WO2022268006A1 - 亚胺还原酶突变体、亚胺还原酶和葡萄糖脱氢酶共表达酶及其应用 - Google Patents

亚胺还原酶突变体、亚胺还原酶和葡萄糖脱氢酶共表达酶及其应用 Download PDF

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WO2022268006A1
WO2022268006A1 PCT/CN2022/099711 CN2022099711W WO2022268006A1 WO 2022268006 A1 WO2022268006 A1 WO 2022268006A1 CN 2022099711 W CN2022099711 W CN 2022099711W WO 2022268006 A1 WO2022268006 A1 WO 2022268006A1
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imine reductase
nicotine
glucose dehydrogenase
mutant
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French (fr)
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陈泽聪
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陈泽聪
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Priority to EP22827500.4A priority Critical patent/EP4361260A1/en
Priority to KR1020237044009A priority patent/KR20240011764A/ko
Priority to CN202280035192.2A priority patent/CN117751184A/zh
Publication of WO2022268006A1 publication Critical patent/WO2022268006A1/zh
Priority to US18/389,868 priority patent/US20240141306A1/en

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    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • 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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    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)

Definitions

  • the present invention relates to the field of medicine and chemical industry, in particular to an imine reductase mutant and the application of the mutant, and also relates to the co-expression enzyme of the imine reductase mutant and glucose dehydrogenase and the co-expression Enzyme application.
  • (S)-Nicotine is an alkaloid found in plants of the Solanaceae family and an important component of tobacco. Although there are relevant reports on chemical synthesis methods, (R,S)-nicotine is synthesized, and (S)-nicotine can only be obtained after splitting. Therefore, the cost of chemical synthesis is higher than the cost of extracting nicotine from tobacco leaves.
  • (S)-nicotine is catalyzed by enzyme catalysis, which greatly reduces the cost of preparation and is environmentally friendly.
  • Patent WO2014174505 discloses a method for preparing nicotine using sp.GF3587 and 3546.
  • the reaction substrate is 4-(methylamino)-1-(pyridin-3-yl)butanone, the conversion rate is only 23%, and only (R)-nicotine can be prepared.
  • Patent WO2020098978 discloses a method for preparing (S)-nicotine using imine reductase.
  • Imine reductase catalyzes mesmin into nornicotine, which is methylated to obtain (S)-nicotine.
  • the conversion rate of the enzyme used can reach 77% after 8 hours and 99% after 24 hours.
  • the enzyme used in the patent comes from Enzymicals.
  • imine reductase is an oxidase that relies on the coenzyme NADPH
  • imine reductase and glucose dehydrogenase in patents WO2014174505 and WO2020098978 are prepared separately and used in combination. This method increases the steps of enzyme preparation. Increased the cost of obtaining enzymes. Li Jixuan et al.
  • the first object of the present invention is to provide a mutant of imine reductase, which solves the problem that nicotine in the S configuration cannot be obtained by using the enzyme in the prior art, and the enzyme activity in the prior art is low.
  • This mutant can convert The substrate Mesmin or 4-(methylamino)-1-(pyridin-3-yl)butanone is catalytically reduced to (S)-nicotine.
  • the second object of the present invention is to provide the co-expression enzyme of this imine reductase and glucose dehydrogenase, specifically the co-expression enzyme of imine reductase mutant enzyme and glucose dehydrogenase, the application and preparation of this co-expression enzyme (S)-nicotine reduces the cost of enzyme-catalyzed preparation of (S)-nicotine.
  • the imine reductase mutant Compared with the amino acid sequence of SEQ ID NO: 1, the imine reductase mutant has residue differences including one or more of V171, A172, and Y230.
  • SEQ ID NO: 1 is a wild-type imine reductase derived from Aeromonas veronii, which is mutated to obtain the imine reductase mutant of the present invention, and the mutation points include one or more of V171, A172, and Y230 .
  • the activity of the mutated imine reductase is 31 to 50 times that of the wild-type enzyme.
  • the residue difference includes one or more of V171Y/N/A/S, A172V/F, Y230G/A/T.
  • Residue differences also include:
  • the amino acid sequence of the imine reductase mutant is selected from the group consisting of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 , 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 , 89, 91, 93, 95, 97, 99, 101, 105, 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO : 135, SEQ ID NO: 137, SEQ ID NO:
  • the imine reductase mutant of the present invention is used for preparing (S)-nicotine, and under suitable conditions, the imine reductase mutant catalyzes the substrate I
  • Suitable conditions refer to that when the substrate is Max, after mixing the substrate I, coenzyme, glucose, glucose dehydrogenase, buffer and imine reductase, the compound III(S)-nornicotine is reacted.
  • the load of substrate I is 10g/L to 300g/L; the concentration of enzyme is 1g/L to 10g/L; the amount of coenzyme is 1g/L; the amount of glucose dehydrogenase is 2g/L; The range is 6 to 7; the reaction temperature is 23°C to 30°C; the reaction time is 15 hours to 24 hours.
  • the load of substrate I includes 10g/L, 50g/L, 100g/L, 150g/L, 200g/L, 250g/L, 300g/L;
  • the concentration of the enzyme used to catalyze substrate I is 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L.
  • the reaction temperature when catalyzing the substrate I is 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C.
  • the reaction time when catalyzing substrate I is 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours.
  • Compound III (S)-nornicotine is methylated to obtain (S)-nicotine.
  • (S)-nornicotine reacts with formic acid/formaldehyde to obtain (S)-nicotine.
  • Substrate II is cyclized and catalyzed by imine reductase to obtain (S)-nicotine;
  • the salt of the substrate II includes hydrochloride, dihydrochloride, hydrobromide, dihydrobromide, sulfate or bisulfate.
  • Substrate II can be cyclized into enamine compound or iminium compound, the structure is as follows:
  • the suitable condition when imine reductase is catalyzed is after substrate II, coenzyme, glucose, glucose dehydrogenase, buffer solution and imine reductase are mixed, react to obtain (S)-nicotine.
  • the load of substrate II is 10g/L to 300g/L; the concentration of enzyme is 1g/L to 10g/L; the amount of coenzyme is 1g/L; the amount of glucose dehydrogenase is 2g/L; The range is 6 to 7; the reaction temperature is 23°C to 30°C; the reaction time is 15 hours to 24 hours.
  • the load of substrate II includes 10g/L, 50g/L, 100g/L, 150g/L, 200g/L, 250g/L, 300g/L;
  • the concentration of the enzyme used to catalyze substrate II is 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L.
  • the reaction temperature when catalyzing the substrate II is 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C.
  • the reaction time when catalyzing the substrate II is 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours.
  • Amino acid sequence SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 105, 107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO: 121.
  • the corresponding nucleic acid sequence is selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 , 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 , 96, 98, 100, 102, 104, 106, 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120 , SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID
  • An expression vector including the above nucleic acid.
  • a carrier cell the above nucleotide or a vector comprising the above nucleic acid.
  • a method for producing an imine reductase mutant The imine reductase mutant and glucose dehydrogenase are co-expressed. Coexpressed enzymes are produced using cells containing both the imine reductase mutant gene and the glucose dehydrogenase gene.
