Imine reductase and method for preparing (S)-nicotine using the same

Abstract

The present invention discloses an imine reductase and a method for preparing (S)-nicotine using the enzyme. The amino acid sequence of the imine reductase IR-55 is shown in SEQ ID NO.1. The present invention also provides two mutants of the imine reductase IR-55, which are SEQ ID NO.3 and SEQ ID NO.4 respectively. The novel imine reductase R-55 or its mutant is used as a catalytic enzyme, which can effectively catalyze the conversion of N-methyl-[4-(pyridine-3-yl)-4-oxo-butylamine dihydrochloride into (S)-nicotine; with a Tris-HCl buffer of pH 8.5, the reaction substrate concentration can be significantly increased, the bioconversion efficiency can be improved, the reaction time can be shortened, and finally the (S)-nicotine with an optical purity ee value of up to 99.1% can be synthesized within a relatively short reaction time (6h), and the yield is 77%, and the conversion rate is> 99.9%, which is significantly better than the existing technical level.

Description

Technical Field

[0001] The invention belongs to the field of biological enzyme catalysis, and specifically relates to an imine reductase and a method for preparing (S)-nicotine by using the enzyme. Background Art

[0002] Nicotine is an important chemical component of tobacco products, and its content is closely related to the quality and taste of tobacco products. Natural nicotine (>99%, (S)-nicotine) mainly comes from tobacco plants, such as flue-cured tobacco and cigarettes. At present, (S)-nicotine is obtained by extracting tobacco plant leaves, but this method has many impurities, low purity, is greatly affected by geographical factors, and has the problem of difficult separation of impurities. (S)-Nicotine can also be obtained by synthetic methods, mainly chemical methods and biocatalytic methods. The chemical synthesis method has harsh reaction conditions and requires multi-stage reactions under high temperature and high pressure conditions. It has high energy consumption and strict requirements on the reactor. The biocatalytic synthesis of (S)-nicotine has the advantages of high efficiency, high stereoselectivity, low cost, and green environmental protection, and has received widespread attention in recent years.

[0003] Although there are reports on the synthesis of (S)-nicotine using imine reductase, these technologies mostly use mycosamine as a raw material for preparation, and need to first synthesize (S)-nornicotine, and then methylate (S)-nornicotine to obtain (S)-nicotine. Not only are the reaction steps complicated, but the reaction time is long, and the yield of the final product (S)-nicotine is not high. In addition, relevant studies have shown that imine reductase can be used to catalyze N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride to synthesize (S)-nicotine in one step, but the imine reductase used in these studies has poor catalytic activity, a long reaction time, and the yield of the final product (S)-nicotine is not high.

[0004] Therefore, it is necessary to provide an imine reductase with high catalytic activity to increase the synthesis rate of (S)-nicotine and improve the yield. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the deficiencies of the prior art, and to provide an imine reductase with high catalytic activity and a mutant thereof, as well as a method for synthesizing (S)-nicotine by catalyzing N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride using the imine reductase and the mutant thereof. The method of the present invention has a high yield of (S)-nicotine, a short reaction time, simple steps, low production cost, low pollution, and little impact on the environment, and can achieve large-scale production of (S)-nicotine.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] An imine reductase IR-55, whose amino acid sequence is shown as SEQ ID NO.1.

[0008] >SEQ ID NO.1

[0009] MNTKQITIIGLGQMGTKLAELLVAKGYTVSVWNRTRAKGDSIAGVKVFEDVEEAVRQSPLI

[0010] VICVLDYAAVDHVLDHISDKSILGGKTLINFTTADPQEAETLETTLARISAGYINGAIQVAPD

[0011] QMGLPETTIIVSGNQDHFQRSRDALAVFGGNIKYLGNRAALASAMDLATLTWLYGSYTGL

[0012] IHGIVLAQKTGLPLDAFSEILGEITPGFTEFFKHQIGVVEKGDFRVSQSPLSISIAATQRIHHAFRKAGAQEDFVKAISDLLQKAESLGLADQEVAALVKVVG.

[0013] The present invention also provides a gene encoding imine reductase IR-55, characterized in that the nucleotide sequence of the gene encoding is shown as SEQ ID NO.2.

