A method for preparing S-nicotine

Abstract

The present invention relates to the field of biosynthesis technology, and in particular to a method for preparing S-nicotine. The present invention pioneeringly utilizes amine oxidase to oxidize 1-methylpyrrolidine to the corresponding imine, and then condenses and decarboxylates the imine with nicotinic acid under the catalysis of nicotine synthase to obtain the final product S-nicotine. The method can obtain chiral-specific 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 material source is wide, the price is low, the production cost is low, and it is environmentally friendly, which significantly reduces the production cost of nicotine and also makes it more in line with the needs of today's green industrial production.

Description

[0001] This application is a divisional application with an application date of August 10, 2021, application number 202110914222.4, and invention name “A method for preparing S-nicotine”. Technical Field

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

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

[0004] Traditional tobacco cultivation, extraction, and purification methods occupy a large area, take a long time, and cause great harm to the human body because other highly toxic substances are inevitably contained in the extraction process. Therefore, direct synthesis using chemical or biological processes has become an important way to prepare S-nicotine.

[0005] Several common methods for preparing S-nicotine:

[0006]

[0007] Route I: Chemical preparation of racemic nicotine. Using pyridine acetaldehyde as raw material, nicotine racemate is prepared through three-step chemical reaction, and then chiral separation is performed using chemical reagents or enzymes to obtain S-nicotine. The above chemical reactions require the use of complex and dangerous production processes such as highly toxic (NaCN, etc.) and explosive (RaneyNi hydrogenation). (Reference: International Patent WO2017 / 119003AI; "A PROCESS FOR THE PREPARATION OF NICOTINE").

[0008] Route II: Direct preparation of S-nicotine by chemical method. Using pyridineethylamine as the starting material, S-nicotine is obtained by three-step chemical conversion. The catalysts for the first two steps 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 nicotine precursor Myosmine as raw material, and then using enzyme chiral reduction and chemical reagent methylation to prepare S-nicotine. Although this route is relatively short and has a high yield, the production cost is high because expensive Myosmine is used as the starting material.

[0010] From the above three classic S-nicotine preparation processes, it can be concluded that although the two chemical preparation methods of route I and route II also use relatively cheap raw materials and the overall route is not long (three or four steps), the factors such as expensive chemical catalysts (BF3, LDA, NaBH4) and dangerous operations (LDA, NaCN) involved in the reaction lead to high environmental and safety costs in large-scale production. Route III uses mesamine as a raw material, uses imine reductase to chirally reduce S-nornicotine, and then uses a chemical method for methylation; the defect of this route is that it needs to use expensive mesamine as a raw material, which greatly increases the overall production cost. At the same time, with the development of science and technology, the country has higher and higher requirements for the green production index of the chemical industry. Therefore, it is of great significance to provide a production process for 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 converts them into S-nicotine at one time, has a simple process, a high reaction yield, a low cost, and is environmentally friendly.

[0012] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0013] The present invention provides an amine oxidase mutant, the amino acid sequence of which is:

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

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

[0016] An amino acid sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and has 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, amine oxidase mutant 1 (AO1) and amine oxidase mutant 2 (AO2) are both derived from a monoamine oxidase in Aspergillus niger, and the amino acid sequence number of the wild-type enzyme is: Uniprot ID: P46882, EC1.4.3.4.

[0018] The amine oxidase mutant comprises five mutation sites: M242R, W230I, T354S, Y365V and W430R, and is named as amine oxidase mutant 1 (abbreviated as AO1), and its amino acid sequence is shown in SEQ ID NO:1.

[0019] In some embodiments, the amine oxidase mutant comprises 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 shown in SEQ ID NO:2.

[0020] The present invention also provides a nucleic acid encoding the amine oxidase mutant.

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

[0022] The present invention provides a nicotine synthase mutant, the amino acid sequence of which is:

[0023] The amino acid sequence shown in SEQ ID NO: 3; or

[0024] An amino acid sequence having the same or similar function as SEQ ID NO:5 obtained by substitution, deletion or addition of one or more amino acids to SEQ ID NO:5; or

[0025] An amino acid sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO:5 and has 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 the Solanaceae plant Anisodus acutangulus, and the amino acid sequence number of the wild-type nicotine synthase is 6J1M.