  • these nucleotide sequences can be inserted into a plasmid, such as pET-30a(+), to prepare a vector.
  • vectors are transformed into engineering bacteria, such as Escherichia coli BL21(DE3), to prepare vector cells.
  • the imine reductase mutant and the glucose dehydrogenase gene are expressed in the same engineering bacterium to prepare co-expressed enzymes.
  • the cost of separately preparing the glucose dehydrogenase is solved, and the co-expression of the imine reductase and the glucose dehydrogenase realizes the repeated use of the coenzyme in the system.
  • the glucose dehydrogenase gene is amplified from the vector by primers with restriction endonuclease sites, respectively.
  • the vector with imine reductase gene fragment and the amplified glucose dehydrogenase gene fragment were double-digested with BamHI and XhoI endonucleases, the digested fragments were recovered by gel, and transformed into Escherichia coli BL21 after ligation with T4 DNA ligase Competent cells, obtained recombinant bacteria containing both imine reductase gene and glucose dehydrogenase gene, cultured the above recombinant bacteria, then induced, centrifuged to collect the bacteria, resuspended, and after resuspended, the cells were ultrasonically disrupted, and then After crushing, the solution is freeze-dried to obtain the co-expression enzyme powder of imine reductase
  • coenzyme, glucose, buffer are mixed with imine reductase and glucose dehydrogenase co-expression enzyme, react to obtain compound III (S)-nornicotine, then compound III (S) - methylation of nornicotine to give (S)-nicotine,
  • (S)-nicotine is catalyzed by the co-expression enzyme of imine reductase and glucose dehydrogenase;
  • the salt of the substrate II includes hydrochloride, dihydrochloride, hydrobromide, dihydrobromide, sulfate or bisulfate.
  • Substrate II or its salt is catalyzed by the imine reductase mutant of the present invention into (S)-nicotine, with a conversion rate of 99.9%, a chemical purity of 99.9%, and a substrate load of 300 g/L.
  • the imine reductase mutant of the present invention can catalyze the substrate I into (S)-nornicotine, the enzyme activity is higher, and the loading capacity of the substrate is increased.
  • Fig. 1 is the synthetic route diagram of (S)-(3-pyrrolidin-2-yl)pyridine
  • Fig. 2 is the reverse phase achiral HPLC collection of patterns of (S)-(3-pyrrolidin-2-yl)pyridine standard
  • Fig. 3 is the reverse phase achiral HPLC collection of patterns of substrate mesmin standard substance
  • Fig. 4 is the reverse-phase achiral HPLC collection of illustrative plates (SEQ ID NO:1 sample) that control is controlled in embodiment 5 reaction;
  • Fig. 5 is the normal-phase chiral HPLC spectrum of (R, S)-(3-pyrrolidin-2-yl)pyridine;
  • Fig. 6 is the normal-phase chiral HPLC spectrum of (S)-(3-pyrrolidin-2-yl)pyridine standard substance
  • Fig. 7 is the normal phase chiral HPLC collection of illustrative plates (SEQ ID NO:1 sample) of (S)-(3-pyrrolidin-2-yl) pyridine synthesized in embodiment 5;
  • Fig. 8 is the normal phase chiral HPLC collection of illustrative plates (SEQ ID NO:17 sample) of (S)-(3-pyrrolidin-2-yl) pyridine synthesized in embodiment 5;
  • Figure 9 is a co-expression plasmid map of imine reductase and glucose dehydrogenase
  • the coding gene SEQ ID NO: 1 of wild-type imine reductase was used as a template, and error-prone PCR was carried out using a random mutation kit (Beiyuntian).
  • the primers are shown in the table below, and a DNA fragment recovery kit (Shanghai Sangong ) and reclaim the above-mentioned error-prone PCR product according to the instructions.
  • the error-prone PCR product and the pET-30a(+) plasmid were double digested with restriction endonucleases NdeI and BamHI respectively, and the recovered digested products were ligated with T4 DNA ligase to linearize the pET-30a(+) plasmid medium, at 22°C for 1 hour.
  • the ligation product was transformed into Escherichia coli BL21(DE3) competent cells (Shanghai Sangong), cultured overnight at 37°C, and a random mutation library was obtained.
  • the obtained beneficial mutant enzyme gene SEQ ID NO: 37 was used as a template for site-directed saturation mutation and error-prone PCR again for multiple rounds of library construction and screening.
  • each well of the culture plate contains 150 ⁇ L LB liquid medium and 50 ⁇ g/mL kanamycin, and culture the bacteria in a shaker at 37°C and 220rpm Overnight; transfer 20 ⁇ L of the bacterial solution to a 96-well plate containing 380 ⁇ L of LB liquid medium and 50 ⁇ g/mL of kanamycin, continue to culture in a shaker at 37 °C and 220 rpm for 2 to 3 h, and add IPTG to a final concentration of 0.4mM for induction, and then cooled to 25°C for overnight culture (at least 16 hours).
  • enzyme activity the amount of enzyme required to generate 1 ⁇ mol of product per unit time is an enzyme activity unit.
  • V1 the amount of enzyme added
  • the cells were broken with an ultrasonic breaker (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), centrifuged (4°C, 10000rpm, 20min) to obtain the supernatant, part of which was used for activity detection, and the remaining supernatant was freeze-dried , to prepare enzyme powder.
  • an ultrasonic breaker JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.
  • ++ indicates that the enzyme activity is 1.2 to 3 times that of SEQ ID NO:1; +++ indicates that the enzyme activity is 5 to 10 times that of SEQ ID NO:1; ++++ indicates that the enzyme activity is SEQ ID NO:1 11 to 20 times; +++++ indicates that the enzyme activity is 21 to 30 times that of SEQ ID NO:1; ++++++ indicates that the enzyme activity is 31 to 50 times that of SEQ ID NO:1;
  • reaction bottle Take a 10mL reaction bottle, add 100mg of the substrate mesmin, add 4mL of phosphate buffer (pH6.0 0.1M), and adjust the pH to 6.0. Then add 2.5mg NADP and 180mg glucose to the reaction bottle, stir until completely dissolved, then add 10mg glucose dehydrogenase and 50mg imine reductase mutant enzyme powder to it, mix well and heat up to 25°C, at 300r/h Min stirring reaction 24h. After the reaction, 100 ⁇ L of the reaction solution was added to 900 ⁇ L of acetonitrile for full shaking, filtered with a 0.22 ⁇ m filter membrane, and analyzed by HPLC.
  • phosphate buffer pH6.0 0.1M
  • Example 8 The application of the imine reductase mutant marked as "+++++" in the preparation of S-(3-pyrrolidin-2-yl)pyridine in Table 2
  • SEQ ID NO:43 99.7 99.4 SEQ ID NO:45 99.1 99.5 SEQ ID NO:47 100 99.8 SEQ ID NO:61 100 100 SEQ ID NO:65 99.8 99.6 SEQ ID NO:73 99.7 99.8 SEQ ID NO:85 99.9 100
  • the prepared (S)-nicotine is characterized by proton nuclear magnetic spectrum, and the data are:
  • Example 13 The application of the imine reductase mutant shown in SEQ ID NO:61 in the preparation of (S)-nicotine
  • Example 14 The application of the imine reductase mutant shown in SEQ ID NO:85 in the preparation of (S)-nicotine
  • the glucose dehydrogenase gene was amplified from the pET30a-GDH (see SEQ ID NO: 171 for the sequence) vector by primers (see the table below) with restriction endonuclease sites.
