CN117965479B

CN117965479B - Imine reductase mutant and application thereof

Info

Publication number: CN117965479B
Application number: CN202311704978.1A
Authority: CN
Inventors: 李杉; 邝小霜; 张雷
Original assignee: South China University of Technology SCUT; Zhongshan Institute of Modern Industrial Technology of South China University of Technology
Current assignee: South China University of Technology SCUT; Zhongshan Institute of Modern Industrial Technology of South China University of Technology
Priority date: 2023-12-13
Filing date: 2023-12-13
Publication date: 2024-08-06
Anticipated expiration: 2043-12-13
Also published as: CN117965479A

Abstract

The invention belongs to the technical field of biocatalysis synthesis, and discloses an imine reductase mutant and application thereof, wherein the amino acid sequence of the imine reductase mutant is the amino acid sequence of SEQ ID NO.1 subjected to combined mutation, and the mutation points are as follows: alanine at position 126 was mutated to asparagine and methionine at position 181 was mutated to leucine, the amino acid sequence of which is shown in SEQ ID NO. 7. The iminoreductase mutant has high catalytic activity, high stereoselectivity and low consumption of a coenzyme regeneration system, and can be applied to the preparation of chiral (S) -nornicotine. The imine reductase provided by the invention is used for catalyzing the process of preparing (S) -nornicotine by using the myosmine, the conversion rate and the enantiomeric excess percentage are higher, the pH of the catalytic reaction is wider, the relative activity of the enzyme is more than 90% at pH6.0-8.5, the enzyme activity property is more stable, and the cost can be obviously reduced.

Description

Imine reductase mutant and application thereof

Technical Field

The invention belongs to the field of biocatalysis, and relates to an imine reductase mutant from nocardia and application thereof.

Background

Chiral compounds refer to organic compounds having chiral carbon atoms, and the two enantiomers in chiral drugs may have completely different pharmacodynamic effects, resulting in serious side effects, such as the different effects of stereoisomers exhibited by the anti-pregnancy reaction drug thalidomide used in the "reaction arrest" event occurring in the 20 th century. Chiral amine compounds, particularly cyclic chiral amine drugs, are important pharmaceutical intermediates, and asymmetric synthesis technology thereof has been attracting attention, and is one of important research contents of biocatalysis.

With the development of enzyme catalysis technology, various enzymes such as transaminase, imine reductase, reductive amination enzyme, amine dehydrogenase and the like are discovered and can be applied to the synthesis process of chiral amine. However, different enzymes have different characteristics. For example, certain enzymes have relatively low activities and a narrow substrate spectrum, requiring the involvement of complex coenzyme regeneration systems. For this reason, it is particularly important to find a highly active and highly stereoselective bio-enzyme catalyst for preparing chiral amines.

(S) -nornicotine, also known as (S) -nornicotine, english name (S) - (-) -Nornicotine, CAS number: 494-97-3, the molecular formula C ₉H₁₂N₂ is shown in the specification, is a nicotine metabolite and tobacco alkaloid, and can be used as a tobacco withdrawal agent in electronic cigarettes.

The compound is synthesized by a chemical method, which is mostly prepared by taking myosmine as a raw material and using a chiral derivatizing agent or a metal catalyst through multi-step reaction, the optical purity and the yield of the product are low, the environmental pollution is easy to cause, and the compound has a plurality of limitations in actual mass production. In recent years, the catalytic synthesis of (S) -nornicotine by using a biological enzyme method has been attracting attention, and although a lot of reports about the catalytic reduction of myosmine by using the imine reductase and the industrial application and production are available, in the current industrial application of enzyme catalysis, the problems of weak enzyme specificity, large use amount of enzyme and coenzyme thereof, low yield and the like still exist, and a biological enzyme and coenzyme system suitable for the industrial application still needs to be further excavated, so that the production cost is reduced.

