A method for preparing S-nicotine

Abstract

The present invention relates to the field of biosynthesis technology, and particularly to a method for preparing S-nicotine. The present invention creatively uses amine oxidase to oxidize 1-methylpyrrolidine into the corresponding imine, and then under the catalysis of nicotine synthase, condenses and decarboxylates the imine with nicotinic acid to obtain the final product S-nicotine. This method can obtain chirally specific S-nicotine through two-step reactions in a single reaction system, with a short synthesis route, high yield, mild reaction conditions and easy large-scale production; at the same time, the raw materials are widely sourced, inexpensive, with low production costs, environmentally friendly, significantly reducing the production cost of nicotine while also making it more in line with the requirements of today's green industrial production.

Description

[0001] This application is a divisional application of the application with the application date of August 10, 2021, application number 202110914222.4, and invention name "A Preparation Method of S-Nicotine". Technical Field

[0002] The present invention relates to the field of biosynthesis technology, and in particular to a preparation method of S-nicotine. Background Art

[0003] Nicotine is an important component in tobacco and is also the core raw material for e-cigarette formulations and the synthesis of certain nicotine-based drugs.

[0004] Traditional methods for cultivating, extracting, and purifying tobacco leaves cover a large area, consume a long cycle, and cause great harm to the human body due to the inevitable presence of other highly toxic components in the extraction. Therefore, direct synthesis using chemical or biological processes has become an important approach for the preparation of S-nicotine.

[0005] Several common preparation methods of S-nicotine:

[0006]

[0007] Route I: Preparation of racemic nicotine by chemical method. Using pyridine acetaldehyde as a raw material, nicotine racemate is prepared through three-step chemical reactions, and then S-nicotine is obtained by chiral resolution using chemical reagents or enzymes. The above several chemical reactions require the use of highly toxic (such as NaCN) and explosive (such as Raney Ni hydrogenation) and other complex and dangerous production processes. (Reference: International Patent WO2017 / 119003AI; "A PROCESS FOR THE PREPARATION OF NICOTINE").

[0008] Route II: Direct preparation of S-nicotine by chemical method. Using pyridine ethylamine as a starting material, S-nicotine is obtained through three-step chemical conversion. The catalysts for the first two steps of the reaction are expensive and the reaction conditions are harsh; the final yield is also low (<50%). (Reference: Josha T. Ayers, AAPS 2005, "A General Procedure for the Enantioselective Synthesis of the Minor Tobacco Alkaloids Nornicotine, Anabasine, and Anatabine").

[0009] Route III: Using myosmine, a nicotine precursor, as the raw material, and then using enzymatic chiral reduction and methylation with chemical reagents to prepare S-nicotine. Although this route is relatively short and has a high yield, due to the use of expensive myosmine as the starting material, the production cost is high.

[0010] From the above three classic S-nicotine preparation processes, it can be seen that although the two chemical preparation methods of Route I and Route II also use relatively inexpensive raw materials and the overall route is not long (three or four-step reactions), factors such as the expensive chemical catalysts involved in the reactions (BF3, LDA, NaBH4) and the dangerous operations (LDA, NaCN) result in high environmental costs and safety costs in large-scale production. Route III uses myosmine as the raw material, and uses imine reductase for chiral reduction to obtain S-nornicotine, and then uses chemical methods for methylation; the defect of this route is that it needs to use expensive myosmine as the raw material, thus greatly increasing the overall production cost. At the same time, with the development of science and technology, the country's requirements for the green production index of the chemical industry are getting higher and higher. Therefore, it is of great significance to provide a production process of S-nicotine with simple operation steps, low cost, safety and environmental protection. Summary of the Invention

[0011] In view of this, the present invention provides a method for preparing S-nicotine, which uses 1-methylpyrrolidine and nicotinic acid as raw materials and is directly converted into S-nicotine at one time. The process is simple, the reaction yield is high, the cost is low, and it is environmentally friendly.