  • the pET30a vector carrying the imine reductase gene fragment and the amplified glucose dehydrogenase gene fragment were double digested with restriction endonucleases BamHI and XhoI respectively.
  • the enzyme-digested fragments were recovered by gel, ligated with T4 DNA ligase, and then transformed into Escherichia coli BL21 (DE3) competent cells to obtain recombinant bacteria containing both the imine reductase gene and the glucose dehydrogenase gene (see Figure 9 for a schematic diagram of the plasmid).
  • reaction bottle Take a 10mL reaction bottle, add 750mg of the substrate mesmin, add 4mL of phosphate buffer (pH6.0 0.1M), and adjust the pH to 6.0. Then add 5mg NADP and 1200mg glucose in the reaction flask, stir until fully dissolved, then add co-expression enzyme powder (herein, the imine reductase is a mutant enzyme shown in SEQ ID NO:37, and the nucleotide sequence of the co-expression enzyme is Shown in SEQ ID NO: 173), mixed and heated to 25°C, stirred and reacted at 300r/min for 16h.
  • co-expression enzyme powder herein, the imine reductase is a mutant enzyme shown in SEQ ID NO:37, and the nucleotide sequence of the co-expression enzyme is Shown in SEQ ID NO: 173
  • reaction solution 100 ⁇ L was added to 900 ⁇ L of acetonitrile for full shaking, filtered with a 0.22 ⁇ m filter membrane, and analyzed by HPLC. Take 500 ⁇ L of the reaction solution, adjust the pH above 11, add 1000 ⁇ L of n-hexane for extraction, take the n-hexane layer and pass it through the membrane for normal phase HPLC analysis of chirality. The conversion rate was 99.6%, and the ee value was 99.8%.
  • the (S)-nornicotine prepared in Example 5-9 or 16 is used to prepare (S)-nicotine.
  • the obtained product was characterized by proton nuclear magnetic spectrum, and the result of nuclear magnetic data was:
  • the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

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Abstract

本发明公开了一种亚胺还原酶突变体，以及这种亚胺还原酶突变体用于制备(S)-尼古丁的制备方法。发明第一个目的是提供了一种亚胺还原酶突变体，解决现有技术使用酶得不到S构型的尼古丁，以及现有技术中酶活性低的问题，这种突变体可以将底物麦思明或4-(甲胺基)-1-(吡啶-3-基)丁酮催化还原为(S)-尼古丁。本发明第二个目的是提供这种亚胺还原酶与葡萄糖脱氢酶的共表达酶，具体是亚胺还原酶突变酶与葡萄糖脱氢酶的共表达酶，这种共表达酶应用与制备(S)-尼古丁，降低了酶催化制备(S)-尼古丁的成本。

Description

亚胺还原酶突变体、亚胺还原酶和葡萄糖脱氢酶共表达酶及其应用 [技术领域]
本发明涉及医药化工领域，具体涉及一种亚胺还原酶突变体，以及这种突变体的应用，还涉及这种亚胺还原酶突变体与葡萄糖脱氢酶的共表达酶以及这种共表达酶的应用。
[背景技术]
(S)-尼古丁是一种存在茄科植物的生物碱，也是烟草的重要成分。虽然有化学合成法的相关报道，但是合成的是(R,S)-尼古丁，经拆分后才能获得(S)-尼古丁。所以化学合成成本比从烟叶中提取烟碱的成本要高。
使用酶催化的方式催化制备(S)-尼古丁，大大降低制备的成本，而且绿色环保。
Koichi Mitsukura等人在2010年发现Streptomyces sp.GF3587和3546的亚胺还原酶(Org.Biomol.Chem.,2010,8,4533-4535)。使用这两种酶将2-MPN还原为S-2MP或者R-2MP。
专利WO2014174505公开使用sp.GF3587和3546制备尼古丁的方法。反应底物为4-(甲胺基)-1-(吡啶-3-基)丁酮，转化率仅有23％，而且仅能制备(R)-尼古丁。
专利WO2020098978中公开了使用亚胺还原酶制备(S)-尼古丁方法。亚胺还原酶催化麦思明为去甲烟碱，去甲烟碱甲基化后得到(S)-尼古丁。使用的酶转化率在8小时后的可以达到77％，24小时后达 到99％，专利中使用的酶来自Enzymicals。
现在公开使用酶催化制备(S)-尼古丁的技术，酶活性不够，或者仅能制备(R)-尼古丁。为此需要一种新的亚胺还原酶，能够催化底物制备(S)-尼古丁，而且酶的活性更高，在较高的底物起始浓度下，具有较高的转化率和光学纯度。
另外，亚胺还原酶是一种依赖辅酶NADPH的氧化酶，而专利WO2014174505、WO2020098978中的亚胺还原酶和葡萄糖脱氢酶都是单独制备后混合使用，这种方式增加了制备酶的步骤，提高了获得酶的成本。李骥璇等(生物技术通报，2019,35(1)：105-111)公开了S-亚胺还原酶和葡萄糖脱氢酶共表达系统的构建及手性胺的合成，可以将亚胺还原酶和葡萄糖脱氢酶共同表达，降低了使用酶的成本。但是李骥璇等公开的共表达系统仅公开了催化2-MPN还原为S-2MP的反应，而且一般的葡萄糖脱氢酶的活性要大于亚胺还原酶，共表达系统的亚胺还原酶与葡萄糖脱氢酶是等量的，这使得葡萄糖脱氢酶过量，在实际生产中，过量的酶易引起乳化，给后面的提纯带来不便。为了解决这个问题，需要一种活性更高的亚胺还原酶与葡萄糖脱氢酶一起形成共表达酶，并且能够催化底物生成(S)-尼古丁。
[发明内容]
本发明第一个目的是提供了一种亚胺还原酶突变体，解决现有技术使用酶得不到S构型的尼古丁，以及现有技术中酶活性低的问题，这种突变体可以将底物麦思明或4-(甲胺基)-1-(吡啶-3-基)丁酮催化还原为(S)-尼古丁。
本发明第二个目的是提供这种亚胺还原酶与葡萄糖脱氢酶的共表达酶，具体是亚胺还原酶突变酶与葡萄糖脱氢酶的共表达酶，这种共表达酶应用与制备(S)-尼古丁，降低了酶催化制备(S)-尼古丁的成本。
为了达到发明目的，本发明的技术方案如下：
亚胺还原酶突变体，与氨基酸序列SEQ ID NO：1相比，存在残基差异包括V171、A172、Y230中的一个或多个。