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

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

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

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

[0031] The amino acid sequence 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 substitution, deletion or addition of one or more amino acids to SEQ ID NO:7; or

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

[0034] In the present invention, the phosphite dehydrogenase (PTDH) mutant is transformed from a phosphite dehydrogenase in Pseudomonas stutzeri, and the wild-type amino acid sequence is Uniprot ID: O69054, EC1.20.1.1.

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

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

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

[0038] The present invention provides a composite enzyme, comprising at least two of the following as shown in (a) to (b):

[0039] (a) amine oxidase or a mutant thereof;

[0040] (b) nicotine synthase or a mutant thereof;

[0041] (c) phosphite dehydrogenase or a mutant thereof;

[0042] (d) catalase or a mutant thereof.

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

[0044] In some embodiments, the composite enzyme of the present invention includes the enzymes shown in (a) to (b):

[0045] (a) amine oxidase or a mutant thereof; and

[0046] (b) nicotine synthase or a mutant thereof; and

[0047] (c) phosphite dehydrogenase or a mutant thereof; and

[0048] (d) catalase or a mutant thereof.

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

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

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

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

[0053] The catalase was purchased. In a specific embodiment of the present invention, the catalase was purchased from Terminox Ultra.

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

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

[0056] In the presence of a solvent, oxygen and NADPH, 1-methylpyrrolidine, nicotinic acid and the complex enzyme are mixed and reacted to generate S-nicotine.

[0057] In the preparation method provided by the present invention, 1-methylpyrrolidine is oxidized to the corresponding imine by using amine oxidase or its mutant in the complex enzyme, 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 is shown in Figure 1 .

[0058] Among them, oxygen is required in the first step of the oxidation reaction, and the reaction will also produce hydrogen peroxide as a by-product. Therefore, in some embodiments, the present invention can effectively remove hydrogen peroxide in the system by adding a small amount of catalase, while also recycling O2.

[0059] The second step of the condensation decarboxylation reaction requires the participation of the coenzyme NADPH. Since this coenzyme is relatively expensive, by adding an NADPH regeneration system (phosphite oxidase PTDH) into the same system, the coenzyme can be effectively regenerated, thereby greatly reducing its usage and reducing production costs.

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

[0061] In some embodiments, the solvent is a tris(hydroxymethyl)aminomethane hydrochloric acid solution or a tris(hydroxymethyl)aminomethane hydrochloric acid solution containing a cosolvent. The cosolvent can promote the dissolution of each substrate in the solvent, which is conducive to the reaction. The present invention believes that the commonly used feasible cosolvent types can include but are not limited to isopropanol, acetone, DMSO, among which, in the specific embodiment of the present invention, isopropanol is used as the substrate cosolvent, and the effect is better.

[0062] Specifically, the method for preparing S-nicotine of the present invention comprises:

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

[0064] The reaction system is slowly stirred and reacted 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, extracted with ethyl acetate, and the organic phases are combined, dried, filtered, and concentrated to obtain S-nicotine.

[0065] In some specific embodiments, the method for preparing S-nicotine comprises:

[0066] 1-Methylpyrrolidine nicotinic acid, β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and isopropanol are sequentially added to a tris(hydroxymethylaminomethane) hydrochloric acid solution, and after adjusting the pH to 8.0, a composite enzyme is 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, the pH was adjusted to 10.0. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried, filtered, and concentrated to obtain S-nicotine.

[0068] In some embodiments, the composite enzyme of the present invention comprises an amine oxidase mutant, a phosphite dehydrogenase mutant, a nicotine synthase mutant and a catalase. The ratio of the enzyme activities 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 ratio of the enzyme activities 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, specifically 150 mM, 200 mM or 250 mM;

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

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

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

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

[0075] The present invention pioneered the use of amine oxidase to oxidize 1-methylpyrrolidine to the corresponding imine, and then condensed and decarboxylated the imine with nicotinic acid under the catalysis of nicotine synthase to obtain the final product S-nicotine. This method can obtain chiral-specific S-nicotine through two-step reactions in one 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, low in price, low in production cost, and environmentally friendly, which significantly reduces the production cost of nicotine and also makes it more in line with the needs of today's green industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The synthetic route of S-nicotine of the present invention is shown;

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

[0078] Figure 3 Example 1 (S) - Nicotine 1 H-NMR spectrum, 400M Varian, D2O solvent. DETAILED DESCRIPTION

[0079] The present invention provides a method for preparing S-nicotine. Those skilled in the art can refer to the content of this article and appropriately improve 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 are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of 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 common commercial products and can be purchased in the market.