[0012] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0013] The present invention provides an amine oxidase mutant, and its amino acid sequence is:

[0014] The amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; or

[0015] An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 1 or SEQ ID NO: 2 and having the same or similar function as SEQ ID NO: 2; or

[0016] An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having the same or similar function as the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In the present invention, both amine oxidase mutant 1 (AO1) and amine oxidase mutant 2 (AO2) are derived from a monoamine oxidase in Aspergillus niger. The amino acid sequence number of this wild-type enzyme is: Uniprot ID: P46882, EC 1.4.3.4.

[0018] Among them, the amine oxidase mutant contains 5 site mutations: M242R, W230I, T354S, Y365V, W430R, named amine oxidase mutant 1 (abbreviated as AO1), and its amino acid sequence is as shown in SEQ ID NO: 1.

[0019] In some embodiments, the amine oxidase mutant contains 10 site mutations: F210M, L213C, M242V, I246T, R259K, R260K, N336S, T384N, D385S, W430G, named amine oxidase mutant 2 (abbreviated as AO2), and its amino acid sequence is as shown in SEQ ID NO: 2.

[0020] The present invention also provides nucleic acids encoding the amine oxidase mutants.

[0021] In some embodiments, the nucleotide sequences encoding the oxidase mutants are as shown in SEQ ID NO: 3 or SEQ ID NO: 4. Among them, the nucleotide sequence encoding amine oxidase mutant 1 (AO1) is as shown in SEQ ID NO: 3, and the nucleotide sequence encoding amine oxidase mutant 2 (AO2) is as shown in SEQ ID NO: 4.

[0022] The present invention provides a nicotine synthase mutant, and its amino acid sequence is:

[0023] The amino acid sequence as shown in SEQ ID NO: 5; or

[0024] An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 5 and having the same or similar function as SEQ ID NO: 5; or

[0025] An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO: 5 and having the same or similar function as SEQ ID NO: 5.

[0026] In the present invention, the nicotine synthase mutant is derived from a redox condensing enzyme of Anisodus acutangulus in the Solanaceae family, and the amino acid sequence number of the wild-type nicotine synthase is 6J1M.

[0027] In some embodiments, the nicotine synthase mutant comprises 14 site mutations: M17H, R112T, Q113F, L162A, Q180E, F183A, S212K, A229P, P248L, V254R, A261H, K341V, R346T, G394T, and its amino acid sequence is as shown in SEQ ID NO:5.

[0028] The present invention also provides a nucleic acid encoding the nicotine synthase mutant described above.

[0029] In some embodiments, the nucleotide sequence encoding the nicotine synthase mutant is as shown in SEQ ID NO:6.

[0030] The present invention provides an amino acid sequence as follows:

[0031] The amino acid sequence as shown in SEQ ID NO:7; or

[0032] An amino acid sequence having the same or similar function as SEQ ID NO:7 obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:7; or

[0033] An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO:7 and having the same or similar function as SEQ ID NO:7.

[0034] In the present invention, the phosphite dehydrogenase (PTDH) mutant is modified from a phosphite dehydrogenase in Pseudomonas stutzeri, and the wild-type amino acid sequence is Uniprot ID: O69054, EC 1.20.1.1.

[0035] In some embodiments, the phosphite dehydrogenase mutant comprises 13 site mutations: V71I, Q132R, E130K, Q137R, I150F, A176R, Q215L, R275Q, L276Q, I313L, V315A, A319E, A325V, and its amino acid sequence is as shown in SEQ ID NO:7.

[0036] The present invention also provides a nucleic acid encoding the phosphite dehydrogenase mutant described above.

[0037] In some embodiments, the nucleotide sequence encoding the phosphite dehydrogenase mutant is as shown in SEQ ID NO:8.

[0038] The present invention provides a complex enzyme comprising at least two of the following (a) to (b):

[0039] (a) Amine oxidase or its mutant;

[0040] (b) Nicotine synthase or its mutant;

[0041] (c) Phosphite dehydrogenase or its mutant;

[0042] (d) Catalase or its mutant.

[0043] In some embodiments of the present invention, the complex enzyme comprises two kinds shown in (a) - (b), namely, two enzymes: amine oxidase mutant and phosphite dehydrogenase mutant.

