Imine reductase mutant and application thereof

Abstract

The invention discloses an imine reductase mutant and application thereof, and belongs to the technical field of biological enzyme catalysis. According to the imine reductase mutant disclosed by the invention, the conversion rate of reducing a substrate 6-methylmyosmine into 6-methyl nornicotine within 6 hours under the catalysis of the imine reductase mutant can reach 99.8%, and the optical purity ee value of a product reaches 99.5%. The imine reductase mutant disclosed by the invention can be used for reducing myosmin to generate 6-methyl nornicotine, the process completely depends on imine reductase, reduction is efficiently completed within a relatively short time, and the imine reductase mutant has the following advantages of short reaction time, safety in operation, greenness, environmental protection, high raw material utilization rate and the like.

Description

Technical Field

[0001] The invention relates to an imine reductase mutant and application thereof, belonging to the technical field of bioenzyme catalysis. Background Art

[0002] Nicotine (1-methyl-2-[3-pyridyl]pyrrolidine), also known as nicotine, is a natural substance found in tobacco plant leaves and is widely used in tobacco, agriculture, medicine and other fields. Among them, 6-methylnicotine [(S)-2-methyl-5-(1-methylpyrrolidin-2-yl)]pyridine is an important derivative that can be used as a drug, auxiliary drug, pesticide and insecticide. The sales of drugs with nicotine as the active ingredient in the market are increasing year by year, and most of the 6-methylnicotine produced domestically has a low purity; therefore, the preparation of high-purity 6-methylnicotine has great application value.

[0003] At present, there are two main methods for the synthesis of 6-methylnicotine. One is chemical synthesis, but this method has complicated steps, high cost, and high subsequent separation and extraction costs and is harmful to the environment. The other is biocatalysis, which first synthesizes 6-methylnornicotine, the precursor of 6-methylnicotine, and then synthesizes nicotine. This method has high stereoselectivity, mild reaction conditions, and is environmentally friendly, so it has good industrial application prospects and economic benefits.

[0004] However, the enzymatic synthesis efficiency of 6-methylnicotine still needs to be further improved due to the limitation of enzyme activity. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an imine reductase mutant and its application, the purpose of which is to catalyze 6-methylmycamine to synthesize 6-methylnornicotine with an NADPH-dependent imine reductase mutant, and optimize the reaction conditions in order to efficiently reduce 6-methylmycamine to 6-methylnornicotine.

[0006] The first technical solution provided by the present invention is an imine reductase mutant, which is obtained by mutating the 122nd alanine, the 171st valine, the 174th tyrosine, the 175th phenylalanine, the 213th aspartic acid, the 236th tryptophan, the 241st serine, the 242nd arginine, the 244th alanine and / or the 283rd valine of the imine reductase parent, and the amino acid sequence of the parent is shown in SEQ ID NO.2.

[0007] In certain embodiments, the mutant is a mutant of the parent subjected to any of the following mutations:

[0008] (1) Mutate alanine A at position 122 to serine S;

[0009] (2) mutating valine V at position 171 to glycine G;

[0010] (3) mutating tyrosine Y at position 174 to methionine M;

[0011] (4) mutating phenylalanine F at position 175 to arginine R;

[0012] (5) mutating aspartic acid D at position 213 to methionine M;

[0013] (6) mutating the tryptophan W at position 236 to arginine R;

[0014] (7) mutating serine S at position 241 to arginine R;

[0015] (8) mutating arginine R at position 242 to methionine M;

[0016] (9) mutating alanine A at position 244 to arginine R;

[0017] (10) mutating valine V at position 283 to arginine R;

[0018] (11) The alanine A at position 122 was mutated to serine S, and the aspartic acid D at position 213 was mutated to methionine M;

[0019] (12) mutating the valine V at position 171 to glycine G and the phenylalanine F at position 175 to arginine R;

[0020] (13) mutating the tyrosine Y at position 174 to methionine M and the arginine R at position 242 to methionine M;

[0021] (14) The tryptophan W at position 236 was mutated to arginine R, and the valine V at position 283 was mutated to arginine R.

