WO2023015712A1 - 一种s-尼古丁的制备方法 - Google Patents

一种s-尼古丁的制备方法 Download PDF

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WO2023015712A1
WO2023015712A1 PCT/CN2021/123012 CN2021123012W WO2023015712A1 WO 2023015712 A1 WO2023015712 A1 WO 2023015712A1 CN 2021123012 W CN2021123012 W CN 2021123012W WO 2023015712 A1 WO2023015712 A1 WO 2023015712A1
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mutant
nicotine
amino acid
acid sequence
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赵弘
凌瑞枚
付琳
于铁妹
潘俊锋
刘建
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深圳瑞德林生物技术有限公司
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    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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    • C12Y120/00—Oxidoreductases acting on phosphorus or arsenic in donors (1.20)
    • C12Y120/01—Oxidoreductases acting on phosphorus or arsenic in donors (1.20) with NAD+ or NADP+ as acceptor (1.20.1)
    • C12Y120/01001—Phosphonate dehydrogenase (1.20.1.1)

Definitions

  • the invention relates to the technical field of biosynthesis, in particular to a preparation method of S-nicotine.
  • Nicotine is an important component in tobacco, and it is also the core raw material for e-cigarette formulations and the synthesis of certain nicotine drugs.
  • Route I Preparation of racemic nicotine by chemical method. Using pyridine acetaldehyde as a raw material, a nicotine racemate is prepared through three-step chemical reactions, and then chiral resolution is performed using chemical reagents or enzymes to obtain S-nicotine. The chemical reactions in the above steps require the use of highly toxic (NaCN, etc.), explosive (RaneyNi hydrogenation) and other complex and dangerous production processes. (References: International Patent WO2017/119003 AI; "A PROCESS FOR THE PREPARATION OF NICOTINE").
  • Route III use the nicotine precursor Myosmine (Myosmine) as a raw material, and then use enzymatic chiral reduction and chemical reagent methylation to prepare S-nicotine.
  • Myosmine Myosmine
  • enzymatic chiral reduction and chemical reagent methylation to prepare S-nicotine.
  • the route is relatively short and the yield is high, the production cost is high due to the use of expensive mysmin as the starting material.
  • the present invention provides a method for preparing S-nicotine.
  • the method uses 1-methylpyrrolidine and nicotinic acid as raw materials, and converts it into S-nicotine at one time.
  • the process is simple, the reaction yield is high, and the cost is low. , friendly to the environment.
  • the present invention provides an amine oxidase mutant whose amino acid sequence is:
  • SEQ ID NO: 1 or SEQ ID NO: 2 is substituted, deleted or added one or more amino acids to obtain an amino acid sequence with the same or similar function as SEQ ID NO: 2; or
  • amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 and functionally identical or similar to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.
  • the amine oxidase mutant 1 (AO1) and the amine oxidase mutant 2 (AO2) are all derived from a monoamine oxidase in Aspergillus niger (Aspergillus niger), and the amino acid sequence number of the wild-type enzyme is : Uniprot ID: P46882, EC 1.4.3.4.
  • 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 shown in SEQ ID NO: 1 .
  • the amine oxidase mutant comprises 10 site mutations: F210M, L213C, M242V, I246T, R259K, R260K, N336S, T384N, D385S, W430G, named as amine oxidase mutant 2 (abbreviated as AO2 ), its amino acid sequence is shown in SEQ ID NO:2.
  • the present invention also provides nucleic acid encoding the amine oxidase mutant.
  • the nucleotide sequence encoding the oxidase mutant is shown in SEQ ID NO:3 or SEQ ID NO:4.
  • the nucleotide sequence encoding amine oxidase mutant 1 (AO1) is shown in SEQ ID NO: 3
  • the nucleotide sequence encoding amine oxidase mutant 2 (AO2) is shown in SEQ ID NO: 4.
  • the present invention provides a nicotine synthase mutant whose amino acid sequence is:
  • amino acid sequence with the same or similar function as SEQ ID NO:5 obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:5; or
  • the nicotine synthase mutant is derived from a redox-type condensing enzyme of Anisodus acutangulus, and the amino acid sequence number of the wild-type nicotine synthase is 6J1M.
  • 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 As shown in SEQ ID NO:5.
  • the present invention also provides nucleic acid encoding the nicotine synthase mutant.
  • the nucleotide sequence encoding the nicotine synthase mutant is shown in SEQ ID NO:6.
  • the present invention provides its amino acid sequence as:
  • amino acid sequence with the same or similar function as SEQ ID NO: 7 obtained by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 7; or
  • the phosphorous acid dehydrogenase (PTDH) mutant is transformed from a phosphorous acid dehydrogenase in Pseudomonas stutzeri, and the wild-type amino acid sequence is Uniprot ID: O69054, EC 1.20.1.1 .
  • 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 As shown in SEQ ID NO:7.
  • the present invention also provides nucleic acid encoding the phosphorous acid dehydrogenase mutant.
  • nucleotide sequence encoding the phosphite dehydrogenase mutant is shown in SEQ ID NO:8.
  • the present invention provides a complex enzyme, including at least two of the following as shown in (a) to (b):
  • the complex enzyme includes the two enzymes shown in (a) to (b), ie, an amine oxidase mutant and a phosphorous acid dehydrogenase mutant.
  • the complex enzyme of the present invention includes the enzymes shown in (a) to (b):
  • the compound enzyme provided by the present invention includes mutants of amine oxidase, mutants of phosphorous acid dehydrogenase, mutants of nicotine synthase and catalase.