[0044] In some embodiments, the complex enzyme of the present invention comprises the enzymes shown in (a) - (b):

[0045] (a) Amine oxidase or its mutant; and

[0046] (b) Nicotine synthase or its mutant; and

[0047] (c) Phosphite dehydrogenase or its mutant; and

[0048] (d) Catalase or its mutant.

[0049] In some specific embodiments, the complex enzyme provided by the present invention comprises amine oxidase mutant, phosphite dehydrogenase mutant, nicotine synthase mutant and catalase.

[0050] In some embodiments of the present invention, for the above - mentioned complex enzyme, the amino acid sequence of the amine oxidase mutant is as shown in SEQ ID NO:1 or SEQ ID NO:2;

[0051] The amino acid sequence of the nicotine synthase mutant is as shown in SEQ ID NO:5;

[0052] The amino acid sequence of the phosphite dehydrogenase mutant is as shown in SEQ ID NO:7;

[0053] The catalase is obtained by purchase. In the specific embodiments of the present invention, the catalase is purchased from Novozymes (Terminox Ultra).

[0054] The present invention also provides the application of the complex enzyme in the preparation of S - nicotine.

[0055] The present invention also provides a method for preparing S - nicotine, comprising:

[0056] Mixing 1 - methylpyrrolidine, nicotinic acid with the complex enzyme in the presence of a solvent, oxygen and NADPH, and reacting to generate S - nicotine.

[0057] In the preparation method provided by the present invention, amine oxidase or its mutant in the complex enzyme is used to oxidize 1-methylpyrrolidine into the corresponding imine, and then the imine is condensed and decarboxylated with nicotinic acid under the catalysis of nicotine synthase or its mutant to obtain S-nicotine. The synthetic route map is shown in Figure 1 。

[0058] Among them, oxygen is required in the oxidation reaction of the first step, and hydrogen peroxide by-product will be generated during the reaction. Therefore, in some embodiments, the present invention can effectively remove hydrogen peroxide in the system by adding a small amount of catalase, and at the same time, O2 can be recycled.

[0059] The condensation and decarboxylation reaction of the second step requires the participation of coenzyme NADPH. Since this coenzyme is relatively expensive, by adding a NADPH regeneration system (phosphite dehydrogenase PTDH) in the same system, this coenzyme can be effectively regenerated, thereby greatly reducing its dosage and lowering the production cost.

[0060] In some embodiments, the NADPH is generated by the NADPH regeneration system, and the NADPH regeneration system includes β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and a mutant of phosphite dehydrogenase.

[0061] In some embodiments, the solvent is tris(hydroxymethyl)aminomethane hydrochloride solution or tris(hydroxymethyl)aminomethane hydrochloride solution containing a cosolvent. The cosolvent can promote the dissolution of each substrate in the solvent and is beneficial to the progress of the reaction. The present invention believes that common and feasible types of cosolvents are acceptable, including but not limited to isopropanol, acetone, DMSO. Among them, in the specific embodiments of the present invention, isopropanol is used as the substrate cosolvent, and the effect is better.

[0062] Specifically, the preparation method of S-nicotine of the present invention includes:

[0063] 1-Methylpyrrolidine nicotinic acid, β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and isopropanol are sequentially added to the tris(hydroxymethyl)aminomethane hydrochloride solution, and the pH is adjusted to 6.5-9.0, then the complex enzyme is added to obtain a reaction system;

[0064] The reaction system is slowly stirred at 25-35 °C under an oxygen pressure of 1.0-2.0 atmospheres for 4-8 hours. After the reaction is completed, the pH is adjusted to 9.0-11.0, and then extracted with ethyl acetate. The organic phases are combined, dried, filtered and concentrated to obtain S-nicotine.

[0065] In some specific embodiments, the preparation method of S-nicotine includes:

[0066] 1-Methylpyrrolidine nicotinic acid, β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and isopropanol were successively added to a tris(hydroxymethyl)aminomethane hydrochloride solution, and after adjusting the pH to 8.0, a complex enzyme was added to obtain a reaction system;

[0067] The reaction system was slowly stirred at 30 °C for 6 hours under an oxygen pressure of 1.5 atmospheres. After the reaction was completed, the pH was adjusted to 10.0, and after extraction with ethyl acetate, combination of the organic phases, drying, filtration, and concentration in sequence, S-nicotine was obtained.