[0022] (15) The valine V at position 171 was mutated to glycine G, the aspartic acid D at position 213 was mutated to methionine M, and the valine V at position 283 was mutated to arginine R.

[0023] (16) The phenylalanine F at position 175 was mutated to arginine R, the arginine R at position 242 was mutated to methionine M, and the alanine A at position 244 was mutated to arginine R.

[0024] (17) Aspartic acid D at position 213 was mutated to methionine M, serine S at position 241 was mutated to arginine R, and valine V at position 283 was mutated to arginine R.

[0025] (18) The valine V at position 171 was mutated to glycine G, the aspartic acid D at position 213 was mutated to methionine M, the serine S at position 241 was mutated to arginine R, and the alanine A at position 244 was mutated to arginine R.

[0026] (19) The tyrosine Y at position 174 was mutated to methionine M, the aspartic acid D at position 213 was mutated to methionine M, the alanine A at position 244 was mutated to arginine R, and the valine V at position 283 was mutated to arginine R.

[0027] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0028] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0029] In certain embodiments, pET-30a(+) is used as the expression vector.

[0030] The fourth technical solution provided by the present invention is a recombinant microbial cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0031] In certain embodiments, the recombinant microbial cell includes, but is not limited to, bacteria or fungi.

[0032] In certain embodiments, the microorganism is Escherichia coli.

[0033] In certain embodiments, the recombinant microbial cell uses Escherichia coli BL21 (DE3) as a host cell.

[0034] The fifth technical solution provided by the present invention is a method for improving the enzymatic activity of imine reductase, wherein the method comprises subjecting the imine reductase parent with the amino acid sequence as shown in SEQ ID NO.2 to single point mutation or combined mutation as shown in any one of (a) to (j):

[0035] (a) mutating alanine A at position 122 to serine S;

[0036] (b) mutating the valine V at position 171 to glycine G;

[0037] (c) mutating tyrosine Y at position 174 to methionine M;

[0038] (d) mutating phenylalanine F at position 175 to arginine R;

[0039] (e) mutating aspartic acid D at position 213 to methionine M;

[0040] (f) mutating the tryptophan W at position 236 to arginine R;

[0041] (g) mutating the serine S at position 241 to arginine R;

[0042] (h) mutating arginine R at position 242 to methionine M;

[0043] (i) mutating alanine A at position 244 to arginine R;

[0044] (j) The valine V at position 283 was mutated to arginine R.

[0045] The sixth technical solution provided by the present invention is a method for preparing 6-methylnornicotine, wherein 6-methylmysamine is used as a substrate, and the mutant described in the first technical solution is used as a catalyst to form a catalytic system to catalytically generate 6-methylnornicotine.

[0046] In certain embodiments, in the catalytic system, the concentration of the catalyst is 3 g / L.

[0047] Further, the preparation method of the catalyst is as follows: inoculating the recombinant microbial cells into the fermentation medium, and when the OD 600 IPTG was added at 0.5-0.7 to induce the expression of the catalyst.

[0048] Furthermore, the fermentation medium contains: yeast extract, tryptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and glycerol.

[0049] Furthermore, the induction temperature was 25°C.

[0050] Furthermore, the induction time was 17 hours.

[0051] Further, IPTG was added to a final concentration of 0.1-0.5 mM.

[0052] Furthermore, the recombinant microbial cell uses Escherichia coli BL21 (DE3) as a host bacterium and pET-30a (+) as an expression vector to express the imine reductase mutant.

[0053] In certain embodiments, the catalytic reaction temperature is 28° C. and the reaction pH is 6-10.

[0054] In some embodiments, the catalytic system further includes a cofactor, wherein the cofactor is NADP + .

[0055] In certain embodiments, the 6-methylmysamine is added as a substrate into the reaction system in a dropwise manner, with a final concentration of 20 g / L, at 200 rpm, and the reaction is carried out for 6-12 hours.

[0056] In certain embodiments, after the catalysis is completed, 6-methylnornicotine is obtained by centrifugation, alkalization, and extraction.

[0057] In certain embodiments, the alkalization, i.e., the pH value is adjusted with an inorganic base after the reaction of 6-methylmysamine and imine reductase is completed, and the inorganic base is sodium hydroxide.