U.S. Pat. No. 5,109, 962.B2 reports an example of the formation of (S) -nornicotine by the reduction of myosmine by imine reductase, wherein the substrate concentration can reach 0.4mol/L (58.4 g/L), the substrate concentration is increased, and the conversion rate is obviously reduced. Later, researchers use imine reductase mutants to catalyze the generation of (S) -nornicotine, so as to improve the substrate concentration of a reaction system and improve the conversion efficiency of enzyme catalysis. CN 113774036A reacts for 4 hours in a reaction system of 100g/L substrate concentration, 0.3Wt thallus wet weight, 204g/L glucose concentration and 150mL/L glucose dehydrogenase by utilizing imine reductase mutants (A246V and D285V) derived from Nocardiopsis aiba strain, the conversion rate can reach 99.9%, e.e. can reach 99.0%, good enantioselectivity and activity are shown, but the enzyme stability is poor, the requirement on pH value is higher in the reaction of preparing (S) -nornicotine by catalyzing the myosmine, the reaction conversion rate is only 35.5% in 24 hours under the condition of 8.5 of catalyzing the reaction pH, and the consumption of a coenzyme regeneration system is higher, so that the production cost is higher.

Therefore, there is a need in the industry to find imine reductases of different high activities to accommodate a wider pH environment while reducing the amount of coenzyme regeneration system used to reduce production costs.

Disclosure of Invention

In order to efficiently synthesize (S) -nornicotine under mild reaction conditions, the invention performs directed evolution on imine reductase NaIRED from nocardia (Nocardiopsis aiba), obtains imine reductase mutants with improved enzyme activity and substrate tolerance, reduces the consumption of a coenzyme regeneration system (glucose dehydrogenase/glucose), reduces the consumption of a coenzyme regeneration system (CAS: 532-12-7), and prepares (S) -nornicotine with high chiral purity, the conversion rate reaches more than 99.9%, the optical purity of a product is more than 99.5%, the catalytic reduction activity of more than 85% can be maintained within the pH range of 6.0-9.0, and meanwhile, the production cost is reduced.

The technical scheme of the invention is as follows:

in one aspect, provided is an imine reductase mutein, said mutein being a non-natural protein and said mutein being an imine reductase mutant mutated at one or more positions corresponding to positions 71, 126, 127, 174, 178 and 181 in SEQ ID:

preferably, the cysteine at position 71 is mutated to asparagine.

Preferably, the alanine at position 126 is mutated to asparagine.

Preferably, threonine at position 127 is mutated to proline.

Preferably, aspartic acid at position 174 is mutated to glutamic acid.

Preferably, the leucine at position 178 is mutated to cysteine.

More preferred are the following combinatorial mutations: alanine at position 126 was mutated to asparagine and methionine at position 181 was mutated to leucine. An imine reductase mutant, wherein the amino acid sequence of the imine reductase mutant is the amino acid sequence with combined mutation of SEQ ID NO.1, and the mutation points are as follows: alanine at position 126 was mutated to asparagine and methionine at position 181 was mutated to leucine, the amino acid sequence of which is shown in SEQ ID NO. 7.

The nucleotide sequence of the coding gene of the imine reductase mutant is shown as SEQ ID NO. 14.

The invention discloses an imine reductase and an encoding gene thereof, which are improved based on Nocardiopsis aiba strain natural imine reductase, wherein an imine reductase mutant is used as a biological enzyme catalyst to catalyze a substrate of myosmine to synthesize (S) -nornicotine, and the reaction formula is as follows:

Wherein: NAD is a biological proper noun, chinese name: nicotinamide adenine dinucleotide, referred to as coenzyme for short; (P) indicates the presence or absence of P in parentheses and, in some cases, NADP. The chinese name of NADP is nicotinamide adenine dinucleotide phosphate, also known as pyridine nucleotide Triphosphate (TPN) or co-dehydrogenase pi or oxidized coenzyme pi.

A recombinant plasmid containing the coding gene of the imine reductase mutant. Preferably, the plasmid is pET-28a (+).

A host cell comprising said recombinant plasmid. Preferably, the host cell is an E.coli BL21 (DE 3) cell.

The preparation method of the imine reductase and the mutant thereof comprises the following steps:

Activating the strain: inoculating genetically engineered bacteria loaded with SEQ ID NO.1 gene into LB resistant culture medium, controlling the temperature of a shaking table to be 35-38 ℃, and culturing at the rotating speed of 200-220rpm for 10-12h to obtain seed liquid; preparing crude enzyme solution; inoculating the seed liquid into LB resistant culture medium, controlling the temperature of a shaking table to be 35-38 ℃, controlling the rotating speed to be 200-220rpm, culturing bacterial liquid to be 2.5-3.5h, adding an inducer when the OD ₆₀₀ value is 0.6-0.8, controlling the temperature of the shaking table to be 16-25 ℃ and the rotating speed to be 180-200rpm, continuously inducing and culturing for 18-20h, centrifuging, collecting sediment, and performing ultrasonic crushing to obtain the imine reductase crude enzyme liquid.