[0068] In some embodiments, the complex enzyme of the present invention includes an amine oxidase mutant, a phosphite dehydrogenase mutant, a nicotine synthase mutant, and a catalase. Among them, the enzyme activity ratio of the amine oxidase mutant, the nicotine synthase mutant, the phosphite dehydrogenase mutant, and the catalase is preferably (1.5-2.5):(2.5-5):(4-8):1. In some specific embodiments, the enzyme activity ratio of the amine oxidase mutant, the nicotine synthase mutant, the phosphite dehydrogenase mutant, and the catalase is 2:4:6:1.

[0069] In some embodiments, in the reaction system:

[0070] The concentration of 1-methylpyrrolidine is 150-250 mM, and specifically can be 150 mM, 200 mM or 250 mM;

[0071] The concentration of nicotinic acid is 150-250 mM, and specifically can be 150 mM, 200 mM or 250 mM;

[0072] The concentration of β-nicotinamide adenine dinucleotide phosphate monosodium salt is 0.2-0.6 mM, and specifically can be 0.2 mM, 0.4 mM or 0.6 mM;

[0073] The concentration of sodium phosphite pentahydrate is 200-300 mM, and specifically can be 200 mM, 240 mM or 300 mM;

[0074] The volume fraction of isopropanol is 1-5%, and specifically can be 1% or 5%.

[0075] The present invention innovatively uses amine oxidase to oxidize 1-methylpyrrolidine into the corresponding imine, and then under the catalysis of nicotine synthase, condenses and decarboxylates the imine with nicotinic acid to obtain the final product S-nicotine. This method can obtain enantiomerically pure S-nicotine through two-step reactions in a reaction system, with a short synthesis route, high yield, mild reaction conditions and easy large-scale production; at the same time, the raw materials are widely available, inexpensive, the production cost is low, and it is environmentally friendly, which not only significantly reduces the production cost of nicotine but also makes it more in line with the requirements of today's green industrial production. Brief Description of the Drawings

[0076] Figure 1 Shows the synthetic route diagram of S-nicotine of the present invention;

[0077] Figure 2 Shows the mass spectrum of (S)-nicotine of Example 1 of the present invention;

[0078] Figure 3 Shows (S)-nicotine of Example 1 of the present invention 1 1H-NMR spectrum, 400M Varian NMR, D2O solvent. Detailed Description of the Invention

[0079] The present invention provides a method for preparing S-nicotine. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The method and application of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0080] Unless otherwise specified, the test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0081] In the present invention, catalase was purchased from Novozymes (Terminox Ultra), and the other three enzymes, amine oxidase AO1 mutant (SEQ ID NO:1), amine oxidase AO2 mutant (SEQ ID NO:2), nicotine synthase NS mutant (SEQ ID NO:5), and phosphite oxidase PTDH mutant (SEQ ID NO:7) were all prepared by the present invention through constructing engineering strains for fermentation. The specific method includes:

[0082] First, synthesize the genes corresponding to the above enzyme mutants (synthesized by General Biosystems (Anhui)), and then subclone them into the pET28a plasmid at the NdeI / XhoI restriction sites. The constructed plasmid is transferred into the E. coli (BL21) strain (GenScript) for plate culture, and finally, single colonies are picked for stepwise liquid culture. First, transfer it into 5 ml of LB culture medium containing 50 μM kanamycin and culture it at 37 °C. When the cells grow to the logarithmic phase, inoculate them into 250 ml of LB culture medium containing the same antibiotic, and finally transfer it into a 5 L culture fermenter for culture; when the cell OD reaches about 15, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and induce protein expression at 30 °C for 10 hours. Finally, collect the cells by high-speed centrifugation (6000 rpm, 15 min) to obtain 40 - 60 g of wet cells. Take a small amount of cells and mix them evenly with Tris-HCl buffer (50 mM, pH 8.0), and then break the cells by the freeze-thaw method. After high-speed centrifugation, the supernatant is used to determine protein expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The remaining cells with correct protein expression confirmation are also mixed evenly with the above buffer on ice (10 g of wet cells are mixed with about 200 ml of buffer), and then the cell wall is broken by high pressure. After high-speed centrifugation (16000 rpm, 45 min), the enzyme-containing clear liquid is obtained and used directly (when the liquid enzyme solution reacts, the enzyme activity is 200 - 350 U / ml, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature) or used after further purification and immobilization (when the solid enzyme reacts). The composition of the LB medium is: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 5% glycerol.