SEQ ID NO：1是来源于气单胞菌Aeromonas veronii的野生型亚胺还原酶，经过突变后得到本发明的亚胺还原酶突变体，突变点包括V171、A172、Y230中的一个或多个。通过对比催化底物麦思明的活性，突变后的亚胺还原酶的活性是野生型的酶的活性31到50倍。
所述残基差异包括V171Y/N/A/S、A172V/F、Y230G/A/T中的一个或多个。
残基差异还包括：
亚胺还原酶突变体的氨基酸序列选自SEQ ID NO:3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、105、107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137、 SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ ID NO:149、SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165或SEQ ID NO:167。
。本发明的亚胺还原酶突变体用于制备(S)-尼古丁，在合适的条件下，亚胺还原酶突变体催化底物I
Figure PCTCN2022099711-appb-000001
或底物II
Figure PCTCN2022099711-appb-000002
直接或经过化学反应得到(S)-尼古丁。
合适的条件是指当底物为麦斯时，将底物I、辅酶、葡萄糖、葡萄糖脱氢酶、缓冲液与亚胺还原酶混合后，反应得到化合物III(S)-去甲烟碱。
优选的，底物I的载量为10g/L至300g/L；酶的浓度为1g/L至10g/L；辅酶的用量为1g/L；葡萄糖脱氢酶的用量为2g/L；pH的范围为6至7；反应的温度为23℃至30℃；反应时间为15小时至24小时。
优选的，底物I的载量包括10g/L、50g/L、100g/L、150g/L、200g/L、250g/L、300g/L；
催化底物I使用的酶的浓度为1g/L、2g/L、3g/L、4g/L、5g/L、6g/L、 7g/L、8g/L、9g/L、10g/L。
催化底物I时的反应温度为23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃。
催化底物I时的反应时间15小时、16小时、17小时、18小时、19小时、20小时、21小时、22小时、23小时、24小时。
化合物III(S)-去甲烟碱甲基化得到(S)-尼古丁。如(S)-去甲烟碱与甲酸/甲醛反应得到(S)-尼古丁。
催化麦思明得到(S)-去甲烟碱，再经甲基化得到(S)-尼古丁的反应如下：
Figure PCTCN2022099711-appb-000003
底物II环化后经亚胺还原酶的催化得到(S)-尼古丁；
或底物II的盐经脱盐、环化经亚胺还原酶的催化得到(S)-尼古丁；
所述底物II的盐包括盐酸盐、二盐酸盐、氢溴酸盐、二氢溴酸盐、硫酸盐或硫酸氢盐。
底物II可环化为烯胺化合物或亚胺正离子化合物，结构如下：
Figure PCTCN2022099711-appb-000004
亚胺还原酶催化时的合适条件为将底物II、辅酶、葡萄糖、葡萄 糖脱氢酶、缓冲液与亚胺还原酶混合后，反应得到(S)-尼古丁。
优选的，底物II的载量为10g/L至300g/L；酶的浓度为1g/L至10g/L；辅酶的用量为1g/L；葡萄糖脱氢酶的用量为2g/L；pH的范围为6至7；反应的温度为23℃至30℃；反应时间为15小时至24小时。
优选的，底物II的载量包括10g/L、50g/L、100g/L、150g/L、200g/L、250g/L、300g/L；
催化底物II使用的酶的浓度为1g/L、2g/L、3g/L、4g/L、5g/L、6g/L、7g/L、8g/L、9g/L、10g/L。
催化底物II时的反应温度为23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃。
催化底物II时的反应时间15小时、16小时、17小时、18小时、19小时、20小时、21小时、22小时、23小时、24小时。
氨基酸序列SEQ ID NO:3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、105、107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:139、SEQ ID NO:141SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ ID NO:149、 SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165或SEQ ID NO:167。
对应核酸序列选自SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:122、SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128、SEQ ID NO:130、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:148、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:166或SEQ ID NO:168。
一种表达载体，包括以上的核酸。
一种载体细胞，以上的核苷酸或包括以上核酸的载体。
一种亚胺还原酶突变体的生产方法。将所述的亚胺还原酶突变体和葡萄糖脱氢酶共表达。用同时含有亚胺还原酶突变体基因与葡萄糖脱氢酶基因的细胞生产共表达酶。
具体来说，可以将这些核苷酸序列接入质粒中，如pET-30a(+)， 制备成载体。
将这些重组后的载体转入工程细菌，如大肠杆菌BL21(DE3)，制备得到载体细胞。
亚胺还原酶突变体与葡萄糖脱氢酶的基因在同一个工程细菌中表达，制备成共表达酶。解决了单独制备葡萄糖脱氢酶的成本，亚胺还原酶与葡萄糖脱氢酶共同表达实现体系中辅酶的重复使用。
一种具体的制备亚胺还原酶突变体和葡萄糖脱氢酶共表达酶的方法：通过带有限制性内切酶位点的引物将葡萄糖脱氢酶基因从载体上扩增下来，分别用限制性内切酶BamHI和XhoI对带有亚胺还原酶基因片段的载体和扩增的葡萄糖脱氢酶基因片段进行双酶切，胶回收酶切片段，并用T4 DNA连接酶连接后转化大肠杆菌BL21感受态细胞，获得同时含有亚胺还原酶基因和葡萄糖脱氢酶基因的重组菌，将上述重组菌培养，然后诱导、离心收集菌体、重悬，重悬后对细胞进行超声破碎，再将破碎后溶液进行冷冻干燥，获得亚胺还原酶和葡萄糖脱氢酶共表达酶粉。
将亚胺还原酶突变体和葡萄糖脱氢酶共表达酶应用于制备(S)-尼古丁：在合适条件下，亚胺还原酶突变体和葡萄糖脱氢酶共表达酶催化底物I
Figure PCTCN2022099711-appb-000005
或底物II
Figure PCTCN2022099711-appb-000006
直接或经过化学反应得到(S)-尼古丁。
优选的，将底物I、辅酶、葡萄糖、缓冲液与亚胺还原酶和葡萄糖脱氢酶共表达酶混合后，反应得到化合物III(S)-去甲烟碱，然后将化合物III(S)-去甲烟碱甲基化得到(S)-尼古丁，
Figure PCTCN2022099711-appb-000007
底物II环化后经亚胺还原酶和葡萄糖脱氢酶共表达酶催化得到(S)-尼古丁；
或底物II的盐经脱盐、环化经亚胺还原酶的催化得到(S)-尼古丁；
所述底物II的盐包括盐酸盐、二盐酸盐、氢溴酸盐、二氢溴酸盐、硫酸盐或硫酸氢盐。
相对于现有技术，本发明取得的有益效果：
1、经本发明亚胺还原酶突变体催化底物II或其盐为(S)-尼古丁，转化率为99.9％，化学纯度为99.9％，底物的载量为300g/L。
2、本发明的亚胺还原酶突变体可以催化底物I为(S)-去甲烟碱，酶的活性更高，且提高了底物的载量。
3、通过本发明的亚胺还原酶和葡萄糖脱氢酶共表达酶，在同一个工程菌中一起表达两种酶，在使用亚胺还原酶制备(S)-尼古丁的 体系中实现辅酶循环再生的同时，减少了再次制备葡萄糖脱氢酶的步骤，降低了成本。
[附图说明]
图1为(S)-(3-吡咯烷-2-基)吡啶的合成路线图；
图2为(S)-(3-吡咯烷-2-基)吡啶标准品的反相非手性HPLC图谱；
图3为底物麦斯明标准品的反相非手性HPLC图谱；
图4为实施例5反应中控的反相非手性HPLC图谱(SEQ ID NO:1样品)；