  • the amino acid sequence of the amine oxidase mutant is as shown in SEQ ID NO: 1 or SEQ ID NO: 2;
  • the amino acid sequence of the nicotine synthase mutant is shown in SEQ ID NO:5;
  • amino acid sequence of the phosphorous acid dehydrogenase mutant is shown in SEQ ID NO:7;
  • the catalase (catalase) was purchased, and in the specific embodiment of the present invention, the catalase was purchased from Novozymes Enzyme Preparation Company (Terminox Ultra).
  • the invention also provides the application of the compound enzyme in preparing S-nicotine.
  • the present invention also provides a preparation method of S-nicotine, comprising:
  • 1-methylpyrrolidine and nicotinic acid are mixed with the compound enzyme to react to generate S-nicotine.
  • 1-methylpyrrolidine is oxidized to the corresponding imine by using the amine oxidase in the complex enzyme or its mutant, and then the imine is catalyzed by nicotine synthase or its mutant Condensation and decarboxylation with nicotinic acid to obtain S-nicotine, the synthetic route diagram is shown in Figure 1.
  • the present invention can effectively remove hydrogen peroxide in the system by adding a small amount of catalase (catalase). Hydrogen peroxide, while recycling O 2 .
  • catalase catalase
  • the condensation decarboxylation reaction in the second step requires the participation of the coenzyme NADPH. Since the coenzyme is relatively expensive, the coenzyme can be effectively regenerated by adding the NADPH regeneration system (phosphorous acid oxidase PTDH) in the same system, thereby greatly reducing its consumption and reducing the cost of the coenzyme. Cost of production.
  • NADPH regeneration system phosphorous acid oxidase PTDH
  • the NADPH is produced by a NADPH regeneration system comprising ⁇ -nicotinamide adenine dinucleotide phosphate monosodium salt, sodium phosphite pentahydrate, and a phosphite dehydrogenase mutant.
  • the solvent is a tris hydrochloric acid solution or a tris hydrochloric acid solution containing a co-solvent.
  • the co-solvent can promote the dissolution of each substrate in the solvent, which is beneficial to the reaction.
  • commonly used feasible co-solvent types can be used, including but not limited to isopropanol, acetone, DMSO, wherein, in the specific embodiments of the present invention, isopropanol is used as the substrate co-solvent, and the effect is better.
  • the preparation method of S-nicotine of the present invention comprises:
  • the preparation method of S-nicotine includes:
  • the reaction system was slowly stirred and reacted at 30°C under an oxygen pressure of 1.5 atmospheres for 6 hours. After the reaction was completed, the pH was adjusted to 10.0, extracted with ethyl acetate, combined with organic phases, dried, filtered, and concentrated to obtain S-nicotine .
  • the complex enzymes of the present invention include mutants of amine oxidase, mutants of phosphorous acid dehydrogenase, mutants of nicotine synthase and catalase.
  • the ratio of enzyme activity of the amine oxidase mutant, nicotine synthase mutant, phosphorous acid dehydrogenase mutant and catalase is preferably (1.5 ⁇ 2.5):(2.5-5):(4 ⁇ 8): 1.
  • the enzyme activity ratio of the amine oxidase mutant, the nicotine synthase mutant, the phosphorous acid dehydrogenase mutant and the catalase is 2:4:6:1.
  • reaction system in the reaction system:
  • the concentration of the 1-methylpyrrolidine is 150-250mM, specifically 150mM, 200mM or 250mM;
  • the concentration of niacin per gram is 150-250mM, specifically 150mM, 200mM or 250mM;
  • concentration of ⁇ -nicotinamide adenine dinucleotide phosphate monosodium salt is 0.2-0.6mM, specifically 0.2mM, 0.4mM or 0.6mM;
  • the concentration of sodium phosphite pentahydrate is 200-300mM, specifically 200mM, 240mM or 300mM;
  • the volume fraction of the isopropanol is 1-5%, specifically 1% or 5%.
  • the invention creatively utilizes amine oxidase to oxidize 1-methylpyrrolidine to corresponding imine, and then condenses and decarboxylates the imine and nicotinic acid under the catalysis of nicotine synthase to obtain the final product S-nicotine.
  • chiral specific S-nicotine can be obtained through two-step reactions in a reaction system, and the synthetic route is short, the yield is high, the reaction conditions are mild, and the large-scale production is easy; at the same time, the source of raw materials is wide, the price is low, and the production The cost is low, and it is friendly to the environment. While significantly reducing the cost of nicotine production, it also makes it more in line with the needs of today's green industrial production.
  • Fig. 1 shows the synthetic route figure of S-nicotine of the present invention
  • Fig. 2 shows the mass spectrogram of embodiment 1 of the present invention (S)-nicotine
  • Fig. 3 shows the (S)-nicotine 1 H-NMR chart of Example 1 of the present invention, 400M Varian NMR, D 2 O solvent.
  • the invention provides a preparation method of S-nicotine.
  • Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all 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 make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • test materials used in the present invention are all common commercial products, which can be purchased in the market.
  • catalase is purchased from Novozyme (Terminox Ultra), three other enzymes, amino oxidase AO1 mutant (SEQ ID NO: 1), amino oxidase AO2 mutant (SEQ ID NO: 1) ID NO: 2), nicotine synthase NS mutant (SEQ ID NO: 5) and phosphorous acid oxidase PTDH mutant (SEQ ID NO: 7) are all produced by the present invention through the construction of engineering strains. Specific methods include:
  • the genes corresponding to the mutant enzymes above were synthesized (produced by Anhui General Biosynthesis), and then subcloned into the pET28a plasmid with NdeI/XhoI restriction sites.
  • the constructed plasmid was transferred into E.coli (BL21) strain (Qingke Biology) for plate culture, and finally a single clone was selected for step-by-step liquid culture.
  • Tris.HCl tris-hydrochloride
  • SDS-PAGE polyacrylamide gel electrophoresis
  • the composition of LB medium was: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.