[0083] In the present invention, the complex enzyme can be in a liquid form or a solid form of an immobilized enzyme. The immobilized enzyme can be recovered after the reaction and can be reused. In some embodiments, a liquid complex enzyme is used to prepare S-nicotine. In other embodiments, an immobilized complex enzyme is used to prepare S-nicotine, and the immobilized complex enzyme is prepared according to the following steps:

[0084] Ammonium sulfate solid is gradually added to the crude amine oxidase solution (AO1 or AO2), nicotine synthase crude solution (NS), and phosphite oxidase crude solution (PTDH) obtained by fermentation of the present invention until precipitation (25%-60%, w / v ammonium sulfate / buffer). The enzyme solid is then collected by centrifugation (10000rpm, 12min), and slowly dissolved in 25mM Tris buffer of pH 8.0, and then desalted by G25 size exclusion chromatography column (purchased from Sigma) and separated by DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain pre-purified liquid enzymes AO1, AO2, NS, and PTDH. Finally, AO1 / AO2, NS, PTDH and Novozymes' Catalase are mixed and fixed once using LX-1000EP epoxy resin (Xi'an Lanxiao Company) according to the activity unit ratio of 2:4:6:1. The immobilization method is as follows: 1000U mixed enzyme is dissolved in 1L 50mM pH 8.0 potassium phosphate solution, then 40mM phenoxyacetic acid and 300g LX-1000EP epoxy resin are added to the buffer solution, and the immobilized enzyme is filtered out after stirring at room temperature for 4 hours, and finally washed three times with clean water and 25mM pH 8.0 phosphate buffer solution, and then dried at low temperature for use. The immobilized mixed enzyme has 65-92% of the activity of the corresponding liquid enzyme.

[0085] The present invention will be further described below in conjunction with embodiments:

[0086] Example 1 Preparation of S-nicotine by one-pot method using liquid enzyme (AO1, NS)

[0087] 17 g 1-methylpyrrolidine (200 mM), 24.6 g nicotinic acid (200 mM), 3.0 g β-nicotinamide adenine dinucleotide phosphate (NADP) were added to 1 L 50 mM Tris-HCl solution (pH 8.0). + ) monosodium salt (0.4mM), 52g sodium phosphite pentahydrate (240mM) and 100ml isopropanol (substrate cosolvent). After adjusting the pH value of the reaction solution to 8.0 with NaOH aqueous solution, add the composite enzyme to obtain a reaction solution; wherein the composite enzyme composition is: 2000U AO1 (SEQID NO: 1), 4000U NS (SEQ ID NO: 5), 1000U Catalase, 6000U PTDH (SEQ ID NO: 7);

[0088] Subsequently, the reaction solution was transferred to a pressure-resistant reactor and slowly stirred at 30 °C for 6 hours under an oxygen pressure of 1.5 atmospheres. After the reaction was completed, the pH of the solution was adjusted to 10, and then extracted three times with 700 ml of ethyl acetate. The extracted organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 22 g of a pale yellow liquid (yield 68%, HPLC purity 91%).