[0081] In the present invention, catalase was purchased from 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 constructing an engineered strain and fermenting. The specific method includes:

[0082] First, the gene corresponding to the above enzyme mutant enzyme was synthesized (synthesized by Anhui General Biotechnology), and then subcloned into the pET28a plasmid with NdeI / XhoI restriction sites. The constructed plasmid was transferred into the E. coli (BL21) strain (Qingke Biotechnology) for plate culture, and finally a single clone was selected for step-by-step liquid culture. First, it was transferred into 5ml LB culture medium (37℃) containing 50μM kanamycin for culture. When the cells grew to the logarithmic phase, they were inoculated into 250ml LB culture medium containing the same antibiotics, and finally transferred to a 5L culture fermenter for culture; when the cell OD was ~15, 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added at 30℃ to induce protein expression for 10 hours, and finally the cells were collected by high-speed centrifugation (6000rpm, 15min) to obtain 40-60g of wet cells. Take a small amount of cells and mix them evenly with Tris.HCl buffer (50mM, pH8.0), then break the cells by freeze-thaw method, and use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine protein expression in the supernatant after high-speed centrifugation. The remaining cells that have confirmed correct protein expression are also mixed evenly with the above buffer on ice (10 grams of wet cells are mixed with about 200ml buffer), and then the cell wall is broken by high pressure. After high-speed centrifugation (16000rpm, 45min), the enzyme-containing clear liquid is obtained and used directly (in the case of liquid enzyme reaction, the enzyme activity is 200-350U / ml, U is the amount of enzyme required to convert 1μmol of substrate in one minute at room temperature) or further purified and immobilized for use (in the case of solid enzyme reaction). LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0083] In the present invention, the complex enzyme can be in liquid form or in the solid form of an immobilized enzyme. The immobilized enzyme can be recovered after the reaction is completed and can be reused. In some embodiments, liquid complex enzymes are used to prepare S-nicotine. In other embodiments, immobilized complex enzymes are used to prepare S-nicotine, and the immobilized complex enzymes are 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 pH8.0 Tris buffer, 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 (SEQ ID NO: 1), 4000U NS (SEQ ID NO: 5), 1000U Catalase, 6000U PTDH (SEQ ID NO: 7);

[0088] The reaction solution was then transferred into a pressure-resistant reactor and maintained at 1.5 atmospheres of oxygen pressure at 30°C for 6 hours with slow stirring. After the reaction, the pH of the solution was adjusted to 10 and 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 grams of light yellow liquid (yield 68%, HPLC purity 91%).

[0089] Example 2: One-pot preparation of S-nicotine using liquid enzyme (AO2, NS)

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

[0091] Subsequently, the reaction solution was transferred into a pressure-resistant reactor and stirred at 30° C. for 4 hours under an oxygen pressure of 1.5 atmospheres. The reaction was completed by HPLC detection. 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 29.8 g of a light yellow liquid (yield 92%). The chromatographic purity of S-nicotine was 95% after detection.

[0092] Example 3: One-pot preparation of S-nicotine by immobilized composite enzyme (AO2, NS, PTDH, Catalase)

[0093] Similar to Example 2, the difference is that an immobilized complex enzyme is used (immobilized mixed enzyme is prepared according to the method of the present invention), and the immobilized enzyme can be recycled after the reaction is 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) are added to 1 L of 50 mM Tris.HCl solution at pH 8.0. + ) monosodium salt (0.2mM), 26g sodium phosphite pentahydrate (120mM) and 100ml isopropanol. After adjusting the pH value of the reaction solution to 8.0 with NaOH aqueous solution, 6000-8000U mixed immobilized enzyme (i.e. 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 into a pressure-resistant reactor and maintained at 30° C. for a slight shaking reaction for 12 hours under an oxygen pressure of 1.5 atmospheres. After the reaction was completed, the immobilized complex enzyme was filtered and recovered (the immobilized enzyme was washed three times with 50mM pH 8.0 Tris buffer and stored at 4° C. for standby use). The pH value of the filtrate 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 13.6 grams of light yellow liquid (yield 84%, purity 98%). The immobilized mixed enzyme recovered by filtration had an initial enzyme activity of 75-90%.