  • the complex enzyme can be in a liquid form or in a solid form of an immobilized enzyme, and the immobilized enzyme can be recovered after the reaction and can be reused.
  • liquid compound enzyme is used to prepare S-nicotine.
  • S-nicotine is prepared by using immobilized compound enzyme, and the immobilized compound enzyme is prepared according to the following steps:
  • Ammonium sulfate solids are gradually added to the amine oxidase crude liquid (AO1 or AO2), nicotine synthase crude liquid (NS) and phosphorous acid oxidase crude liquid (PTDH) obtained by fermentation of the present invention until precipitation (25%-60% , w/v ammonium sulfate/buffer).
  • the enzyme solid was then collected by centrifugation (10000rpm, 12min), and slowly dissolved into 25mM Tris buffer at pH 8.0, desalted through a G25 size exclusion chromatography column (purchased from Sigma) and desalted using DEAE Seplite FF (Xi'an Lanxiao Company) ) anion exchange column separation to obtain primary purified liquid enzymes AO1, AO2, NS, PTDH. Finally, AO1/AO2, NS, PTDH and Novozymes’ Catalase were mixed and fixed at one time using LX-1000EP epoxy resin (Xi’an Lanxiao Company) according to the activity unit 2:4:6:1.
  • the immobilization method is as follows: 1000U of mixed enzyme was dissolved in 1L of 50mM potassium phosphate solution with pH 8.0, then 40mM phenoxyacetic acid and 300g of LX-1000EP epoxy resin were added to the buffer solution, and the immobilized enzyme was filtered out after stirring at room temperature for 4 hours. Wash with water and 25mM pH 8.0 phosphate buffer three times each, then dry at low temperature for use. The immobilized mixed enzymes had 65-92% of the activity of the corresponding liquid enzymes.
  • Example 1 One-pot method for preparing S-nicotine by liquid enzyme (AO1, NS)
  • the compound enzyme consists of: 2000U AO1 (SEQ ID NO:1), 4000U NS (SEQ ID NO:5), 1000U Catalase, 6000U PTDH (SEQ ID NO: 7);
  • reaction solution was transferred to a pressure-resistant reactor to maintain an oxygen pressure of 1.5 atmospheres at 30° C. and slowly stirred for 6 hours.
  • pH of the solution was adjusted to 10 and extracted three times with 700 ml of ethyl acetate, and the extracted organic phases were combined. After drying over anhydrous sodium sulfate, filter and concentrate to obtain 22 g of light yellow liquid (68% yield, 91% HPLC purity).
  • Embodiment 2 liquid enzyme (AO2, NS) prepares S-nicotine in one pot
  • Example 2 The difference from Example 1 is that the amino 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), and 3.0 grams of ⁇ -nicotinamide were successively added to 1L of 50 mM pH 8.0 trishydrochloride (Tris.HCl) solution. Adenine dinucleotide phosphate (NADP + ) monosodium salt (0.4mM), 52g sodium phosphite pentahydrate (240mM) and 100ml isopropanol.
  • NADP + Adenine dinucleotide phosphate
  • 0.4mM 52g sodium phosphite pentahydrate
  • 100ml isopropanol.
  • the compound enzyme consists of: 2000U AO2 (SEQ ID NO:2), 4000UNS (SEQ ID NO:5), 1000U Catalase, 6000U PTDH (SEQ ID NO: 7);
  • reaction solution was transferred to a pressure-resistant reactor to maintain 1.5 atmospheric pressure of oxygen at 30° C. and stirred for 4 hours.
  • the pH of the solution was adjusted to 10
  • the phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 29.8 g of light yellow liquid (yield 92%), and the chromatographic purity of S-nicotine was 95%.
  • Example 3 One-pot preparation of S-nicotine by immobilized complex enzymes (AO2, NS, PTDH, Catalase)
  • the immobilized complex enzyme is used (the immobilized mixed enzyme is prepared according to the method of the present invention), and the immobilized enzyme can be recycled after the reaction.
  • 8.5 grams of 1-methylpyrrolidine (100 mM), 12.3 grams of nicotinic acid (100 mM), and 1.5 grams of ⁇ -nicotinamide adenine were added to 1L of 50 mM pH 8.0 trishydrochloride (Tris.HCl) solution.
  • reaction solution After adjusting the pH value of the reaction solution to 8.0 with NaOH aqueous solution, add 6000-8000 U of mixed immobilized enzyme (i.e. compound enzyme) to obtain the reaction solution; wherein the compound enzyme is: AO2 mutant (SEQ ID NO: 2), NS mutant (SEQ ID NO:5), Catalase, PTDH mutant (SEQ ID NO:7);
  • the reaction solution was transferred to a pressure-resistant reactor to maintain an oxygen pressure of 1.5 atmospheric pressure at 30°C for 12 hours of slight shaking reaction, and after the reaction was completed, the immobilized complex enzyme was filtered and recovered (the immobilized enzyme was washed with 50mM pH 8.0 Tris buffer solution) After three times, it was stored at 4°C for later use), the pH value of the filtrate was adjusted to 10 and then extracted three times with 700ml 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%) , the purity is 98%), and the immobilized mixed enzyme recovered by filtration has 75-90% of the initial enzyme activity.
  • Example 2 Similar to Example (2), 8.5 grams of 1-methylpyrrolidine (100 mM), 12.3 grams of nicotinic acid (100 mM), 3.0 gram of ⁇ -nicotinamide adenine dinucleotide phosphate (NADP + ) monosodium salt (0.4mM), 26 grams of sodium phosphite pentahydrate (120mM).
  • reaction solution was transferred into a pressure-resistant reactor to maintain 1.5 atmospheric pressure of oxygen pressure at 30° C. and stirred for 8 hours.