Further, the construction method of the genetically engineered bacterium comprises the following steps:

The gene of SEQ ID NO.1 is taken as a target gene, a plasmid pET28a (+) is taken as an expression vector, the target gene is inserted into the expression vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into a host strain to obtain the genetically engineered bacterium.

The imine reductase mutant is applied to the preparation of chiral (S) -nornicotine.

The imine reductase mutants described above catalyze the following reactions: specifically, the catalytic reaction takes bacterial whole cells or broken liquid supernatant of the cultured genetically engineered bacteria containing the imine reductase mutant as a catalyst, wheat ston as a substrate, NAD (P ⁺) as coenzyme, glucose dehydrogenase/glucose as a regeneration system, and the catalytic reaction is carried out for 12+/-4 hours under the stirring conditions of 25-35 ℃ and pH6.0-9.0 and 150-250rpm, so as to synthesize the (S) -nornicotine.

Preferably, the concentration of NAD (P ⁺) is 0.1-1.0g/L.

The reaction has one or more characteristics selected from the group consisting of:

(i) The reaction system contains 20-100g/L of thallus, more preferably 20-40g/L;

(ii) The pH of the reaction system is 6.0 to 9.0, preferably 6.5 to 8.0, more preferably 7.2;

(iii) The reaction system temperature is 15 ℃ to 45 ℃, preferably 25 ℃ to 35 ℃, more preferably 30 ℃.

(Iv) The concentration of the catalytic substrate is 2.5-100g/L, more preferably 40-60g/L;

(v) The ee value of the (S) -nornicotine obtained by catalysis is >99.5%.

(Vi) The concentration of GDH is 50-500mL/L, preferably 60-100mL/L; more preferably 80mL/L.

(Vii) The concentration of glucose is 30-100g/L, preferably 30-50g/L; more preferably 40g/L.

Compared with the prior art, the invention has the following beneficial effects:

The preparation method of the imine reductase adopts a genetic engineering technology to obtain the genetic engineering bacteria for expressing the imine reductase, and the genetic engineering bacteria are used for producing the imine reductase, thus being suitable for the current industrial production. The imine reductase provided by the invention is used for catalyzing the process of preparing (S) -nornicotine by using the myosmine, the conversion rate and the enantiomeric excess percentage are higher, the pH of the catalytic reaction is wider, the relative activity of the enzyme is more than 90% at pH6.0-8.5, the enzyme activity property is more stable, and the cost can be obviously reduced.

Drawings

FIG. 1 is an electrophoretogram of NaIRED and mutant proteins after induced expression. Wherein M represents Marker,1 is wild-type NaIRED soluble protein expression, and 2-7 are soluble protein expression of mutants C71N, A126N, T127P, D174E, L178C, A N/M181L respectively.

Figure 2 is an HPLC profile of a mixed standard of myosmine and (S) -nornicotine.

FIG. 3 is an HPLC detection pattern of NaIRED mutant A126N/M181L catalytic Massa.

FIG. 4 is a chiral analysis of NaIRED mutant A126N/M181L catalytic Massa.

FIG. 5 is a graph showing the conversion rate, e.e.value, of NaIRED mutant A126N/M181L as a function of pH.

Detailed Description

The following examples further illustrate the invention but are not to be construed as limiting the invention.

As used herein, the term "AxxB" means that amino acid a at position xx is changed to amino acid B, e.g., C71N means that amino acid C at position 71 is mutated to amino acid N, and so on.