[0058] In certain embodiments, the extracting is combining the organic phases with an organic solvent, and the organic solvent is ethyl acetate.

[0059] The seventh technical solution provided by the present invention is the use of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant microbial cell described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution in the production of 6-methylnornicotine or a product containing 6-methylnornicotine.

[0060] The technical effects of the present invention are as follows:

[0061] The imine reductase mutant involved in the present invention can reduce the substrate 6-methyl myosamine to 6-methylnornicotine within 6 hours through catalysis, and the conversion rate can reach 99.8%, and the optical purity ee value of the product reaches 99.5%. The imine reductase mutant involved in the present invention can reduce myosamine to generate 6-methylnornicotine, and the process completely depends on the imine reductase, and the reduction is completed efficiently in a short time, and has the following advantages: short reaction time, safe operation, green environmental protection, high raw material utilization rate, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is the product ratio of the reaction under different temperature conditions.

[0063] Figure 2 is the product ratio of the reaction under different substrate concentration conditions.

[0064] Figure 3 It is the product ratio of the reaction under the conditions of different IR enzyme whole cell addition amounts.

[0065] Figure 4 It is the product ratio of the reaction under different glucose addition conditions. DETAILED DESCRIPTION

[0066] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0067] The recombinant Escherichia coli with the imine reductase gene involved in the present invention is a pET-30a (+) vector purchased from General Biosystems (Anhui) Co., Ltd., and the host is BL21 (DE3) purchased from Sangon Biotech (Shanghai, China). The restriction endonucleases used were purchased from ThermoFisher, and high-fidelity polymerase, DNA ligase, plasmid extraction kit, and DNA gel recovery kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd. The cofactor NADP in the reagents used for enzyme catalysis was purchased from Sangon Biotech (Shanghai, China), and other required reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The rest of the materials and reagents used in the present invention, unless otherwise specified, can be obtained from commercial sources.

[0068] In the following examples, unless otherwise specified, various molecular biological operations, including PCR system conditions, enzyme digestion system conditions, DNA ligation system conditions, DNA recovery and purification operations, competent cell transformation and other operations were performed in accordance with the instructions of the commercial kits or conventional methods in the art.

[0069] Example 1 Construction of an engineered bacterium that efficiently expresses imine reductase

[0070] The imine reductase gene sequence was obtained by querying NCBI, and the whole gene synthesis was completed by General Biosystems (Anhui) Co., Ltd. The codon of the target gene imine reductase was optimized so that it could be correctly expressed in the host bacteria and named TIR (as shown in SEQ ID NO.1). The corresponding amino acid sequence (as shown in SEQ ID NO.2) is:

[0071] MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEEGVAQGFGQQEVSALIKVLRARNGAAQ

[0072] To facilitate subsequent molecular biology operations, six bases, CATATG, were added to the 5' end of the gene fragment to form an NdeI restriction site, and six bases, GGTACC, were added to the 3' end to form a KpnI restriction site.

[0073] The synthesized TIR gene and vector pET-30a(+) were double-digested and the fragments were recovered using a DNA gel recovery kit (purchased from Nanjing Novozymes Biotech Co., Ltd.). The recovered fragments were ligated with T4 DNA ligase and transformed into the TOP10 strain, using kanamycin resistance as a screening marker to obtain positive clones.

[0074] The clones were picked from the plate, and the plasmids were extracted using a plasmid extraction kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.), and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The imine reductase gene expression vector pET-30a-TIR without mutation and correctly connected to the vector was obtained.

[0075] Example 2 Expression and preparation of TIR wild-type enzyme

[0076] The recombinant pET-30a-TIR vector obtained in Example 1 was transformed into Escherichia coli BL21 (DE3), and the target strain BL21 (DE3) / pET-30a-TIR in which the plasmid was successfully introduced into the host bacteria was preliminarily screened out and stored in the form of 15% wt glycerol bacteria.