  • HPLC detected that the reaction was completed and the pH of the solution was adjusted to 10.
  • it was extracted three times with 500 ml of ethyl acetate, and the extracted organic The 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.
  • the compound enzyme consists of: 6000U AO (wild type, Uniprot ID: P46882, EC 1.4.3.4), 2000U NS (SEQ ID NO :5), 1000U Catalase, 3000U PTDH (SEQ ID NO:7);
  • reaction solution was transferred to a pressure-resistant reactor to maintain an oxygen pressure of 1.5 atmospheres at 30° C. and slowly stirred for 12 hours.
  • pH of the solution was adjusted to 10 and extracted three times with 800 ml of ethyl acetate, and the extracted organic phases were combined. After drying over anhydrous sodium sulfate, filter and concentrate to obtain 2.6 g of light yellow liquid (yield 16%, HPLC purity 71%)

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Abstract

涉及生物合成技术领域，具体涉及一种S-烟碱的制备方法。利用胺氧化酶将1-甲基吡咯烷氧化成相应的亚胺，然后在烟碱合成酶的催化下，亚胺与烟酸缩合脱羧得到最终产物S-烟碱。在一个反应体系中通过两步反应即可得到具有特定手性的S-烟碱，合成路线短，收率高，反应条件温，易于实现规模化生产；同时，原料来源广泛，价格低廉，生产成本低，环境友好，显著降低了烟碱的生产成本，同时也较好的满足了目前绿色工业化生产的要求。

Description

一种S-尼古丁的制备方法
本申请要求于2021年8月10日提交中国专利局、申请号为202110914222.4、发明名称为“一种S-尼古丁的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物合成技术领域，尤其涉及一种S-尼古丁的制备方法。
背景技术
尼古丁是烟草中的重要成分，同时也是电子烟配方、某些烟碱类药物合成的核心原料。
传统的烟叶栽培、提取、纯化的方法占地面积大、耗费周期长，且由于提取中不可避免的会含有其它剧毒成本而导致人体危害大。因此利用化学或生物工艺直接合成成为S-尼古丁制备的重要途径。
几种常见的S-尼古丁制备方法：
Figure PCTCN2021123012-appb-000001
路线I:化学法制备外消旋尼古丁。通过吡啶乙醛做原料，通过三步化学反应制备尼古丁消旋体，然后利用化学试剂或酶进行手性拆分得S-尼古丁。以上几步化学反应需要用到剧毒(NaCN等),易爆(RaneyNi氢化)等复杂危险生产工艺。(参考文献：国际专利WO2017/119003 AI；“A  PROCESS FOR THE PREPARATION OF NICOTINE”)。
路线II:化学法直接制备S-尼古丁。利用吡啶乙胺作为起始原料，三步化学转化得到S-尼古丁。前两步反应催化剂昂贵，反应条件苛刻；最终收率也偏低(<50％)。(参考文献:Josha T.Ayers,AAPS 2005,“A General Procedure for the Enantioselective Synthesis of the Minor Tobacco Alkaloids Nornicotine,Anabasine,and Anatabine”).
路线III:利用尼古丁前体麦司明(Myosmine)作原料，然后利用酶手性还原、化学试剂甲基化制备S-尼古丁。虽然该路线比较短，收率高，但由于使用昂贵的麦司明作为起始原料，故生产成本高。
以上三种经典的S-尼古丁制备工艺可以得出，路线I、路线II两种化学制备方法虽然也采用比较便宜的原料，整体路线也不长(三步或四步反应),但是反应所涉及到的化学催化剂昂贵(BF 3,LDA,NaBH 4)、操作危险(LDA,NaCN)等因素导致其规模化生产中的环境成本与安全成本都很高。路线III以麦司明作为原料，利用亚胺还原酶手性还原得S-去甲烟碱(nornicotine),然后采用化学法进行甲基化；该路线的缺陷是需要利用昂贵的麦司明作为原料，从而使整体生产成本大大增加。同时，随着科学技术的发展，国家对化学工业的绿色生产指数要求越来越高。因此，提供一种操作步骤简单、成本低、安全环保的S-尼古丁的生产工艺具有重要的意义。
发明内容
有鉴于此，本发明提供了一种S-尼古丁的制备方法，该方法以1-甲基吡咯烷与烟酸为原料，一次性转化成S-尼古丁，工艺简单，反应收率高，成本低，对环境友好。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了胺氧化酶突变体，其氨基酸序列为：
如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；或