[0014] >SEQ ID NO.2

[0015] ATGAACACCAAACAGATTACCATTATCGGTCTGGGCCAGATGGGCACCAAAACTGGCCGA

[0016] ACTGCTGGTTGCCAAAGGCTATACCGTTAGTGTGTGGAATCGCACCCGCGCAAAAGGCG

[0017] ATAGCATTGCCGGTGTGAAAGTGTTTGAAGATGTTGAAGAAGCCGTTCGCCAGAGCCCG

[0018] CTGATTGTGATTTGCGTTCTGGATTATGCCGCAGTGGATCATGTTCTGGATCATATTAGTG

[0019] ATAAAAGCATTCTGGGTGGCAAAACCCTGATTAATTTTACCACCGCCGATCCGCAGGAA

[0020] GCCGAAACCCTGGAAACCACCCTGGCCCGTATTAGCGCAGGTTATATTAATGGTGCCATT

[0021] CAGGTTGCACCTGATCAGATGGGCCTGCCGGAAACCACCATTATTGTTAGTGGCAATCA

[0022] GGATCATTTTCAGCGCAGCCGGATGCACTGGCCGTGTTTGGGTGGCAATATTAAATATCT

[0023] GGGCAATCGCGCCGCACTGGCAAGCGCAATGGATCTGGCCACCCTGACCTGGCTGTATG

[0024] GCAGTTATACCGGTCTGATTCATGGTATTGTGCTGGCACAGAAAACCGGCCTGCCGCTGG

[0025] ATGCATTTAGTGAAATTCTGGGTGAAATTACACCTGGCTTTACCGAATTTTTTAAACATCA

[0026] GATTGGCGTTGTGGAAAAAGGTGATTTTCGCGTTAGTCAGAGCCCGTTAAGTATTAGTAT

[0027] TGCAGCAACCCAGCGCATTCATCATGCATTTCGCAAAGCAGGCGCACAGGAAGATTTTG

[0028] TTAAAGCAATTAGTGATCTGCTGCAGAAAGCAGAAAGTCTGGGCCTGGCCGATCAGGAAGTGGCAGCACTGGTGAAAGTTGTTGGT.

[0029] The present invention also provides a mutant of imine reductase IR-55, including IR-55-S233G and IR-55-S233G-C64A, wherein the amino acid sequence of the mutant IR-55-S233G is as follows: the amino acid at position 233 of the amino acid sequence shown in SEQ ID NO.1 is mutated from S to G, and the other amino acid residues remain unchanged;

[0030] The amino acid sequence of the mutant IR-55-S233G-C64A is as follows: the 64th amino acid in the amino acid sequence shown in SEQ ID NO.1 is mutated from C to A, and the 233rd amino acid is mutated from S to G, and the other amino acid residues remain unchanged.

[0031] The present invention also provides a recombinant vector or recombinant bacterium containing the coding gene of the above-mentioned imine reductase IR-55 or its mutant.

[0032] The present invention also provides the use of the above-mentioned imine reductase or its mutant, recombinant vector or recombinant bacteria in catalyzing the conversion of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride to prepare (S)-nicotine.

[0033] The present invention also provides a method for preparing (S)-nicotine, comprising the following steps: dissolving N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride in Tris-HCl buffer, freeze-dried powder of an engineered bacterium expressing imine reductase IR-55 or a mutant thereof, glucose dehydrogenase, glucose and NADP + Add to Tris-HCl buffer to obtain an aqueous phase; adjust the pH value and stir the reaction at room temperature; extract with ethyl acetate after the reaction is completed, and distill the extract under reduced pressure to remove the organic solvent to obtain the target compound (S)-nicotine.

[0034] Preferably, the method for constructing the lyophilized powder of engineered bacteria expressing imine reductase IR-55 or its mutant is as follows: introducing the coding gene of imine reductase IR-55 or its mutant into pET28a, with restriction sites NdeI and EcoRI to obtain a recombinant plasmid, and then introducing the recombinant plasmid into E.coli BL21 (DE3).

[0035] Preferably, the glucose dehydrogenase is Glucose dehydrogenase (GDH) (CDX-901) from Codexis, purchased from Sigma.