[0089] Example 2: Preparation of S-nicotine by one-pot method using liquid enzymes (AO2, NS)

[0090] The difference from Example 1 was that amine oxidase AO2 was replaced with AO1, and other procedures were the same. Similarly, 17 g of 1-methylpyrrolidine (200 mM), 24.6 g of nicotinic acid (200 mM), 3.0 g of β-nicotinamide adenine dinucleotide phosphate (NADP + ) monosodium salt (0.4 mM), 52 g of sodium phosphite pentahydrate (240 mM), and 100 ml of isopropanol were successively added to 1 L of 50 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. After adjusting the pH value of the reaction solution to 8.0 with an aqueous NaOH solution, a complex enzyme was added to obtain a reaction solution; wherein, the complex enzyme composition was: 2000 U of AO2 (SEQ ID NO: 2), 4000 U of NS (SEQ ID NO: 5), 1000 U of Catalase, and 6000 U of PTDH (SEQ ID NO: 7);

[0091] Subsequently, the reaction solution was transferred to a pressure-resistant reactor and stirred at 30 °C for 4 hours under an oxygen pressure of 1.5 atmospheres. After the reaction was completed as detected by HPLC and the pH of the solution was adjusted to 10, it was extracted three times with 700 ml of ethyl acetate. The extracted organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 29.8 g of a pale yellow liquid (yield 92%), and the chromatographic purity of S-nicotine was detected to be 95%.

[0092] Example 3: Preparation of S-nicotine by one-pot method using immobilized complex enzyme (AO2, NS, PTDH, Catalase)

[0093] Similar to Example 2, the difference was that an immobilized complex enzyme (prepared by the method of the present invention to prepare an immobilized mixed enzyme) was used, and the immobilized enzyme could be recycled after the reaction was completed. Similarly, 8.5 g of 1-methylpyrrolidine (100 mM), 12.3 g of nicotinic acid (100 mM), 1.5 g of β-nicotinamide adenine dinucleotide phosphate (NADP +) Sodium monophosphate (0.2 mM), 26 g of sodium phosphite pentahydrate (120 mM), and 100 ml of isopropanol. After adjusting the pH of the reaction solution to 8.0 with an aqueous NaOH solution, 6000 - 8000 U of a mixed immobilized enzyme (i.e., a composite enzyme) was added to obtain a reaction solution; wherein, the composite enzyme is: AO2 mutant (SEQ ID NO: 2), NS mutant (SEQ ID NO: 5), Catalase, PTDH mutant (SEQ ID NO: 7);

[0094] Subsequently, the reaction solution was transferred to a pressure-resistant reactor and reacted with gentle shaking at 30 °C for 12 hours under an oxygen pressure of 1.5 atmospheres. After the reaction was completed, the immobilized composite enzyme was recovered by filtration (the immobilized enzyme was washed three times with 50 mM pH 8.0 Tris buffer and stored at 4 °C for later use). After adjusting the pH of the filtrate to 10, it was extracted three times with 700 ml of ethyl acetate. The extracted organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 13.6 g of a light yellow liquid (yield 84%, purity 98%). The immobilized mixed enzyme recovered by filtration had 75 - 90% of its initial enzyme activity.

[0095] Comparative Example 1 (without co-solvent)

[0096] Similar to Example (2), 8.5 g of 1-methylpyrrolidine (100 mM), 12.3 g of nicotinic acid (100 mM), and 3.0 g of β-nicotinamide adenine dinucleotide phosphate (NADP + ) Sodium monophosphate (0.4 mM), 26 g of sodium phosphite pentahydrate (120 mM) were successively added to 1 L of a 50 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. After adjusting the pH of the reaction solution to 8.0 with an aqueous NaOH solution, a composite enzyme was added to obtain a reaction solution; wherein, the composition of the composite enzyme is: 1000 U of AO2 (SEQ ID NO: 2), 2000 U of NS (SEQ ID NO: 5), 1000 U of Catalase, 3000 U of PTDH (SEQ ID NO: 7);

[0097] Subsequently, the reaction solution was transferred to a pressure-resistant reactor and reacted with stirring at 30 °C for 8 hours under an oxygen pressure of 1.5 atmospheres. After the reaction was completed as detected by HPLC and the pH of the solution was adjusted to 10, it was extracted three times with 500 ml of ethyl acetate. The extracted organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 6.8 g of a light yellow liquid (yield 43%). The chromatographic purity of S-nicotine was detected to be 84%.