The technical scheme of the invention is that on the basis of wild type imine reductase shown in SEQ ID NO. 1 (the nucleotide sequence corresponding to the coding gene is shown in SEQ ID NO. 8), the gene of the imine reductase is mutated by adopting a full-plasmid PCR amplified molecular biological method, so that the amino acid sequence of the enzyme is changed, the change of the structure and the function of the enzyme is realized, and the imine reductase with at least one mutated site is obtained by a directional screening method. Specifically, the preparation method comprises the following steps: (1) The gene of the corresponding mutation site of the imine reductase NaIRED is constructed on a pET28a expression vector to obtain a recombinant plasmid with the target enzyme gene. (2) The recombinant plasmid is transferred into a host bacterial cell, preferably escherichia coli BL21 (DE 3), and the corresponding engineering strain is obtained. (3) Inoculating engineering strain into LB resistant culture medium for culturing, amplifying and culturing according to a certain proportion, and adding inducer IPTG or lactose or their mixture for inducing and culturing for a certain time. And (4) centrifugally collecting thalli, and carrying out high-pressure crushing.

The conversion rate of the imine reductase mutant C71N, A126, 126N, T127,127, 127P, D174,174E and L178C for catalyzing the synthesis of (S) -nornicotine is improved by 99.9 percent from 75.4 percent compared with the wild type imine reductase female parent, and the e.e.% is more than 99.0 percent (example 4), so the imine reductase mutant has very high stereoselectivity and conversion rate. The combined mutant A126N/M181L is reacted for 12 hours under 50g/L substrate myosmine and 0.3Wt imine reductase crude enzyme liquid (calculated according to cell wet weight), the substrate conversion rate under 40g/L glucose and 80mL/L GDH regeneration system can reach more than 99.9%, and the product e.e. value reaches 99.5% (example 5).

EXAMPLE 1 construction of the library of imine reductase NaIRED mutants

The present example relates to an imine reductase excavation and mutation process comprising the steps of:

(1) Selecting template sequences

The amino acid sequence of the imine reductase reported in the prior literature is searched, and the species source, catalytic activity data (substrate spectrum, activity, enzymatic property and the like), protein structure information, key amino acid in the structure of the protein and a catalytic mechanism are finished. 5 candidate template enzymes are arranged, a evolutionary tree is constructed by using MEGA6, the genetic relationship among sequences is analyzed, and the amino acid sequence of IRED from Nocardiopsis alba is finally selected as the template sequence by combining the catalytic data.

(2) Prediction of imine reductase mutant hot spots

According to the invention, a NaIRED three-dimensional structure prediction file is obtained from a AlphaFold protein structure prediction database, and mutation sites are further determined through analysis of substrate channels, binding pockets and molecular docking structures. The simulated NaIRED protein structure is further coupled and aligned with the protein crystal structures of the reductive amination enzymes AspRedAm (PDB: 5g6 r) and AtRedAm (PDB: 6h7 p) with higher sequence similarity respectively, and finally 17 key amino acid mutation sites are determined.

(3) Site-directed mutagenesis

Amino acids reported in the literature to play a key role in enzyme catalytic efficiency are determined to be degenerate codon NNK designed to mutate a primer, and pET28a-NaIRED is used as a template to carry out site-directed mutation on an amino acid sequence of imine reductase from Nocardiopsis alba strain. The site of the library-building mutation is 71, 98, 99, 126, 127, 174, 175, 178, 182, 209, 212, 215, 216, 241, 242, 245, 284, respectively, and the beneficial mutation site for obtaining the improvement of the enzyme activity is 71, 126, 127, 174, 178.

EXAMPLE 2 Induction of expression of imine reductase mutants

And carrying out protein induction expression on the constructed imine reductase mutant and preparing crude enzyme solution. The method specifically comprises the following steps:

Activating the strain: mu.L of pET28a-NaIRED recombinant mutant cells were inoculated into 5mL of liquid LB medium containing 50mg/mL kanamycin, and subjected to activation culture in a shaker at 220rpm at 37℃for 12-16 hours.

Preparing crude enzyme solution: inoculating the activated bacterial liquid into LB liquid medium containing 50mL and 50mg/mL kanamycin at 1% inoculation amount, culturing for 2.5-3.5h at 37 ℃ in a shaking table at 220rpm, adding inducer IPTG with final concentration of 0.1mM when the bacterial liquid concentration OD ₆₀₀ is 0.6-0.8, and inducing expression for 18-20h at 25 ℃ in a shaking table at 180 rpm. Centrifuging the cultured bacterial liquid at 10000rpm for 5min, discarding the supernatant, and collecting bacterial precipitate. The collected cells were suspended in a suitable amount of phosphate buffer (100 mM, pH 7.2), and crushed by an ultrasonic crusher for 8-10min. And (3) centrifuging again to obtain intracellular supernatant and precipitate, wherein the supernatant is the obtained crude enzyme solution of the imine reductase.