[0077] The E. coli containing the target gene was inoculated into 50 mL LB liquid medium (yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L) containing 50 μg / mL kanamycin at a 1% inoculation rate, and cultured overnight in a shaker at 180 rpm and 37°C. The overnight cultured bacterial liquid was transferred to 250 mL TB liquid medium (yeast extract 24 g / L, tryptone 12 g / L, glycerol 4 g / L, KH2PO4 2.3 g / L, K2HPO4 12.5 g / L) containing 50 μg / mL kanamycin at a 2% inoculation rate, and cultured in a shaker at 200 rpm and 37°C until OD 600 When the pH value was about 1.5, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2 mM to induce protein expression, and the culture was carried out at 200 rpm and 25°C for 17 hours to obtain a fermentation broth. After the fermentation culture was completed, the bacterial broth was centrifuged at 4°C and 4000 rpm for 15 minutes to collect the bacterial cells, and after adding 35-40 mL of 0.01 M PBS buffer with a pH value of 7.4, it was placed in an ice bath and broken with a cell disruptor, and then freeze-dried into enzyme powder, which was the wild-type imine reductase, for later use.

[0078] Example 3 Site-directed mutagenesis of gene sequence:

[0079] Discovery Studio software was used to perform molecular docking between TIR and the substrate hexamethylmysamine, and the amino acids around hexamethylmysamine were analyzed based on the key sites of enzyme-substrate interaction. 28 amino acid sites were selected for site-directed saturation mutagenesis and catalytic activity change detection.

[0080] The mutation sites are methionine M at position 12, glycine G at position 13, proline P at position 95, phenylalanine F at position 120, proline P at position 121, alanine A at position 122, glutamine Q at position 123, isoleucine I at position 124, valine V at position 171, tyrosine Y at position 174, phenylalanine F at position 175, glycine G at position 178, leucine L at position 179, alanine A at position 182, Isoleucine I at position 209, glycine G at position 212, aspartic acid D at position 213, histidine H at position 216, tryptophan W at position 236, serine S at position 237, alanine A at position 238, serine S at position 241, arginine R at position 242, leucine L at position 243, alanine A at position 244, glycine G at position 245, histidine H at position 246, and valine V at position 283 mutated into 19 other amino acids.

[0081] Primers were designed according to the selected mutation sites as shown in Table 1:

[0082] Table 1 Primer information

[0083]

[0084]

[0085] N=A,T,C,G; M=A,C; K=G,T

[0086] PCR amplification system (50 μL): 20 ng of plasmid template, 2 μL of upstream and downstream primers (10 μM), 25 μL of high-fidelity enzyme, and add ddH2O to make up to 50 μL.

[0087] PCR amplification conditions: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 72°C for 40 s, for a total of 35 cycles, and complete extension at 72°C for 1 min.

[0088] 10 μL of each PCR product was mixed, and the restriction endonuclease DpnI was added to digest at 37°C. After recovery using a DNA gel recovery kit, the product was transformed into Escherichia coli BL21 (DE3) competent cells, and the bacterial solution was spread on a kanamycin-resistant LB plate and cultured overnight at 37°C.

[0089] After culturing for about 16 h, a sterile toothpick was used to pick up a mutant single colony (mutant library strain) and the recombinant strain BL21 (DE3) / pET-30a-TIR obtained in Example 1 and placed in a 96-well plate. The plates were cultured at 37 ° C and 220 rpm for 16 h. An equal volume of 30% sterile glycerol was added to the plate with a pipette, and the plates were evenly pipetted and stored in a -80 ° C refrigerator for later use.

[0090] Example 4 TIR single point mutation conversion rate detection

[0091] The mutant library strains obtained in Example 3 were activated in the first stage. 20 μL of bacterial solution was transferred from the first-stage well plate to a 96-well plate filled with TB medium (kanamycin concentration was 50 μg / mL) using a pipette and cultured at 37°C, 220 rpm for 4 h. The OD 600 When the pH reaches 0.6-0.8, add IPTG to a final concentration of 0.2mM and continue culturing at 25℃ for 18h. After the culture is completed, centrifuge the plate at 4℃, 4000rpm for 15min, retain the bacteria, and place the plate in a -80℃ refrigerator for 30min. Use a pipette to draw 200μL of lysozyme solution into the well with bacteria, shake and mix, and place it in a 37℃ shaker for 1h. Then centrifuge the plate at 4℃, 4000rpm for 15min, and retain the supernatant.