[0098] Comparative Example 2 (AO is wild type)

[0099] Similar to Example 1 above, 8.5 g of 1-methylpyrrolidine (100 mM), 12.3 g of nicotinic acid (100 mM), 3.0 g of β-nicotinamide adenine dinucleotide phosphate (NADP + ) monosodium salt (0.4 mM), 26 g of sodium phosphite pentahydrate (120 mM) and 100 ml of isopropanol (substrate cosolvent) were successively added to 1 L of 50 mM Tris.HCl solution at pH 8.0. After adjusting the pH of the reaction solution to 8.0 with an aqueous NaOH solution, a complex enzyme was added to obtain a reaction solution; wherein, the complex enzyme composition is: 6000 U AO (wild type, Uniprot ID: P46882, EC 1.4.3.4), 2000 U NS (SEQ ID NO: 5), 1000 U Catalase, 3000 U PTDH (SEQ ID NO: 7);

[0100] Subsequently, the reaction solution was transferred to a pressure-resistant reactor and slowly stirred at 30 °C under an oxygen pressure of 1.5 atmospheres for 12 hours. After the reaction, the pH of the solution was adjusted to 10 and then extracted three times with 800 ml of ethyl acetate. The extracted organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 2.6 g of a light yellow liquid (yield 16%, HPLC purity 71%)

[0101] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A composite enzyme, which consists of an amine oxidase mutant, a nicotine synthase mutant, a phosphite dehydrogenase mutant, and a catalase; The amino acid sequence of the phosphite dehydrogenase mutant is shown in SEQ ID NO:7; The amino acid sequence of the nicotine synthase mutant is shown in SEQ ID NO:5; The amino acid sequence of the amine oxidase mutant is shown in SEQ ID NO:1 or SEQ ID NO:2; The enzyme activity ratio of the amine oxidase mutant, the nicotine synthase mutant, the phosphite dehydrogenase mutant, and the catalase is (1.5 - 2.5):(2.5 - 5):(4 - 8):

1.

2. The composite enzyme according to claim 1, wherein, The enzyme activity ratio of the amine oxidase mutant, nicotine synthase mutant, phosphite dehydrogenase mutant and catalase is 2:4:6:

1.

3. Use of the composite enzyme according to claim 1 in the preparation of S-nicotine.

4. A method for preparing S-nicotine, characterized in that, Comprising: Under the conditions of the presence of a solvent, oxygen and NADPH, mixing 1-methylpyrrolidine and nicotinic acid with the complex enzyme according to claim 1 or 2, and reacting to generate S-nicotine.

5. The preparation method according to claim 4, wherein, The NADPH is generated by an NADPH regeneration system, and the NADPH regeneration system comprises β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and the phosphite dehydrogenase mutant according to claim 1; The solvent is tris(hydroxymethyl)aminomethane hydrochloride or tris(hydroxymethyl)aminomethane hydrochloride containing a cosolvent.

6. The preparation method according to claim 5, wherein, Comprising: Sequentially adding 1-methylpyrrolidine nicotinic acid, β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and isopropanol into a tris(hydroxymethyl)aminomethane hydrochloride solution, adjusting the pH to 6.5-9.0, and adding the complex enzyme to obtain a reaction system; Reacting the reaction system under slow stirring at 25-35 °C under an oxygen pressure of 1.0-2.0 atmospheres for 4-8 hours. After the reaction is completed, adjusting the pH to 9.0-11.0, extracting with ethyl acetate, combining the organic phases, drying, filtering and concentrating to obtain S-nicotine.

7. The preparation method according to claim 6, wherein, In the reaction system: The concentration of the 1-methylpyrrolidine is 150-250 mM; The concentration of the nicotinic acid is 100-300 mM; The concentration of the β-nicotinamide adenine dinucleotide phosphate monosodium salt is 0.2-0.6 mM; The concentration of the sodium phosphite pentahydrate is 200-300 mM; The volume fraction of the isopropanol is 1-5%.

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