SEQ ID NO:1或SEQ ID NO:2经取代、缺失或添加一个或多个氨基酸获得的功能与SEQ ID NO:2相同或相似的氨基酸序列；或
与SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列至少有90％同源性且功能与SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列相同或相似的氨基酸序列。
本发明中，胺氧化酶突变体1(AO1)与胺氧化酶突变体2(AO2)均来源于黑曲霉(Aspergillus niger)体内的一个单胺基氧化酶，该野生型酶的氨基酸序列号为：Uniprot ID:P46882,EC 1.4.3.4。
其中，所述胺氧化酶突变体包含5个位点突变：M242R、W230I、T354S、Y365V、W430R，命名为胺氧化酶突变体1(简称AO1)其，氨基酸序列如SEQ ID NO:1所示。
一些实施方案中，所述胺氧化酶突变体包含10个位点突变：F210M,L213C,M242V,I246T,R259K,R260K，N336S,T384N,D385S,W430G，命名为胺氧化酶突变体2(简称AO2)，其氨基酸序列如SEQ ID NO:2所示。
本发明还提供了编码所述胺氧化酶突变体的核酸。
一些实施方案中，编码所述的氧化酶突变体的核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示。其中，编码胺氧化酶突变体1(AO1)的核苷酸序列如SEQ ID NO:3所示，编码胺氧化酶突变体2(AO2)的核苷酸序列如SEQ ID NO:4所示。
本发明提供了尼古丁合成酶突变体，其氨基酸序列为：
如SEQ ID NO:3所示的氨基酸序列；或
SEQ ID NO:5经取代、缺失或添加一个或多个氨基酸获得的功能与SEQ ID NO:5相同或相似的氨基酸序列；或
与SEQ ID NO:5所示氨基酸序列至少有90％同源性且功能与SEQ ID NO:5相同或相似的氨基酸序列。
本发明中，尼古丁合成酶突变体来源于茄科植物三分三(Anisodus acutangulus)的一种氧化还原型缩合酶，野生型尼古丁合成酶的氨基酸序列号为6J1M。
一些实施方案中，所述尼古丁合成酶突变体包含14个位点突变：M17H,R112T,Q113F,L162A,Q180E,F183A,S212K,A229P,P248L,V254R,A261H,K341V,R346T,G394T，其氨基酸序列如SEQ ID NO:5所 示。
本发明还提供了编码所述的尼古丁合成酶突变体的核酸。
一些实施方案中，编码所述的尼古丁合成酶突变体的核苷酸序列如SEQ ID NO:6所示。
本发明提供了其氨基酸序列为：
如SEQ ID NO:7所示的氨基酸序列；或
SEQ ID NO:7经取代、缺失或添加一个或多个氨基酸获得的功能与SEQ ID NO:7相同或相似的氨基酸序列；或
与SEQ ID NO:7所示氨基酸序列至少有90％同源性且功能与SEQ ID NO:7相同或相似的氨基酸序列。
本发明中，亚磷酸脱氢酶(PTDH)突变体由施氏假单胞菌(Pseudomonas stutzeri)体内一个亚磷酸脱氢酶改造而来，野生型氨基酸序列为Uniprot ID:O69054,EC 1.20.1.1。
一些实施方案中，所述亚磷酸脱氢酶突变体包含13个位点突变：V71I,Q132R,E130K,Q137R,I150F,A176R,Q215L,R275Q,L276Q,I313L,V315A,A319E,A325V，其氨基酸序列如SEQ ID NO:7所示。
本发明还提供了编码所述亚磷酸脱氢酶突变体的核酸。
一些实施方案中，编码所述亚磷酸脱氢酶突变体的核苷酸序列如SEQ ID NO:8所示。
本发明提供一种复合酶，包括如下至少如(a)～(b)所示的两种：
(a)胺氧化酶或其突变体；
(b)尼古丁合成酶或其突变体；
(c)亚磷酸脱氢酶或其突变体；
(d)过氧化氢酶或其突变体。
本发明中，一些实施方案中，所述复合酶包括(a)～(b)所示的两种，即胺氧化酶突变体和亚磷酸脱氢酶突变体两种酶。
一些实施方案中，本发明所述复合酶包括(a)～(b)所示的酶：
(a)胺氧化酶或其突变体；和
(b)尼古丁合成酶或其突变体；和
(c)亚磷酸脱氢酶或其突变体；和
(d)过氧化氢酶或其突变体。
一些具体实施例中，本发明提供的复合酶包括胺氧化酶突变体、亚磷酸脱氢酶突变体、尼古丁合成酶突变体和过氧化氢酶。
一些实施方案中，本发明提供的上述复合酶中，所述胺氧化酶突变体的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
所述尼古丁合成酶突变体的氨基酸序列如SEQ ID NO:5所示；
所述亚磷酸脱氢酶突变体的氨基酸序列如SEQ ID NO:7所示；
所述过氧化氢酶(catalase)购买获得，本发明具体实施例中，过氧化氢酶购于诺维信酶制剂公司(Terminox Ultra)。
本发明还提供了所述复合酶在制备S-尼古丁中的应用。
本发明还提供一种S-尼古丁的制备方法，包括：
在溶剂、氧气和NADPH存在的条件下，将1-甲基吡咯烷、烟酸与所述复合酶混合，反应，生成S-尼古丁。
本发明提供的制备方法中，利用复合酶中的胺氧化酶或其突变体将1-甲基吡咯烷氧化成对应的亚胺，然后再尼古丁合成酶或其突变体催化下将所述亚胺与烟酸缩合脱羧，得到S-尼古丁，合成路线图见图1。
其中，第一步的氧化反应中需要用到氧气，反应同时会产生过氧化氢副产物，因此，在一些实施方案中，本发明通过添加少量的过氧化氢酶(catalase)可以有效除去体系内的过氧化氢，同时还能循环利用O 2。
第二步的缩合脱羧反应需要辅酶NADPH的参与，由于该辅酶比较昂贵，通过在同一体系内添加NADPH再生系统(亚磷酸氧化酶PTDH)，可有效再生该辅酶，从而得以大大减少其用量，降低生产成本。