[0092] Add 200 μL of the reaction solution to the well plate containing the enzyme supernatant (the reaction solution formula is: 50 mM potassium phosphate buffer solution, pH 6.0, 2.7 mM hexamethylmycamine, 1.8 mM glucose, 0.5 mM NADP + , 0.5 mg glucose dehydrogenase bacteria), react at 28°C for 6 h. After the reaction, add 800 μL of ethyl acetate to mix, centrifuge at 4000 rpm for 10 min, and pipette the supernatant into a well plate for enzyme activity and product chirality analysis.

[0093] Reaction enzyme activity determination method: The gas phase detection of 6-methylnornicotine selected Agilent DB-35 chromatographic column (column length 250mm, column inner diameter 4.6mm, column thickness 0.255μm), heating program: initial temperature 100℃, hold for 1min, increase to 130℃ at a rate of 8.0℃ / min and hold for 3min, then increase to 220℃ at a rate of 5℃ / min and hold for 8min. SPL1 temperature: 250℃; FID temperature: 275℃; split ratio: 50:1; injection volume: 0.5μL;

[0094] Column flow rate: 1.0 mL / min; pressure: 94.2 kPa; carrier gas: N2; linear velocity: 28.2 cm / sec;

[0095] Chromatographic conditions for detecting chiral purity: Chromatographic column: OD-H polysaccharide derivative normal phase coated chiral chromatographic column (5μm, 4.6mmI.D.×250mmL); (UV) detection wavelength: 254nm; flow rate: 0.8ml / min; column temperature: 35℃; injection volume: 20μl.

[0096] The conversion rate was calculated by measuring the peak area of ​​6-methylnornicotine by GC, and the formula is as follows:

[0097] Con=A Nor / (A Nor +A Nme )*100%, A Nor is the peak area of ​​6-methylnornicotine, A Nme is the peak area of ​​hexamethylmysmine.

[0098] The chirality was calculated by measuring the peak area of ​​6-methylnornicotine by HPLC, and the formula is as follows:

[0099] ee%=(A Nor -A Nme ) / (A Nor +A Nme )*100%, A Nor is the peak area of ​​the main configuration of 6-methylnornicotine, A Nme It is the peak area of ​​another configuration of 6-methylnornicotine.

[0100] Single point mutation sites with higher conversion rates than the parental WT were selected and sent to a sequencing company for sequencing. The results are shown in Table 2.

[0101] Table 2 Single point mutants

[0102]

[0103]

[0104] According to Table 2, after single-point mutations of amino acids at positions 122, 171, 174, 175, 213, 236, 241, 242, 244, and 283, the conversion rates were higher than those of the wild type, and the conversion rate of the mutant F175R was as high as 86.4%.

[0105] Example 5 TIR multi-point mutation screening

[0106] Based on the results of Example 4, single-point mutations were combined to screen out multi-site mutants of imine reductase with improved catalytic activity.

[0107] The mutation sites are obtained by randomly combining the single-point mutations at positions 122, 171, 174, 175, 213, 236, 241, 242, 244, and 283 obtained in Example 4 into multi-site mutants.

[0108] The primers designed according to the mutation sites are shown in Table 3:

[0109] Table 3 Primer information

[0110]

[0111] Multiplex PCR amplification system (50 μL): 20 ng of plasmid template, 2 μL of each pair of upstream and downstream primers (10 μM), 25 μL of high-fidelity enzyme, and add ddH2O to make up to 50 μL.

[0112] PCR amplification conditions were the same as in Example 3

[0113] The obtained PCR product was digested with restriction endonuclease DpnI at 37°C, recovered with a DNA gel recovery kit, transformed into E. coli TOP10 competent cells, and then spread on a kanamycin-resistant plate and cultured overnight at 37°C. The single clone site was picked and sent to a sequencing company for sequencing.