图5为(R，S)-(3-吡咯烷-2-基)吡啶的正相手性HPLC图谱；
图6为(S)-(3-吡咯烷-2-基)吡啶标准品的正相手性HPLC图谱；
图7为实施例5合成的(S)-(3-吡咯烷-2-基)吡啶的正相手性HPLC图谱(SEQ ID NO:1样品)；
图8为实施例5合成的(S)-(3-吡咯烷-2-基)吡啶的正相手性HPLC图谱(SEQ ID NO:17样品)；
图9为亚胺还原酶和葡萄糖脱氢酶共表达质粒图谱；
[具体实施例]
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述 的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 亚胺还原酶的表达及酶粉制备
(1)将来源于气单胞菌Aeromonas veronii的野生型亚胺还原酶的编码基因SEQ ID NO:2送至南京金斯瑞公司进行密码子优化和全基因合成，连接入质粒pET-30a(+)中；将重组质粒转入大肠杆菌BL21(DE3)中，将菌液涂布于含50μg/mL卡那霉素的LB琼脂平板上，置于37℃下培养过夜；挑取转化子进行测序验证，将验证正确的转化子命名为E.coli BL21/pET30a-No.2。
(2)将上述重组菌E.coli BL21/pET30a-No.2接种于5mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养过夜；取1mL菌液接种于100mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养3h，然后加入50μL 1M IPTG，25℃下诱导16小时；离心(4000rpm，4℃，10min)收集菌体，加入4倍体积的PBS缓冲液(pH＝7.0)重悬，重悬后对细胞进行超声破碎，再将溶液进行冷冻干燥，获得亚胺还原酶酶粉，野生型亚胺还原酶的氨基酸序列如SEQ ID NO:1所示。
实施例2 亚胺还原酶突变体文库的构建
本实施例以野生型亚胺还原酶的编码基因SEQ ID NO:1为模板，使用随机突变试剂盒(碧云天)进行易错PCR，引物见下表，使用DNA片段回收试剂盒(上海生工)并按照说明书回收上述易错PCR产物。
分别用限制性内切酶NdeI和BamHI对易错PCR产物和pET-30a(+)质粒进行双酶切，将回收的酶切产物用T4 DNA连接酶连接在线性化pET-30a(+)质粒中，22℃下连接1小时。将连接产物转化入大肠杆菌BL21(DE3)感受态细胞(上海生工)，37℃过夜培养，获得随机突变文库。
表1 易错PCR引物序列
序列编号 引物序列
SEQ ID NO:169 TATA CATATGCGCCATCTGAGCGTGATTGG
SEQ ID NO:170 TTC GGATCCTTACTGCGCCGCGCCGTTGC
突变文库经过筛选后，将获得的有益突变酶基因SEQ ID NO:37作为模板进行定点饱和突变及再次进行易错PCR，进行多轮次的建库与筛选。
实施例3 亚胺还原酶突变体的初筛
(1)挑取文库中的菌落至96孔微量培养板上，培养板的每孔含有150μL LB液体培养基和50μg/mL卡那霉素，将菌体置于37℃、220rpm摇床中培养过夜；将20μL菌液转接至含有380μL LB液体培养基和50μg/mL卡那霉素的96孔板中，继续在37℃、220rpm的摇床中培养2～3h，添加IPTG至终浓度为0.4mM进行诱导，然后降温至25℃培养过夜(至少16小时)。
(2)将96孔板离心(10min，4000rpm，25℃)收集菌体，用200μL裂解液(含1g/L溶菌酶和0.5g/L硫酸多粘菌素B)重悬菌体，于25℃、600rpm下振荡2h进行裂解，裂解后的细胞碎片通过离心 (4000rpm，4℃，10min)进行沉淀，获得上清液即粗酶液，用于酶标仪进行活性初筛。
(3)反应混合液的配制：取1mL麦斯明溶液(pH6.0 50mM)，0.25mL NADPH溶液(20mM)，13.75mL磷酸盐缓冲液(pH6.0 0.1M)置于加样槽中，混匀即得反应混合液。用液体工作站进行液体分装，取酶标板每孔分装150μL反应混合液，然后加入上述粗酶液50μL，并立即于25℃检测340nm处吸光光度值的变化。
酶活性定义：单位时间内生成1μmol产物所需要的酶的用量为一个酶活单位。
Figure PCTCN2022099711-appb-000008
其中：
ΔA340：吸光值变化值；
V0：反应总体积；
N：酶液稀释倍数；
T：反应时间；
V1：加酶量；
6.22为NADPH斜率常数
实施例4 亚胺还原酶突变体的复筛
(1)将实施例3中酶活性高于母本的突变体接种于100mL含50μg/mL卡那霉素的LB液体培养基中，37℃振荡培养至OD600＝0.6时，加入IPTG至终浓度为0.5mM，在25℃下进行诱导表达。诱导16h后，离心(10min，4000rpm，25℃)收集菌体。菌体用超声 破碎仪(JY92-2D，宁波新芝生物科技股份有限公司)破碎细胞，离心(4℃，10000rpm，20min)获得上清液，部分用于活性检测，剩余上清液进行冷冻干燥，制备酶粉。
(2)取5mL离心管，加入50mg底物麦斯明，加入2mL磷酸盐缓冲液(pH6.0 0.1M)。再向反应瓶中加入0.5mg NADP和90mg葡萄糖，搅拌至完全溶解，再向其中分别加入葡萄糖脱氢酶5mg和待筛选粗酶液0.2mL，混匀并升温至25℃，以300r/min搅拌反应24h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。
选取催化活性优于母本的突变体进行测序，分析突变位点，获得亚胺还原酶突变体的编码基因SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106或108，相应的转氨酶突变体的氨基酸序列分别如SEQ ID NO:3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、105、107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO: 137、SEQ ID NO:139、SEQ ID NO:141
SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ ID NO:149、SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:167所示。
不同亚胺还原酶突变体的活性检测结果见表2。
表2 亚胺还原酶突变体的突变位点与活性检测
Figure PCTCN2022099711-appb-000009
Figure PCTCN2022099711-appb-000010
Figure PCTCN2022099711-appb-000011
Figure PCTCN2022099711-appb-000012
Figure PCTCN2022099711-appb-000013
Figure PCTCN2022099711-appb-000014
Figure PCTCN2022099711-appb-000015
注：++表示酶活性是SEQ ID NO:1的1.2至3倍；+++表示酶活性是SEQ ID NO:1的5至10倍；++++表示酶活性是SEQ ID NO:1的11至20倍；+++++表示酶活性是SEQ ID NO:1的21至30倍；++++++表示酶活性是SEQ ID NO:1的31至50倍；
实施例5 表2中标为“++”的亚胺还原酶突变体在制备S-(3-吡 咯烷-2-基)吡啶中的应用
取10mL反应瓶，加入100mg底物麦斯明，加入4mL磷酸盐缓冲液(pH6.0 0.1M)，调节pH至6.0。再向反应瓶中加入2.5mg NADP和180mg葡萄糖，搅拌至完全溶解，再向其中分别加入10mg葡萄糖脱氢酶和50mg亚胺还原酶突变体酶粉，混匀并升温至25℃，以300r/min搅拌反应24h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。取反应液500μL，调pH11以上，加1000μL正己烷萃取，取正己烷层过膜后供正相HPLC分析手性。SEQ ID NO:1转化率图谱见图4，SEQ ID NO:1手性检测图谱见图7，SEQ ID NO:17手性检测图谱见图8.