EXAMPLE 3 preliminary screening of the imine reductase wild type NaIRED

Imine reductase catalyzes the reaction of myosmine to prepare (S) -nornicotine: to a 1L reaction flask, 10g of the main material of Mestigmine, 1.0 Wt% of the crude enzyme solution for the mutant prepared in the above example 2 was added, 0.5L of phosphate buffer (100 mM, pH=7.2) was added, 0.4g of NADP ⁺, 40g of glucose, 0.1L of coenzyme (glucose dehydrogenase, GDH) was added, the pH of the reaction solution was controlled to 7.2, and after 24 hours of reaction at 30℃the conversion was 75.4% and 96.1% of e.e. value were measured by HPLC analysis.

EXAMPLE 4 screening of imine reductase mutants

(1) Imine reductase catalyzes the reaction of myosmine to prepare (S) -nornicotine:

To a 1L reaction flask, 10g of the main material of Mestigmine, 0.3Wt of the crude enzyme solution of the mutant prepared in the above example 2 was added, 0.5L of phosphate buffer (100 mM, pH=7.2) was added, 0.4g of NADP ⁺, 40g of glucose, 0.1L of coenzyme (glucose dehydrogenase, GDH) and an appropriate amount of the crude enzyme solution of the mutant were added, the pH of the reaction solution was controlled to 7.2, and after 24 hours of reaction at 30℃the conversion and e.e value were analyzed by HPLC.

(2) Measurement of catalytic conversion of imine reductase:

Adding a saturated solution of sodium hydroxide into the reaction system in the step (1) to adjust the pH value to be more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, spin-drying to collect a product, and detecting and analyzing by HPLC. The HPLC detection conditions were:

Instrument: agilent 1200

Chromatographic column: agilent poroshell 120C 18, 4.6X105 mm, 4. Mu.M.

Mobile phase: linear gradient elution was performed according to table 1 using 0.01M dipotassium hydrogen phosphate solution (pH adjusted with phosphoric acid 7.8) as mobile phase a and acetonitrile as mobile phase B;

TABLE 1 HPLC detection conditions for biocatalytic Mastigmine

Detection wavelength: 260nm;

flow rate: 1.0mL/min;

Column temperature: 40 ℃;

sample injection volume: 10. Mu.L;

Conversion calculation formula: conversion (%) =a (P)/[ a (P) +a (S) ]%100%

Wherein A (P) is the peak area of (S) -nornicotine; a (S) is the peak area of the starting material Mastigmine.

(3) Imine reductase catalytic product optical purity identification

And (3) carrying out mobile phase treatment on the reaction system in the step (2), and carrying out HPLC direct sample injection analysis after membrane filtration. The HPLC conditions were:

Instrument: waters 2489 uv detector;

chromatographic column: chiralpak-IA, 4.6X105 mm, 5. Mu.M;

Mobile phase: n-hexane: isopropanol: ethanolamine=95:5:0.1;

Detection wavelength: 260nm;

flow rate: 1.0mL/min;

column temperature: 25 ℃;

sample injection volume: 10. Mu.L;

the optical purity of the S-shaped product is calculated as follows: e =a (S) -a (R)/a (S) +a (R) 100%

Wherein A (S) is the peak area of the target product (S) -nornicotine; a (R) is the enantiomer (R) -nornicotine peak area.

The method comprises the steps of carrying out catalytic activity detection on a plurality of mutants with 17 mutation sites, establishing a transformation reaction for a substrate, investigating the transformation rate and the stereoselectivity of the mutants, and screening out five mutants with high transformation rate and high enantioselectivity by using an HPLC (high performance liquid chromatography) detection method, wherein the screening result of the imine reductase mutants is shown in a table 2.

The results in table 2 show that: the five mutants selected are optimal imine reductase mutants, the catalytic efficiency of the optimal imine reductase mutants on the myosmine is about 5 times that of a wild imine reductase female parent, and the product has a higher e.e. value.