[0095] Comparative Example 1 (without solvent)

[0096] Similar to Example (2), 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) were added to 1 L of 50 mM Tris-HCl solution (pH 8.0). + ) monosodium salt (0.4mM), 26g sodium phosphite pentahydrate (120mM). After adjusting the pH value of the reaction solution to 8.0 with a NaOH aqueous solution, a composite enzyme was added to obtain a reaction solution; wherein the composite enzyme composition is: 1000U AO2 (SEQ ID NO: 2), 2000U UNS (SEQ ID NO: 5), 1000U Catalase, 3000U PTDH (SEQ ID NO: 7);

[0097] Subsequently, the reaction solution was transferred into a pressure-resistant reactor and stirred at 30° C. for 8 hours under an oxygen pressure of 1.5 atmospheres. The reaction was completed by HPLC detection. The pH of the solution was adjusted to 10 and then 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 light yellow liquid (yield 43%). The chromatographic purity of S-nicotine was 84% ​​after detection.

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

[0099] Similar to Example 1, 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) were added to 1 L of 50 mM Tris-HCl solution (pH 8.0). +) monosodium salt (0.4mM), 26g sodium phosphite pentahydrate (120mM) 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: 6000U AO (wild type, Uniprot ID: P46882, EC 1.4.3.4), 2000UNS (SEQ ID NO: 5), 1000U Catalase, 3000U PTDH (SEQ ID NO: 7);

[0100] The reaction solution was then transferred to a pressure-resistant reactor and maintained at 1.5 atmospheres of oxygen pressure at 30°C for 12 hours with slow stirring. After the reaction, the pH of the solution was adjusted to 10 and 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 light yellow liquid (yield 16%, HPLC purity 71%).

[0101] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite enzyme consisting of a wild-type amine oxidase, a phosphite dehydrogenase mutant, a nicotine synthase mutant and a catalase; The wild-type amine oxidase Uniprot ID number is P46882, and the EC number is 1.4.3.4; 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.

2. The complex enzyme according to claim 1, characterized in that The nucleotide sequence of the nucleic acid encoding the nicotine synthase mutant is shown in SEQ ID NO:

6.

3. The complex enzyme according to claim 1, characterized in that The nucleotide sequence of the nucleic acid encoding the phosphite dehydrogenase mutant is shown in SEQ ID NO:

8.

4. Use of the complex enzyme according to any one of claims 1 to 3 in the preparation of S-nicotine.

5. A method for preparing S-nicotine, characterized in that: include: In the presence of a solvent, oxygen and NADPH, 1-methylpyrrolidine, nicotinic acid and the composite enzyme according to any one of claims 1 to 3 are mixed and reacted to generate S-nicotine; The solvent is tris(hydroxymethyl)aminomethane hydrochloric acid containing isopropanol.

6. The preparation method according to claim 5, characterized in that: The NADPH is generated by a NADPH regeneration system, which includes β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and the phosphite dehydrogenase mutant.

7. The preparation method according to claim 5, characterized in that: include: 1-methylpyrrolidine, nicotinic acid, β-nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate and isopropanol are sequentially added to a tris(hydroxymethyl)aminomethane hydrochloric acid solution, the pH is adjusted to 6.5-9.0, and a composite enzyme is added to obtain a reaction system; The reaction system is slowly stirred and reacted at 25-35° C. for 4-8 hours under an oxygen pressure of 1.0-2.0 atmospheres. After the reaction, the pH is adjusted to 9.0-11.0, extracted with ethyl acetate, and the organic phases are combined, dried, filtered, and concentrated to obtain S-nicotine.

8. The preparation method according to claim 7, characterized in that: In the reaction system: The concentration of the 1-methylpyrrolidine is 150-250 mM; The concentration of niacin 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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