[0036] Preferably, the mass ratio of the N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride, the freeze-dried powder of the engineered bacteria expressing imine reductase or its mutant, the glucose dehydrogenase and the glucose is 50:30:4:75; the glucose and NADP + The mass ratio is 10:1.

[0037] Preferably, the Tris-HCl buffer is a 0.1 mol / L Tris-HCl buffer with a pH of 8.5.

[0038] Preferably, the pH value is adjusted to 8.5, and the reaction time is 6 hours.

[0039] The present invention has the following beneficial effects:

[0040] The present invention optimizes the factors of the catalytic enzyme, reaction system and pH in the (S)-nicotine synthesis process, uses the novel imine reductase R-55 or its mutant discovered by the inventor's team as the catalytic enzyme, and the enzyme has high catalytic activity and good stereoselectivity for (S)-nicotine, and can effectively catalyze the conversion of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride into (S)-nicotine; with a Tris-HCl buffer of pH 8.5, the reaction substrate concentration can be significantly increased, the bioconversion efficiency is improved, and the reaction time is shortened. Finally, within a relatively short reaction time (6 hours), (S)-nicotine with an optical purity ee value as high as 99.1% is synthesized, and the yield is 77%, and the conversion rate is greater than 99.9%, which is significantly better than the existing technical level. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned purpose of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0042] Unless otherwise specified, the experimental reagents used in the following examples of the present invention are all conventional biochemical reagents.

[0043] The recombinant plasmids and genetically engineered bacteria in the following embodiments of the present invention are all constructed by conventional methods in the art.

[0044] Example 1: Preparation of lyophilized powder of engineered bacteria expressing imine reductase IR-55 or its mutants

[0045] 1. Preparation of lyophilized powder of genetically engineered bacteria that efficiently express imine reductase IR-55:

[0046] The gene sequence of imine reductase IR-55 as shown in SEQ ID NO.2 was cloned into pET28a plasmid with restriction sites of NdeI and EcoRI to obtain recombinant plasmid pET28a-IR-55, and then the recombinant plasmid pET28a-IR-55 was introduced into Escherichia coli E. coli BL21 (DE3) to construct genetically engineered bacteria E. coli BL21 (DE3) / pET28a-IR-55.

[0047] The engineered bacteria E. coli BL21 (DE3) / pET28a-IR-55 constructed above was inoculated into LB medium containing kanamycin (50 μg / mL), and cultured at 37 ° C, 200 rpm. When the absorbance density (OD600) of the bacterial solution reached 0.6-0.8, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added for induction. The induction temperature was 16 ° C and the induction time was 20 hours. After the culture was completed, the genetically engineered bacteria that efficiently expressed imine reductase IR-55 were obtained by centrifugation. The genetically engineered bacteria were freeze-dried to obtain freeze-dried powder.

[0048] 2. Preparation of lyophilized powder of genetically engineered bacteria that efficiently express imine reductase IR-55 mutant:

[0049] The amino acid sequence of the imine reductase IR-55 mutant SEQ ID NO.3 is as follows: the amino acid at position 233 of the amino acid sequence shown in SEQ ID NO.1 is mutated from S to G, and the other amino acid residues remain unchanged;

[0050] Specifically, the amino acid sequence of the imine reductase IR-55 mutant IR-55-S233G is shown in SEQ ID NO.3.

[0051] The amino acid sequence of the imine reductase IR-55 mutant SEQ ID NO.4 is as follows: the 64th amino acid in the amino acid sequence shown in SEQ ID NO.1 is mutated from C to A, and the 233rd amino acid is mutated from S to G, and the other amino acid residues remain unchanged.

[0052] Specifically, the amino acid sequence of the imine reductase IR-55 mutant IR-55-S233G-C64A is shown in SEQ ID NO.4.

[0053] According to the amino acid sequence of the above-mentioned imine reductase mutant SEQ ID NO.3 or SEQ ID NO.4, the corresponding coding gene sequence is obtained.