一些实施方案中，所述NADPH由NADPH再生系统生成，所述NADPH再生系统包括β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐、五水亚磷酸钠和亚磷酸脱氢酶突变体。
一些实施方案中，所述溶剂为三羟甲基氨基甲烷盐酸溶液或含助溶剂的三羟甲基氨基甲烷盐酸溶液。助溶剂可促进各底物在溶剂中的溶解，有利于反应的进行。本发明认为常用的可行的助溶剂种类均可，包括但不仅 限于异丙醇、丙酮、DMSO，其中，本发明具体实施例中，以异丙醇为底物助溶剂，效果更优。
具体地，本发明S-尼古丁的制备方法，包括：
在三羟甲基氨基甲烷盐酸溶液中依次加入1-甲基吡咯烷烟酸、β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐、五水亚磷酸钠和异丙醇，调节pH至6.5～9.0加入复合酶，获得反应体系；
将所述反应体系于1.0～2.0大气压的氧气压力下25～35℃缓慢搅拌反应4～8小时，反应结束后调节pH至9.0～11.0经乙酸乙酯萃取、合并有机相，干燥、过滤、浓缩后，获得S-尼古丁。
一些具体实施例中，S-尼古丁的制备方法，包括：
在三羟甲基氨基甲烷盐酸溶液中依次加入1-甲基吡咯烷烟酸、β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐、五水亚磷酸钠和异丙醇，调节pH至8.0后加入复合酶，获得反应体系；
将所述反应体系于1.5大气压的氧气压力下30℃缓慢搅拌反应6小时，反应结束后调节pH至10.0，依次经乙酸乙酯萃取、合并有机相、干燥、过滤、浓缩后，获得S-尼古丁。
一些实施方案中，本发明所述复合酶包括胺氧化酶突变体、亚磷酸脱氢酶突变体、尼古丁合成酶突变体和过氧化氢酶。其中，所述胺氧化酶突变体、尼古丁合成酶突变体、亚磷酸脱氢酶突变体和过氧化氢酶的酶活力之比优选为(1.5～2.5)：(2.5-5)：(4～8)：1。一些具体实施例中，胺氧化酶突变体、尼古丁合成酶突变体、亚磷酸脱氢酶突变体和过氧化氢酶的酶活力之比为2：4：6：1。
一些实施方案中，所述反应体系中：
所述1-甲基吡咯烷的浓度为150～250mM，具体可为150mM、200mM或250mM；
所述克烟酸的浓度为150～250mM，具体可为150mM、200mM或250mM；
所述β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐的浓度为0.2～0.6mM，具体可为0.2mM、0.4mM或0.6mM；
所述五水亚磷酸钠的浓度为200～300mM，具体可为200mM、240mM或300mM；
所述异丙醇的体积分数为1～5％，具体可为1％或5％。
本发明开创性地利用胺氧化酶将1-甲基吡咯烷氧化成对应的亚胺，然后在尼古丁合成酶催化下将该亚胺与烟酸缩合脱羧得到最终产品S-尼古丁。该方法在一个反应体系中通过两步反应，即可获得手性专一的S-尼古丁，合成路线短、收率高、反应条件温和易规模化生产；同时，原料来源广泛、价格低廉，生产成本较低，对环境友好，在显著降低尼古丁生产成本的同时也使得其更符合当今绿色工业生产的需求。
附图说明
图1示本发明S-尼古丁的合成路线图；
图2示本发明实施例1(S)-尼古丁的质谱图；
图3示本发明实施例1(S)-尼古丁 1H-NMR图，400M Varian核磁,D 2O溶剂。
具体实施方式
本发明提供了一种S-尼古丁的制备方法。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
如无其他特殊说明，本发明采用的试材皆为普通市售品，皆可于市场购得。
本发明中，过氧化氢酶(catalase)购于诺维信(Terminox Ultra)，另外三种酶，胺基氧化酶AO1突变体(SEQ ID NO:1)、胺基氧化酶AO2突变体(SEQ ID NO:2)、尼古丁合成酶NS突变体(SEQ ID NO:5)以及亚磷酸氧化酶PTDH突变体(SEQ ID NO:7)均由本发明通过构建工程菌株发 酵制得。具体方法包括：
首先合成以上酶突变体酶所对应的基因(由安徽通用生物合成),再以NdeI/XhoI酶切位点亚克隆到pET28a质粒上。构建好的质粒转入E.coli(BL21)菌株(擎科生物)进行平板培养，最后挑单克隆进行逐级液体培养。首先转入5ml含50μM卡那霉素的LB培养液中(37℃)进行培养，当细胞生长至对数期后接种到250ml含同样抗菌素的LB培养液中，最后再转入5L培养发酵罐里进行培养；当细胞OD～15加入0.5mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)30℃诱导蛋白表达10小时，最后高速离心收集细胞(6000rpm,15min)获得湿细胞40-60g。取少量细胞与三羟甲基氨基甲烷盐酸(Tris.HCl)缓冲液(50mM,pH 8.0)混合均匀，然后利用冻融法破碎细胞，高速离心后上清液利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定蛋白表达。蛋白表达确认正确的剩余细胞同样与上述缓冲液在冰上混合均匀(10克湿细胞与约200ml缓冲液混合)，然后进行高压破碎细胞壁，高速离心(16000rpm,45min)后获得含酶清液直接使用(液体酶液反应时，酶活力在200-350U/ml,U是室温一分钟转化1μmol的底物所需酶量)或进一步纯化后固定化使用(固体酶反应时)。LB培养基构成为：1％胰蛋白胨、0.5％酵母粉，1％NaCl，1％磷酸氢二钾、1％磷酸氢二钾以及5％的甘油。
在本发明中，复合酶可以是液态形式也可以是固定化酶的固态形式，固定化酶在反应结束后可以回收，能够重复利用。一些实施例中采用液态复合酶制备S-尼古丁。另一些实施例中，采用固定化复合酶制备S-尼古丁，所述固定化复合酶的按照如下步骤制备：