[0114] The sequencing results were analyzed to obtain cloned strains with successful multi-point mutations. The correct gene sequence plasmid was extracted using a plasmid extraction kit and transformed into BL21 (DE3) competent cells. After culturing on a kanamycin-resistant plate at 37°C overnight, a single colony was picked and placed in a 96-well plate and cultured at 37°C, 220rpm for 16 hours. After shaking, an equal volume of 30% sterile glycerol was added, pipetted evenly, and stored in a -80°C refrigerator.

[0115] Example 6 TIR multi-point mutation conversion rate detection

[0116] The multi-point mutant library strains obtained in Example 5 were activated, 20 μL of the bacterial solution was transferred to a 96-well plate, cultured at 37°C, 220 rpm for 4 h, IPTG was added for induction to a final concentration of 0.2 mM, and cultured at 25°C for 18 h. After the culture was completed, the bacterial cells were centrifuged to retain the cells, the well plate was placed in a -80°C refrigerator for 30 min, 200 μL of lysozyme solution was added, the mixture was shaken and placed in a 37°C shaker for 1 h, and then the supernatant was centrifuged to retain the supernatant.

[0117] Add 200 μL of the reaction solution to the well plate containing the enzyme supernatant (the reaction solution formula is: 50 mM potassium phosphate buffer solution, pH 6.0, 2.7 mM hexamethylmycamine, 1.8 mM glucose, 0.5 mM NADP + , 0.5 mg glucose dehydrogenase bacteria), react at 28°C for 6 h. After the reaction, add 800 μL of ethyl acetate to mix, centrifuge at 4000 rpm for 10 min, and pipette the supernatant into a well plate for enzyme activity and product chirality analysis.

[0118] The transformation rates and chirality results of the multi-site mutant strains are shown in Table 4.

[0119] Table 4 Transformation rate and chirality results of multi-site mutant strains

[0120]

[0121] As shown in Table 4, the mutant A122S / D213M had the highest conversion rate of 98.3% and an ee% of 99.4%.

[0122] Example 7 Inducible expression of imine reductase mutants F175R and A122S / D213M recombinant bacteria

[0123] The imine reductase mutant F175R recombinant bacteria obtained in Example 4 and the mutant A122S / D213M recombinant bacteria obtained in Example 6 were inoculated into TB medium at 1% inoculation amount and cultured at 37°C and 220 rpm until OD 600 When the pH value reached about 1.5, the culture temperature was adjusted to 25°C, IPTG (final concentration was 0.2 mM) was added to induce the expression of the target protein, and the expressing bacteria were collected by centrifugation after culturing for 17 hours. The enzyme powder was dissolved and broken with PBS buffer at pH 7.4, and freeze-dried to be used for subsequent experiments.

[0124] Example 8 Functional verification of TIR parent (WT) and mutant strains (F175R, A122S / D213M)

[0125] Reaction system: hexamethylmysamine 1.35mM, glucose 1.8mM, coenzyme NADP + 0.5mM, imine reductase parent and mutant strain enzyme powder 10g / L, glucose dehydrogenase bacteria 20g / L, 10mL 50mM potassium phosphate buffer solution with a pH value of 6.0. Add other substances except enzymes in the reaction system and incubate for 10 minutes, adjust the pH value to 6.0, then add enzyme to start the reaction, add sodium hydroxide solution after the reaction is completed to adjust the pH value to greater than 12, extract with ethyl acetate, centrifuge, dilute a certain number of times, filter with a 0.22μm organic filter membrane and then detect. The conversion rate of hexamethylmycamine to 6-methylnornicotine catalyzed by TIR(WT) imine reductase was 30.3%, and the ee% was 89.2%. The conversion rate of hexamethylmycamine to 6-methylnornicotine catalyzed by TIR(F175R) imine reductase was 90.4%, and the ee% was 99.5%. The conversion rate of hexamethylmycamine to 6-methylnornicotine catalyzed by TIR(A122S / D213M) imine reductase was 99.2%, and the ee% was 99.6%. The above shows that the catalytic activity of TIR(A122S / D213M) is 3.27 times that of TIR(WT) and 1.1 times that of TIR(F175R).