[0054] >SEQ ID NO.3

[0055] MNTKQITIIGLGQMGTKLAELLVAKGYTVSVWNRTRAKGDSIAGVKVFEDVEEAVRQSPLI

[0056] VICVLDYAAVDHVLDHISDKSILGGKTLINFTTADPQEAETLETTLARISAGYINGAIQVAPD

[0057] QMGLPETTIIVSGNQDHFQRSRDALAVFGGNIKYLGNRAALASAMDLATLTWLYGSYTGL

[0058] IHGIVLAQKTGLPLDAFSEILGEITPGFTEFFKHQIGVVEKGDFRVSQGPLSISIAATQRIHHAFRKAGAQEDFVKAISDLLQKAESLGLADQEVAALVKVVG.

[0059] >SEQ ID NO.4

[0060] MNTKQITIIGLGQMGTKLAELLVAKGYTVSVWNRTRAKGDSIAGVKVFEDVEEAVRQSPLI

[0061] VIAVLDYAAVDHVLDHISDKSILGGKTLINFTTADPQEAETLETTLARISAGYINGAIQVAPD

[0062] QMGLPETTIIVSGNQDHFQRSRDALAVFGGNIKYLGNRAALASAMDLATLTWLYGSYTGL

[0063] IHGIVLAQKTGLPLDAFSEILGEITPGFTEFFKHQIGVVEKGDFRVSQGPLSISIAATQRIHHAFRKAGAQEDFVKAISDLLQKAESLGLADQEVAALVKVVG.

[0064] Preparation of freeze-dried powder of mutant SEQ ID NO.2 genetically engineered bacteria: Preparation is carried out according to the method of step 1 above, except that the coding gene sequence of the imine reductase mutant SEQ ID NO.2 is cloned into the pET28a plasmid, and the other steps remain unchanged.

[0065] Preparation of freeze-dried powder of mutant SEQ ID NO.3 genetically engineered bacteria: Preparation is carried out according to the method of step 1 above, except that the coding gene sequence of the imine reductase mutant SEQ ID NO.3 is cloned into the pET28a plasmid, and the other steps remain unchanged.

[0066] Example 2: Synthesis of (S)-nicotine

[0067] The synthetic route of (S)-nicotine is as follows:

[0068]

[0069] The specific steps include:

[0070] (1) 0.0125 g of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride raw material was dissolved in 1 mL of 0.1 mol / L Tris-HCl buffer (pH=8.5), and then IR-55 engineered bacteria freeze-dried bacteria (0.03 g), glucose dehydrogenase pure protein (0.003 g), glucose (0.05 g) and NADP+ (0.0003 g) were added to Tris-HCl buffer to obtain an aqueous phase, and the reaction pH was controlled at about 8.5. The reaction was stirred at room temperature for 6 hours. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate, and the extract was distilled under reduced pressure to remove the organic solvent, and passed through a gel column to obtain (S)-nicotine (yield 40%, ee value 85.3%, conversion rate>99.9%).

[0071] (2) 0.0125 g of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride raw material was dissolved in 1 mL of 0.1 mol / L Tris-HCl buffer (pH = 8.5), and then IR-55 mutant SEQ ID NO.3 engineered bacteria freeze-dried bacteria (0.03 g), glucose dehydrogenase pure protein (0.003 g), glucose (0.05 g) and NADP+ (0.0003 g) were added to Tris-HCl buffer to obtain an aqueous phase, and the reaction pH was controlled at about 8.5. The reaction was stirred at room temperature for 6 hours. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate, and the extract was distilled under reduced pressure to remove the organic solvent, and passed through a gel column to obtain 0.0053 g of (S)-nicotine (yield 42%, ee value 93.2%, conversion rate >99.9%).

[0072] (3) 0.5 g of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride raw material was dissolved in 10 mL of 0.1 mol / L Tris-HCl buffer (pH = 8.5), and then IR-55 mutant SEQ ID NO.4 engineered bacteria freeze-dried bacteria (0.3 g), glucose dehydrogenase pure protein (0.004 g), glucose (0.5 g) and NADP+ (0.0472 g) were added to Tris-HCl buffer to obtain an aqueous phase, and the reaction pH was controlled at about 8.5. The reaction was stirred at room temperature for 6 hours. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate, and the extract was distilled under reduced pressure to remove the organic solvent, and passed through a gel column to obtain 0.35 g of (S)-nicotine (yield 70%, ee value>99%, conversion rate>99.9%).