将本发明发酵获得的胺氧化酶粗液(AO1或AO2)、尼古丁合成酶粗液(NS)、亚磷酸氧化酶粗液(PTDH)中逐量加入硫酸铵固体直至析出(25％-60％,w/v硫酸铵/缓冲液)。该酶固体随后通过离心收集(10000rpm,12min)，并缓慢溶入25mM pH 8.0的Tris缓冲液中,再经G25尺寸排阻色谱柱脱盐(购于Sigma)以及利用DEAE Seplite FF(西安蓝晓公司)阴离子交换柱分离得到初纯化液体酶AO1、AO2、NS、PTDH。最后AO1/AO2、NS、PTDH以及诺维信的Catalase利用LX-1000EP环氧树脂(西安蓝晓公 司)按照活性单位2:4:6:1进行一次性混合固定。固定方法如下:1000U混合酶溶解在1L 50mM pH 8.0的磷酸钾溶液中，随后加入40mM苯氧乙酸以及300克LX-1000EP环氧树脂至缓冲液,室温搅拌4小时后过滤出固定化酶,最后用清水以及25mM pH 8.0磷酸缓冲液各洗涤三次后低温干燥待用。固定化混合酶具有对应液体酶的65-92％的活性。
下面结合实施例，进一步阐述本发明：
实施例1液体酶(AO1,NS)一锅法制备S-尼古丁
在1L 50mM pH 8.0的三羟甲基氨基甲烷盐酸(Tris.HCl)溶液中先后加入17克1-甲基吡咯烷(200mM),24.6克烟酸(200mM),3.0克β-烟酰胺腺嘌呤二核苷酸磷酸(NADP +)单钠盐(0.4mM),52克五水亚磷酸钠(240mM)以及100ml异丙醇(底物助溶剂)。利用NaOH水溶液调节反应液pH值到8.0后，加入复合酶，获得反应液；其中，复合酶组成为：2000U AO1(SEQ ID NO:1)、4000U NS(SEQ ID NO:5)、1000U Catalase、6000U PTDH(SEQ ID NO:7)；
随后将反应液转入耐压反应器中维持1.5大气压的氧气压力下30℃缓慢搅拌反应6小时，反应结束后将溶液pH调节到10后用700ml乙酸乙酯萃取三次，将萃取有机相合并，无水硫酸钠干燥后过滤、浓缩得22克浅黄色液体(收率68％，HPLC纯度91％)。
实施例2：液体酶(AO2,NS)一锅法制备S-尼古丁
与实施例1的区别在于：胺基氧化酶AO2替换AO1，其他过程相同。同样的在1L 50mM pH 8.0的三羟甲基氨基甲烷盐酸(Tris.HCl)溶液中先后加入17克1-甲基吡咯烷(200mM),24.6克烟酸(200mM),3.0克β-烟酰胺腺嘌呤二核苷酸磷酸(NADP +)单钠盐(0.4mM),52克五水亚磷酸钠(240mM)以及100ml异丙醇。利用NaOH水溶液调节反应液pH值到8.0后，加入复合酶，获得反应液；其中，复合酶组成为：2000U AO2(SEQ ID NO:2)、4000UNS(SEQ ID NO:5)、1000U Catalase、6000U PTDH(SEQ ID NO:7)；
随后将所述反应液转入耐压反应器中维持1.5大气压的氧气压力下 30℃搅拌反应4小时，HPLC检测反应完成并将溶液pH调节到10后用700ml乙酸乙酯萃取三次，将萃取有机相合并，无水硫酸钠干燥后过滤、浓缩得29.8克浅黄色液体(收率92％)，经检测S-尼古丁色谱纯度为95％。
实施例3：固定化复合酶(AO2、NS、PTDH、Catalase)一锅法制备S-尼古丁
与实施例2类似，不同之处在于利用固定化复合酶(按照本发明的方法制备固定化混合酶)，反应结束后固定化酶还可以回收使用。同样在1L 50mM pH 8.0的三羟甲基氨基甲烷盐酸(Tris.HCl)溶液中加入8.5克1-甲基吡咯烷(100mM),12.3克烟酸(100mM),1.5克β-烟酰胺腺嘌呤二核苷酸磷酸(NADP +)单钠盐(0.2mM),26克五水亚磷酸钠(120mM)以及100ml异丙醇。利用NaOH水溶液调节反应液pH值到8.0后，加入6000-8000U混合固定化酶(即复合酶)，获得反应液；其中，复合酶为：AO2突变体(SEQ ID NO:2)、NS突变体(SEQ ID NO:5)、Catalase、PTDH突变体(SEQ ID NO:7)；
随后将所述反应液转入耐压反应器中维持1.5大气压的氧气压力下30℃轻微震荡反应12小时，待反应完成后过滤回收固定化复合酶(固定化酶用50mM pH 8.0 Tris缓冲液洗涤三次后4℃保存待用)，滤液pH值调节到10后用700ml乙酸乙酯萃取三次，将萃取有机相合并，无水硫酸钠干燥后过滤、浓缩得13.6克浅黄色液体(收率84％,纯度为98％)，过滤回收的固定化混合酶具有75-90％的初始酶活力。
对比例1(没有助溶剂)
和实施例(2)类似在1L 50mM pH 8.0的三羟甲基氨基甲烷盐酸(Tris.HCl)溶液中先后加入8.5克1-甲基吡咯烷(100mM),12.3克烟酸(100mM),3.0克β-烟酰胺腺嘌呤二核苷酸磷酸(NADP +)单钠盐(0.4mM),26克五水亚磷酸钠(120mM)。利用NaOH水溶液调节反应液pH值到8.0后，加入复合酶，获得反应液；其中，复合酶组成为：1000U AO2(SEQ ID NO:2)、2000U NS(SEQ ID NO:5)、1000U Catalase、3000U PTDH(SEQ ID NO:7)；
随后将所述反应液转入耐压反应器中维持1.5大气压的氧气压力下 30℃搅拌反应8小时，HPLC检测反应完成并将溶液pH调节到10后用500ml乙酸乙酯萃取三次，将萃取有机相合并，无水硫酸钠干燥后过滤、浓缩得6.8克浅黄色液体(收率43％)，经检测S-尼古丁色谱纯度为84％。
对比例2(AO为野生型)
与上述实施例1类似在1L 50mM pH 8.0的三羟甲基氨基甲烷盐酸(Tris.HCl)溶液中先后加入8.5克1-甲基吡咯烷(100mM),12.3克烟酸(100mM),3.0克β-烟酰胺腺嘌呤二核苷酸磷酸(NADP +)单钠盐(0.4mM),26克五水亚磷酸钠(120mM)以及100ml异丙醇(底物助溶剂)。利用NaOH水溶液调节反应液pH值到8.0后，加入复合酶，获得反应液；其中，复合酶组成为：6000U AO(野生型，Uniprot ID:P46882,EC 1.4.3.4)、2000U NS(SEQ ID NO:5)、1000U Catalase、3000U PTDH(SEQ ID NO:7)；