[0126] Example 9 Optimization of reaction conditions for the production of 6-methylnornicotine by TIR (A122S / D213M) whole cell catalysis

[0127] (1) Determine the optimal temperature of the reaction

[0128] The enzyme catalyzed reaction was carried out in 10 mL of 50 mM potassium phosphate buffer solution with a pH value of 6.0, which contained 1.35 mM hexamethylmysamine substrate, 1.8 mM glucose, 10 g / L imine reductase enzyme powder, 20 g / L glucose dehydrogenase bacteria, and coenzyme NADP + 0.5mM.

[0129] Add other substances except enzymes to the reaction system and incubate for 10 minutes. Adjust the pH value to 6.0 before adding enzymes to start the reaction. Set the reaction temperature to 28°C, 32°C, and 36°C. Perform GC gas phase detection after the reaction is completed. Figure 1 It can be seen that the optimal temperature for the reaction is 28°C. After 24 hours of reaction, the product proportion of 6-methylnornicotine can be as high as 99%, and the ee% is 99.5%.

[0130] (2) Determine the optimal substrate concentration for the reaction

[0131] Based on the above reaction temperature optimization, at 28 ° C, the imine reductase catalyzed reaction was carried out in 10 mL of 50 mM potassium phosphate buffer solution with a pH value of 6.0, and different substrate concentrations were set, and the substrate concentration was increased or decreased by 1 / 3. The control group contained substrate hexamethylmysamine 1.35 mM, glucose 1.8 mM, imine reductase enzyme powder 10 g / L, glucose dehydrogenase bacteria 20 g / L, coenzyme NADP + 0.5mM. Add other substances except enzymes to the reaction system and incubate for 10 minutes. Adjust the pH value to 6.0 before adding enzymes to start the reaction. After the reaction is completed, perform GC gas phase detection. Figure 2 It can be seen that when the substrate 6-methylmysamine of the reaction is reduced by 1 / 3, after reacting for 6 hours, the product proportion of 6-methylnornicotine can be as high as 99%, and the ee% is 99.6%.

[0132] (3) Determine the optimal amount of imine reductase added to the reaction

[0133] Based on the optimization of the above reaction temperature and substrate concentration, the imine reductase catalyzed reaction was carried out in 10 mL of 50 mM potassium phosphate buffer solution with a pH value of 6.0 at 28 °C. Different imine reductase whole cell addition amounts were set, including 1 / 3, 1-fold, and 2-fold increase in the imine reductase whole cell addition amount. The control group contained substrate hexamethylmysamine 1.35 mM, glucose 1.8 mM, imine reductase enzyme powder 10 g / L, glucose dehydrogenase bacteria 20 g / L, and coenzyme NADP + 0.5mM. Add other substances except enzymes to the reaction system and incubate for 10 minutes. Adjust the pH value to 6.0 before adding enzymes to start the reaction. After the reaction is completed, perform GC gas phase detection. Figure 3 It can be seen that when the amount of imine reductase added to the whole cell of the reaction is increased by 1 / 3, the product proportion of 6-methylnornicotine can be as high as 99% after 6 hours of reaction, and the ee% is 99.6%.

[0134] (4) Determine the optimal amount of glucose added for the reaction

[0135] Based on the above optimization of reaction temperature, substrate concentration and the amount of imine reductase whole cell added, the imine reductase catalytic reaction was carried out in 10 mL of 50 mM potassium phosphate buffer solution with a pH value of 6.0 at 28 ° C, and different glucose addition amounts were set, with glucose increased by 1 / 2 and decreased by 1 / 2. The control group contained substrate hexamethylmysamine 1.35 mM, glucose 1.8 mM, imine reductase enzyme powder 10 g / L, glucose dehydrogenase bacteria 20 g / L, coenzyme NADP + 0.5mM. Add other substances except enzymes to the reaction system and incubate for 10 minutes. Adjust the pH value to 6.0 before adding enzymes to start the reaction. After the reaction is completed, perform GC gas phase detection. Figure 4 It can be seen that when the glucose in the reaction increases by 1 / 2 or decreases by 1 / 2, the product proportion decreases to 77.3% and 38.9% respectively. Therefore, it can be determined that the optimal addition amount of glucose in the reaction is 1.8 mM.