SEQ ID NO:1及活性最高的三个突变体转化率及手性结果如表3所示。
表3
酶 转化率％ ee％
SEQ ID NO:1 46.0 95.4
SEQ ID NO:9 78.9 99.2
SEQ ID NO:11 83.1 99.6
SEQ ID NO:17 90.3 99.9
实施例6 表2中标为“+++”的亚胺还原酶突变体在制备S-(3-吡咯烷-2-基)吡啶中的应用
取10mL反应瓶，加入250mg底物麦斯明，加入4mL磷酸盐 缓冲液(pH6.0 0.1M)，调节pH至6.0。再向反应瓶中加入2.5mg NADP和450mg葡萄糖，搅拌至完全溶解，再向其中分别加入10mg葡萄糖脱氢酶和50mg亚胺还原酶突变体酶粉，混匀并升温至25℃，以300r/min搅拌反应24h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。取反应液500μL，调pH11以上，加1000μL正己烷萃取，取正己烷层过膜后供正相HPLC分析手性。
SEQ ID NO:1及活性最高的三个突变体转化率及手性结果如表4所示。
表4
酶 转化率％ ee％
SEQ ID NO:1 18.7 95.1
SEQ ID NO:25 98.1 99.3
SEQ ID NO:31 98.1 99.7
SEQ ID NO:33 90.3 99.6
实施例7 表2中标为“++++”的亚胺还原酶突变体在制备S-(3-吡咯烷-2-基)吡啶中的应用
取10mL反应瓶，加入500mg底物麦斯明，加入4mL磷酸盐缓冲液(pH6.0 0.1M)，调节pH至6.0。再向反应瓶中加入2.5mg NADP和900mg葡萄糖，搅拌至完全溶解，再向其中分别加入10mg葡萄糖脱氢酶和50mg亚胺还原酶突变体酶粉，混匀并升温至25℃，以300r/min搅拌反应24h。反应结束后，分别取反应液100μL加入到 900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。取反应液500μL，调pH11以上，加1000μL正己烷萃取，取正己烷层过膜后供正相HPLC分析手性。
SEQ ID NO:1及活性最高的三个突变体转化率及手性结果如表5所示。
表5
酶 转化率％ ee％
SEQ ID NO:1 8.9 95.6
SEQ ID NO:37 99.9 99.6
SEQ ID NO:47 99.8 99.9
SEQ ID NO:53 95.5 99.7
实施例8 表2中标为“+++++”的亚胺还原酶突变体在制备S-(3-吡咯烷-2-基)吡啶中的应用
取10mL反应瓶，加入1000mg底物麦斯明，加入4mL磷酸盐缓冲液(pH6.0 0.1M)，调节pH至6.0。再向反应瓶中加入5mg NADP和1800mg葡萄糖，搅拌至完全溶解，再向其中分别加入10mg葡萄糖脱氢酶和50mg亚胺还原酶突变体酶粉，混匀并升温至25℃，以300r/min搅拌反应16h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。取反应液500μL，调pH11以上，加1000μL正己烷萃取，取正己烷层过膜后供正相HPLC分析手性。
SEQ ID NO:37及活性最高的三个突变体转化率及手性结果如表 6所示。
表6
酶 转化率％ ee％
SEQ ID NO:37 46.8 99.7
SEQ ID NO:61 99.3 99.8
SEQ ID NO:65 99.5 99.7
SEQ ID NO:71 99.9 99.6
实施例9 表2中标为“++++++”的亚胺还原酶突变体在制备S-(3-吡咯烷-2-基)吡啶中的应用
取20mL反应瓶，加入3g底物麦斯明，加入7mL磷酸盐缓冲液(pH6.0 0.1M)，调节pH至6.0。再向反应瓶中加入10mg NADP和5.4g葡萄糖，搅拌至完全溶解，再向其中分别加入20mg葡萄糖脱氢酶和100mg亚胺还原酶突变体酶粉，混匀并升温至25℃，以300r/min搅拌反应16h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。取反应液500μL，调pH11以上，加1000μL正己烷萃取，取正己烷层过膜后供正相HPLC分析手性。
SEQ ID NO:37及活性高突变体转化率及手性结果如表7所示。
表7
酶 转化率％ ee％
SEQ ID NO:37 33.3 99.8
SEQ ID NO:81 99.1 99.9
SEQ ID NO:85 99.6 99.6
SEQ ID NO:95 99.9 99.9
SEQ ID NO:133 99.9 100
实施例10 以4-(甲胺基)-1-(吡啶-3-基)丁酮为底物筛选表2中亚胺还原酶突变体
取5mL离心管，加入30mg底物4-(甲胺基)-1-(吡啶-3-基)丁酮，加入2mL磷酸盐缓冲液(pH6.0 0.1M)。再向反应瓶中加入0.3mg NADP和50mg葡萄糖，搅拌至完全溶解，再向其中分别加入3mg葡萄糖脱氢酶和30mg亚胺还原酶突变体酶粉，混匀并升温至25℃，以300r/min搅拌反应24h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析，以(S)-尼古丁的面积比为转化率。对转化率高的样品分别取反应液1mL进行乙酸乙酯萃取，用于ee值检测。
SEQ ID NO:1及活性较高的部分突变体转化率及手性结果如表8所示。
表8
酶 转化率％ ee％
SEQ ID NO:1 50.8 98.8
SEQ ID NO:23 98.1 99.3
SEQ ID NO:25 99.7 99.8
SEQ ID NO:27 99.7 99.9
SEQ ID NO:37 100 100
SEQ ID NO:43 99.7 99.4
SEQ ID NO:45 99.1 99.5
SEQ ID NO:47 100 99.8
SEQ ID NO:61 100 100
SEQ ID NO:65 99.8 99.6
SEQ ID NO:73 99.7 99.8
SEQ ID NO:85 99.9 100
实施例11 SEQ ID NO:37所示亚胺还原酶突变体在(S)-尼古丁制备中的应用
向50mL三口圆底烧瓶中加入4.5g 4-(甲胺基)-1-(吡啶-3-基)丁酮，加入20mL 0.1M的磷酸盐缓冲液，调节pH至7.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。，0.04g葡萄糖脱氢酶和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至30℃，以300r/min搅拌反应16h。取样检测转化率99％，终止反应，过滤，滤液用氢氧化钠溶液调节至pH＝10后用甲基叔丁基醚萃取，无水硫酸钠干燥，浓缩后得(S)-尼古丁2.6g。产品纯度99％，光学纯度100％。
对制备得到的(S)-尼古丁进行核磁氢谱表征，数据为：
1H-NMR(400MHz,CDCl3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H，m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例12 SEQ ID NO:47所示亚胺还原酶突变体在(S)-尼古丁制备中的应用
向50mL三口圆底烧瓶中加入4.5g 4-(甲胺基)-1-(吡啶-3-基)丁酮，加入20mL 0.1M的磷酸盐缓冲液，调节pH至7.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入10mL 0.1M的磷酸盐缓冲液，0.3g SEQ ID NO:53所示亚胺还原酶，0.04g葡萄糖脱氢酶和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至30℃，以300r/min搅拌反应16h。取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析，以(S)-尼古丁的面积比为转化率。取反应液1mL进行乙酸乙酯萃取，用于ee值检测。转化率99.5％，ee值99.6％。
实施例13 SEQ ID NO:61所示亚胺还原酶突变体在(S)-尼古丁制备中的应用