随后将反应液转入耐压反应器中维持1.5大气压的氧气压力下30℃缓慢搅拌反应12小时，反应结束后将溶液pH调节到10后用800ml乙酸乙酯萃取三次，将萃取有机相合并，无水硫酸钠干燥后过滤、浓缩得2.6克浅黄色液体(收率16％，HPLC纯度71％)
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (19)

  1. 胺氧化酶突变体，其氨基酸序列为：
    如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；或
    SEQ ID NO:1或SEQ ID NO:2经取代、缺失或添加一个或多个氨基酸获得的功能与SEQ ID NO:2相同或相似的氨基酸序列；或
    与SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列至少有90％同源性且功能与SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列相同或相似的氨基酸序列。
  2. 编码权利要求1所述的胺氧化酶突变体的核酸。
  3. 根据权利要求2所述核酸，其特征在于，所述核酸的核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示。
  4. 尼古丁合成酶突变体，其氨基酸序列为：
    如SEQ ID NO:3所示的氨基酸序列；或
    SEQ ID NO:5经取代、缺失或添加一个或多个氨基酸获得的功能与SEQ ID NO:5相同或相似的氨基酸序列；或
    与SEQ ID NO:5所示氨基酸序列至少有90％同源性且功能与SEQ ID NO:5相同或相似的氨基酸序列。
  5. 编码权利要求4所述的尼古丁合成酶突变体的核酸。
  6. 根据权利要求5所述核酸，其特征在于，所述核酸的核苷酸序列如SEQ ID NO:6所示。
  7. 亚磷酸脱氢酶突变体，其氨基酸序列为：
    如SEQ ID NO:7所示的氨基酸序列；或
    SEQ ID NO:7经取代、缺失或添加一个或多个氨基酸获得的功能与SEQ ID NO:7相同或相似的氨基酸序列；或
    与SEQ ID NO:7所示氨基酸序列至少有90％同源性且功能与SEQ ID NO:7相同或相似的氨基酸序列。
  8. 编码权利要求4所述的亚磷酸脱氢酶突变体的核酸。
  9. 根据权利要求5所述核酸，其特征在于，所述核酸的核苷酸序列 如SEQ ID NO:8所示。
  10. 一种复合酶，包括如下至少如(a)～(b)所示的两种：
    (a)胺氧化酶或其突变体；
    (b)尼古丁合成酶或其突变体；
    (c)亚磷酸脱氢酶或其突变体；
    (d)过氧化氢酶或其突变体。
  11. 根据权利要求10所述的复合酶，其特征在于，包括胺氧化酶突变体和尼古丁合成酶突变体；
    所述胺氧化酶突变体的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
    所述尼古丁合成酶突变体的氨基酸序列如SEQ ID NO:5所示。
  12. 根据权利要求11所述的复合酶，其特征在于，所述复合酶还包括亚磷酸脱氢酶突变体和过氧化氢酶；
    所述亚磷酸脱氢酶突变体的氨基酸序列如SEQ ID NO:7所示。
  13. 权利要求10～12任一项所述的复合酶在制备S-尼古丁中的应用。
  14. 一种S-尼古丁的制备方法，其特征在于，包括：
    在溶剂、氧气和NADPH存在的条件下，将1-甲基吡咯烷、烟酸与权利要求10～12任一项所述的复合酶混合，反应，生成S-尼古丁。
  15. 根据权利要求14所述的制备方法，其特征在于，所述NADPH由NADPH再生系统生成，所述NADPH再生系统包括β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐、五水亚磷酸钠和亚磷酸脱氢酶突变体；
    所述溶剂为三羟甲基氨基甲烷盐酸或含助溶剂的三羟甲基氨基甲烷盐酸。
  16. 根据权利要求15所述的制备方法，其特征在于，包括：
    在三羟甲基氨基甲烷盐酸溶液中依次加入1-甲基吡咯烷烟酸、β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐、五水亚磷酸钠和异丙醇，调节pH至6.5～9.0加入复合酶，获得反应体系；
    将所述反应体系于1.0～2.0大气压的氧气压力下25～35℃缓慢搅拌反 应4～8小时，反应结束后调节pH至9.0～11.0经乙酸乙酯萃取、合并有机相，干燥、过滤、浓缩后，获得S-尼古丁。
  17. 根据权利要求14～16任一项所述的制备方法，其特征在于，所述复合酶包括胺氧化酶突变体、亚磷酸脱氢酶突变体、尼古丁合成酶突变体和过氧化氢酶。
  18. 根据权利要求17所述的制备方法，其特征在于，所述胺氧化酶突变体、尼古丁合成酶突变体、亚磷酸脱氢酶突变体和过氧化氢酶的酶活力之比为(1.5～2.5)：(3～5)：(4～8)：1。
  19. 根据权利要求14～18任一项所述的制备方法，其特征在于，所述反应体系中：
    所述1-甲基吡咯烷的浓度为150～250mM；
    所述克烟酸的浓度为100～300mM；
    所述β-烟酰胺腺嘌呤二核苷酸磷酸单钠盐的浓度为0.2～0.6mM；
    所述五水亚磷酸钠的浓度为200～300mM；
    所述异丙醇的体积分数为1～5％。
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