[0136] The above examples provide the technical results of different imine reductase mutants catalyzing the synthesis of 6-methylnornicotine from hexamethylmysamine. From the results, it can be seen that compared with the parent, the conversion rate and optical purity of the imine reductase mutant provided by the present invention to generate 6-methylnornicotine are greatly improved.

[0137] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An imine reductase mutant, characterized in that: The mutant is obtained by mutating the 122nd alanine, the 171st valine, the 174th tyrosine, the 175th phenylalanine, the 213th aspartic acid, the 236th tryptophan, the 241st serine, the 242nd arginine, the 244th alanine and / or the 283rd valine of the imine reductase parent, and the amino acid sequence of the parent is shown in SEQ ID NO.

2.

2. The mutant according to claim 1, characterized in that The mutant is a mutant of the parent subjected to any of the following mutations: (1) Mutate alanine A at position 122 to serine S; (2) mutating valine V at position 171 to glycine G; (3) mutating tyrosine Y at position 174 to methionine M; (4) mutating phenylalanine F at position 175 to arginine R; (5) Mutation of aspartic acid D at position 213 to methionine M; (6) mutating tryptophan W at position 236 to arginine R; (7) mutating serine S at position 241 to arginine R; (8) mutating arginine R at position 242 to methionine M; (9) Mutation of alanine A at position 244 to arginine R; (10) mutating valine V at position 283 to arginine R; (11) The alanine A at position 122 was mutated to serine S, and the aspartic acid D at position 213 was mutated to methionine M; (12) The valine V at position 171 was mutated to glycine G, and the phenylalanine F at position 175 was mutated to arginine R; (13) The tyrosine Y at position 174 was mutated to methionine M, and the arginine R at position 242 was mutated to methionine M; (14) mutating tryptophan W at position 236 to arginine R and valine V at position 283 to arginine R; (15) mutating the valine V at position 171 to glycine G, the aspartic acid D at position 213 to methionine M, and the valine V at position 283 to arginine R; (16) mutating phenylalanine F at position 175 to arginine R, mutating arginine R at position 242 to methionine M, and mutating alanine A at position 244 to arginine R; (17) Aspartic acid D at position 213 was mutated to methionine M, serine S at position 241 was mutated to arginine R, and valine V at position 283 was mutated to arginine R; (18) mutating valine V at position 171 to glycine G, mutating aspartic acid D at position 213 to methionine M, mutating serine S at position 241 to arginine R, and mutating alanine A at position 244 to arginine R; (19) The tyrosine Y at position 174 was mutated to methionine M, the aspartic acid D at position 213 was mutated to methionine M, the alanine A at position 244 was mutated to arginine R, and the valine V at position 283 was mutated to arginine R.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector uses pET-30a(+) as an expression vector.

6. A recombinant microbial cell expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3, or transformed with the recombinant vector according to claim 4 or 5.

7. The recombinant microbial cell according to claim 6, characterized in that The recombinant microbial cell uses Escherichia coli BL21 (DE3) as a host cell.

8. A method for improving the activity of imine reductase, characterized in that: The method comprises subjecting the imine reductase parent with the amino acid sequence as shown in SEQ ID NO.2 to single point mutation or combined mutation as shown in any one of (a) to (j): (a) mutating alanine A at position 122 to serine S; (b) mutating the valine V at position 171 to glycine G; (c) mutating tyrosine Y at position 174 to methionine M; (d) mutating phenylalanine F at position 175 to arginine R; (e) mutating aspartic acid D at position 213 to methionine M; (f) mutating the tryptophan W at position 236 to arginine R; (g) mutating the serine S at position 241 to arginine R; (h) mutating arginine R at position 242 to methionine M; (i) mutating alanine A at position 244 to arginine R; (j) The valine V at position 283 was mutated to arginine R.

9. A method for preparing 6-methylnornicotine, characterized in that: The method uses 6-methylmysamine as a substrate and the mutant described in the first technical solution as a catalyst to form a catalytic system to catalytically generate 6-methylnornicotine.

10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant vector according to claim 4 or 5, or the recombinant microbial cell according to claim 6 or 7, or the method according to claim 8, or the method according to claim 9 in producing 6-methylnornicotine or a product containing 6-methylnornicotine.

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