向50mL三口圆底烧瓶中加入4.5g 4-(甲胺基)-1-(吡啶-3-基)丁酮，加入20mL 0.1M的磷酸盐缓冲液，调节pH至7.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入10mL 0.1M的磷酸盐缓冲液，0.3g SEQ ID NO:61所示亚胺还原酶，0.04g葡萄糖脱氢酶和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至30℃，以300r/min搅拌反应16h。取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析，以(S)-尼古丁的 面积比为转化率。取反应液1mL进行乙酸乙酯萃取，用于ee值检测。转化率93.2％，ee值99.7％。
实施例14 SEQ ID NO:85所示亚胺还原酶突变体在(S)-尼古丁制备中的应用
向50mL三口圆底烧瓶中加入4.5g 4-(甲胺基)-1-(吡啶-3-基)丁酮，加入20mL 0.1M的磷酸盐缓冲液，调节pH至7.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入10mL 0.1M的磷酸盐缓冲液，0.3g SEQ ID NO:85所示亚胺还原酶，0.04g葡萄糖脱氢酶和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至30℃，以300r/min搅拌反应16h。取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析，以(S)-尼古丁的面积比为转化率。取反应液1mL进行乙酸乙酯萃取，用于ee值检测。转化率100％，ee值99.8％。
实施例15 亚胺还原酶与葡萄糖脱氢酶的共表达酶粉制备
通过带有限制性内切酶位点的引物(见下表)将葡萄糖脱氢酶基因从pET30a-GDH(序列见SEQ ID NO:171)载体上扩增下来。分别用限制性内切酶BamHI和XhoI对带有亚胺还原酶基因片段的pET30a载体和扩增的葡萄糖脱氢酶基因片段进行双酶切。胶回收酶切片段，并用T4 DNA连接酶连接后转化大肠杆菌BL21(DE3)感受态细胞，获得同时含有亚胺还原酶基因和葡萄糖脱氢酶基因的重组菌(质粒示意图见图9)。
表9 葡萄糖脱氢酶扩增引物
Figure PCTCN2022099711-appb-000016
将上述重组菌接种于5mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养过夜；取1mL菌液接种于100mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养3h，然后加入50μL 1M IPTG，25℃下诱导16小时；离心(4000rpm，4℃，10min)收集菌体，加入4倍体积的PBS缓冲液(pH＝7.0)重悬，重悬后对细胞进行超声破碎，再将溶液进行冷冻干燥，获得亚胺还原酶和葡萄糖脱氢酶共表达酶粉。
实施例16 亚胺还原酶与葡萄糖脱氢酶的共表达酶粉在S-(3-吡咯烷-2-基)吡啶制备中的应用
取10mL反应瓶，加入750mg底物麦斯明，加入4mL磷酸盐缓冲液(pH6.0 0.1M)，调节pH至6.0。再向反应瓶中加入5mg NADP和1200mg葡萄糖，搅拌至完全溶解，再向其中加入共表达酶粉(此处亚胺还原酶为SEQ ID NO:37所示突变酶，共表达酶的核酸序列为SEQ ID NO:173所示)，混匀并升温至25℃，以300r/min搅拌反应16h。反应结束后，分别取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析。取反应液500μL， 调pH11以上，加1000μL正己烷萃取，取正己烷层过膜后供正相HPLC分析手性。转化率99.6％，ee值99.8％。
实施例17 亚胺还原酶与葡萄糖脱氢酶的共表达酶粉在(S)-尼古丁制备中的应用
向50mL三口圆底烧瓶中加入4.5g 4-(甲胺基)-1-(吡啶-3-基)丁酮，加入30mL 0.1M的磷酸盐缓冲液，调节pH至7.0。再向反应瓶中加入4.8g葡萄糖和0.008g NADP盐，搅拌至完全溶解。直接加入0.3g共表达酶粉(此处亚胺还原酶为SEQ ID NO:37所示突变酶，共表达酶的核酸序列为SEQ ID NO:173所示)，搅拌至完全溶解，升温至30℃，以300r/min搅拌反应16h。取反应液100μL加入到900μL乙腈中充分振荡，使用0.22μm过滤膜过滤，进行HPLC分析，以(S)-尼古丁的面积比为转化率。取反应液1mL进行乙酸乙酯萃取，用于ee值检测。转化率99.6％，ee值99.8％。
实施例18 用(S)-去甲烟碱制备(S)-尼古丁
将实施例5-9或16制备的(S)-去甲烟碱制备(S)-尼古丁。
向三个500mL三口瓶中均加入使用实施例16方法制备的107g(S)-去甲烟碱，80g 37％甲醛溶液，升温到75℃。滴加60g 85％甲酸溶液，滴加完毕后，保温反应24h。反应完成后，加入氢氧化钠，调节pH＝12。水相用甲叔醚萃取，合并萃取液，浓缩，减压蒸馏，得无色液体80g，即(S)-尼古丁。
对所得产物进行核磁氢谱表征，核磁数据结果为：
1H-NMR(400MHz,CDCl3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H, dt),7.24-7.27(1H，m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)，表明(S)-尼古丁被成功合成。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (17)

  1. 亚胺还原酶突变体，其特征在于：与氨基酸序列SEQ ID NO：1相比，存在残基差异包括V171、A172、Y230中的一个或多个。
  2. 根据权利要求1所述的亚胺还原酶突变体，其特征在于：所述残基差异包括V171Y/N/A/S、A172V/F、Y230G/A/T中的一个或多个。
    根据权利要求1或2所述的亚胺还原酶突变体，其特征在于：亚胺还原酶突变体的氨基酸序列选自SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、105、107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:139、SEQ ID NO:141
  3. SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ IDNO:149、SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165或SEQ ID NO:167。
  4. 一种制备(S)-尼古丁的方法，其特征在于：在合适的条件下，如权利要求1至3任意一项所述的亚胺还原酶突变体催化底物I为化合物III(S)-去甲烟碱，化合物III(S)-去甲烟碱经甲基化 得到(S)-尼古丁
    Figure PCTCN2022099711-appb-100001
  5. 根据权利要求4所述的制备(S)-尼古丁的方法，其特征在于：将底物I、辅酶、葡萄糖、葡萄糖脱氢酶、缓冲液与亚胺还原酶混合后，反应得到化合物III(S)-去甲烟碱，然后将化合物III(S)-去甲烟碱甲基化得到(S)-尼古丁。
  6. 一种制备(S)-尼古丁的方法，其特征在于：在合适的条件下，如权利要求1至3任意一项所述的亚胺还原酶突变体催化底物II得到(S)-尼古丁，
    Figure PCTCN2022099711-appb-100002
  7. 根据权利要求6所述的制备(S)-尼古丁的方法，其特征在于：
    底物II环化后经亚胺还原酶的催化得到(S)-尼古丁；
    或底物II的盐经脱盐、环化经亚胺还原酶的催化得到(S)-尼古丁；
    所述底物II的盐包括盐酸盐、二盐酸盐、氢溴酸盐、二氢溴酸盐、硫酸盐或硫酸氢盐。
  8. 一种核酸，其特征在于：所述核酸可以编码权利要求1至3中任一项所述的亚胺还原酶突变体。
  9. 一种表达载体，其特征在于：包括权利要求8的核酸。
