, the acidic condition is the presence of concentrated hydrochloric acid or hydrobromic acid. Preferably, the reaction equivalent of the concentrated hydrochloric acid or hydrobromic acid is 10-12 eq of compound I. (7) In step S2, the reaction is carried out at 110±10℃; (8) In step S4, compound IV reacts with formaldehyde in an Eschweiler-Clarke reaction. Preferably, the molar ratio of compound IV to formaldehyde is 1:3-4. (9) In step S4, the acidic condition is the presence of formic acid, preferably, the reaction equivalent of the formic acid is 4-6 eq of compound IV; (10) In step S4, the reaction is carried out at 40~45°C.

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