[0073] (4) 5 g of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride raw material was dissolved in 30 mL of 0.1 mol / L Tris-HCl buffer (pH = 8.5), and then IR-55 mutant SEQ ID NO.4 engineered bacteria freeze-dried bacteria (0.3 g), glucose dehydrogenase pure protein (0.012 g), glucose (5 g) and NADP+ (0.1416 g) were added to Tris-HCl buffer to obtain an aqueous phase, and the reaction pH was controlled at about 8.5. The reaction was stirred at room temperature for 6 hours. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate, and the extract was distilled under reduced pressure to remove the organic solvent, and passed through a gel column to obtain 3.85 g of (S)-nicotine (yield 77%, ee value> 99%, conversion rate> 99.9%).

[0074] (5) 20 g of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride raw material was dissolved in 50 mL of 0.1 mol / L Tris-HCl buffer (pH = 8.5), and then IR-55 mutant SEQ ID NO.4 engineered bacteria freeze-dried bacteria (0.3 g), glucose dehydrogenase pure protein (0.048 g), glucose (20 g) and NADP+ (0.5664 g) were added to Tris-HCl buffer to obtain an aqueous phase, and the reaction pH was controlled at about 8.5. The reaction was stirred at room temperature for 6 hours. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate. The extract was distilled under reduced pressure to remove the organic solvent, and passed through a gel column to obtain 14.4 g of (S)-nicotine (yield 72%, ee value>99.5%, conversion rate>99.9%).

[0075] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An imine reductase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding imine reductase according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. An imine reductase mutant, characterized in that The amino acid sequence shown in any of the following: (1) the amino acid at position 233 of the amino acid sequence shown in SEQ ID NO.1 is mutated from S to G, and the other amino acid residues remain unchanged; (2) The 64th amino acid in the amino acid sequence shown in SEQ ID NO.1 is mutated from C to A, and the 233rd amino acid is mutated from S to G, while the other amino acid residues remain unchanged.

4. A recombinant vector or recombinant bacterium comprising the gene encoding the imine reductase according to claim 1 or the gene encoding the imine reductase mutant according to claim 3.

5. Use of the imine reductase according to claim 1, the imine reductase mutant according to claim 3, or the recombinant vector or recombinant bacterium according to claim 4 in catalyzing the conversion of N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride to prepare (S)-nicotine.

6. A method for preparing (S)-nicotine, characterized in that: The method comprises the following steps: dissolving N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride in Tris-HCl buffer, and freeze-drying an engineered bacterium powder expressing the imine reductase according to claim 1 or the imine reductase mutant according to claim 3, glucose dehydrogenase, glucose and NADP + Add to Tris-HCl buffer to obtain an aqueous phase; adjust the pH value and stir the reaction at room temperature; extract with ethyl acetate after the reaction is completed, and distill the extract under reduced pressure to remove the organic solvent to obtain the target compound (S)-nicotine.

7. The preparation method according to claim 6, characterized in that: The method for constructing the lyophilized powder of the engineered bacteria expressing the imine reductase according to claim 1 or the imine reductase mutant according to claim 3 is as follows: the coding gene of the imine reductase according to claim 1 or the imine reductase mutant according to claim 3 is introduced into pET28a, the restriction sites are NdeI and EcoRI, a recombinant plasmid is obtained, and then the recombinant plasmid is introduced into E.coli BL21 (DE3).

8. The preparation method according to claim 6, characterized in that: The mass ratio of the N-methyl-[4-(pyridin-3-yl)-4-oxo-butylamine dihydrochloride, the freeze-dried powder of the engineered bacteria expressing the imine reductase according to claim 1 or the imine reductase mutant according to claim 3, glucose dehydrogenase and glucose is 50:30:4:75; the glucose and NADP + The mass ratio is 10:

1.

9. The preparation method according to claim 6, characterized in that: The Tris-HCl buffer is 0.1 mol / L, Tris-HCl buffer, pH = 8.

5.

10. The preparation method according to claim 6, characterized in that: The pH value is adjusted to 8.5, and the reaction time is 6 hours.

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