  10. 一种细胞，其特征在于：包括权利要求8的核酸或权利要求9的载体。
  11. 一种如权利要求1至3中任意一项的亚胺还原酶突变体的生产方法，其特征在于培养权利要求1至3中所述的细胞得到所述的亚胺还原酶突变体。
  12. 如权利要求11所述的生产方法，其特征在于所述的亚胺还原酶突变体和葡萄糖脱氢酶共表达。
  13. 如权利要求12所述的生产方法，其特征在于：用同时含有亚胺还原酶突变体基因与葡萄糖脱氢酶基因的细胞生产共表达酶。
  14. 如权利要求13所述的生产方法，其特征在于：通过带有限制性内切酶位点的引物将葡萄糖脱氢酶基因从载体上扩增下来，分别用限制性内切酶BamHI和XhoI对带有亚胺还原酶基因片段的载体和扩增的葡萄糖脱氢酶基因片段进行双酶切，胶回收酶切片段，并用T4 DNA连接酶连接后转化大肠杆菌BL21感受态细胞，获得同时含有亚胺还原酶基因和葡萄糖脱氢酶基因的重组菌，将上述重组菌培养，然后诱导、离心收集菌体、重悬，重悬后对细胞进行超声破碎，再将破碎后溶液进行冷冻干燥，获得亚胺还原酶和葡萄糖脱氢酶共表达酶粉。
  15. 通过权利要求11-14任意一项所述的生产方法制备得到的亚胺还原酶突变体或者亚胺还原酶突变体与葡萄糖脱氢酶的共表达酶。
  16. 如权利要求15所述的共表达酶的应用，其特征在于：在合适条件下，亚胺还原酶突变体和葡萄糖脱氢酶共表达酶催化底物I 为化合物III(S)-去甲烟碱，化合物III(S)-去甲烟碱经甲基化得到(S)-尼古丁
    Figure PCTCN2022099711-appb-100003
  17. 一种如权利要求15所述的共表达酶的应用，其特征在于：在合适条件下，亚胺还原酶突变体和葡萄糖脱氢酶共表达酶催化底物II为(S)-尼古丁，
    Figure PCTCN2022099711-appb-100004
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117363667A (zh) * 2023-12-07 2024-01-09 欣雅利华生物技术(上海)有限公司 亚胺还原酶在制备达泊西汀中间体和/或达泊西汀中的用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014174505A2 (en) 2013-04-22 2014-10-30 Perrigo Api Ltd. A process for the preparation of nicotine comprising the enzymatic reduction of 4- (methylamino) -1- (pyridin-3- yl) butan-1-one
CN110564788A (zh) * 2019-09-30 2019-12-13 江南大学 一种利用亚胺还原酶生产麻黄碱的方法
WO2020098978A1 (en) 2018-11-16 2020-05-22 Zanoprima Lifesciences Limited Process for the preparation of (s)-nicotin from myosmine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014174505A2 (en) 2013-04-22 2014-10-30 Perrigo Api Ltd. A process for the preparation of nicotine comprising the enzymatic reduction of 4- (methylamino) -1- (pyridin-3- yl) butan-1-one
WO2020098978A1 (en) 2018-11-16 2020-05-22 Zanoprima Lifesciences Limited Process for the preparation of (s)-nicotin from myosmine
CN110564788A (zh) * 2019-09-30 2019-12-13 江南大学 一种利用亚胺还原酶生产麻黄碱的方法

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Thesis Submitted to Tsinghua University in partial fulfillment of the requirement for the degree of Master of Science in Biology", 15 February 2021, TSINGHUA UNIVERSITY, CN, article LI JIXUAN ：: "Co-expressed System Construction and Site-directed Mutagenesis of Imine Reductase", pages: 1 - 54, XP093016960 *
CHENG FENG, LI QINGHUA, LI HENG, XUE YAPING: "NAD(P)H-dependent Oxidoreductases for Synthesis of Chiral Amines by Asymmetric Reductive Amination of Ketones", SHENG WU GONG CHENG XUE BAO = CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 36, no. 9, 25 September 2020 (2020-09-25), pages 1794 - 1816, XP093017118, DOI: 10.13345/j.cjb.190582 *
DATABASE Genbank NCBI; . . : "NAD(P)-binding Domain-containing Protein [Aeromonas veronii]"", XP093016954 *
DATABASE Genbank NCBI; . . : "NAD(P)-binding Domain-containing Protein [Aeromonas veronii]", XP093016919 *
KOICHI MITSUKURA ET AL., ORG. BIOMOL. CHEM., vol. 8, 2010, pages 4533 - 4535
LI JIXUAN ET AL., BIOTECHNOLOGY BULLETIN, vol. 35, no. 1, 2019, pages 105 - 111
MAN HENRY, WELLS ELIZABETH, HUSSAIN SHAHED, LEIPOLD FRIEDEMANN, HART SAM, TURKENBURG JOHAN P., TURNER NICHOLAS J., GROGAN GIDEON: "Structure, Activity and Stereoselectivity of NADPH-Dependent Oxidoreductases Catalysing the S -Selective Reduction of the Imine Substrate 2-Methylpyrroline", CHEMBIOCHEM, vol. 16, no. 7, 4 May 2015 (2015-05-04), pages 1052 - 1059, XP093017123, ISSN: 1439-4227, DOI: 10.1002/cbic.201402625 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117363667A (zh) * 2023-12-07 2024-01-09 欣雅利华生物技术(上海)有限公司 亚胺还原酶在制备达泊西汀中间体和/或达泊西汀中的用途
CN117363667B (zh) * 2023-12-07 2024-03-22 欣雅利华生物技术(上海)有限公司 亚胺还原酶在制备达泊西汀中间体和/或达泊西汀中的用途

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