TABLE 2NaIRED conversion and stereoselectivity of mutant substrates thereof

Example 5 construction of a combination mutant of imine reductase NaIRED

According to the catalytic result of example 4, the above several mutants were subjected to combined mutation prediction simulation by Hotspots on-line service software to construct a two-point combined mutant library. The catalytic results show that the combined mutation of selected sites 126 and 181 further increases substrate concentration while maintaining high conversion and high selectivity of the 126 site mutation in example 4. Specifically: the combined mutant with improved enzyme catalytic efficiency is characterized in that 126 th alanine is mutated into asparagine and 181 th methionine is mutated into leucine, and the amino acid sequence and the nucleotide sequence are shown as SEQ ID NO. 7 and SEQ ID NO. 14. Conversion reaction is established for substrate myosmine to examine the conversion rate and stereoselectivity of mutant: 50g of the main raw material of myosmine, 0.3 wt% of the crude enzyme solution of the mutant, 0.5L of phosphate buffer (100 mM, pH=7.2), 0.4g of NADP ⁺, 40g of glucose and 0.08L of coenzyme (glucose dehydrogenase, GDH) of the crude enzyme solution are added into a 1L reaction bottle, the pH of the reaction solution is controlled to 7.2, after the reaction is carried out for 12 hours at 30 ℃, a saturated solution of sodium hydroxide is added to adjust the pH value to more than 10, denatured proteins are removed centrifugally, the supernatant is extracted by methylene dichloride, dried, the product is collected by spin drying, and HPLC detection is carried out.

The result shows that the imine reductase combined mutant shown in SEQ ID NO. 7 reacts for 12 hours in a reaction system of enzyme catalysis of the myosmine, namely 50g/L substrate and 0.3Wt crude enzyme solution (calculated according to wet weight), the conversion rate can reach more than 99.9%, the e.e. value of the product reaches 99.5%, and the screened imine reductase mutant shows extremely high stereoselectivity and high efficiency in an enzymatic method for preparing (S) -nornicotine. In addition, the catalytic efficiency of the catalyst on the myosmine is 5 times that of the mutant in the example 4, the reaction time is shortened by half, and the high conversion rate and the enantioselectivity can be still maintained.

Compared with the A246V and D285V mutants protected in the prior published patent CN 113774036A, the invention provides a technical scheme that the high conversion rate and high enantioselectivity can be realized by the other five mutation sites, wherein the combined mutant A126N/M181L can further reduce the substrate inhibition and realize the complete conversion of 50g/L of the myosmine, the catalytic reaction of the myosmine only needs 0.3Wt of imine reductase crude enzyme liquid (calculated according to the wet weight of cells), the dosage of glucose added in the coenzyme regeneration system is only 40g/L, the dosage of glucose dehydrogenase GDH is reduced by 5 times compared with the dosage of glucose dehydrogenase GDH added in the patent, the industrial production cost of (S) -nornicotine and nicotine can be further reduced, and the invention has good industrial application value.

EXAMPLE 6 Whole-cell catalytic Synthesis of (S) -nornicotine by mutant A126N/M181L

Comparative example 5: use of the imine reductase mutant shown in SEQ ID NO. 7 in the preparation of (S) -nornicotine.

Adding 50g of myosmine, 0.3Wt of the mutant crude enzyme solution into A1L reaction bottle, adding 0.5L of phosphate buffer (100 mM) under different pH conditions (pH 6.0-9.0), adding 0.4g of NADP ⁺, 40g of glucose and 0.08L of coenzyme (glucose dehydrogenase, GDH) crude enzyme solution, controlling the pH of the reaction solution to be 7.2, reacting for 12 hours at 30 ℃, adding sodium hydroxide saturated solution to adjust the pH value to be more than 10, centrifuging to remove denatured proteins, extracting the supernatant by using dichloromethane, drying, and spin-drying to collect products, wherein the catalytic reduction activity of the mutant A126N/M181L can be maintained to be more than 90% in the pH range of 6.0-8.5 through HPLC detection, as shown in a graph of the catalytic efficiency influence of different pH on the mutant A126N/M181L, the conversion rate at the pH of 6.0 is 98.1%, and the conversion rate at the pH of 8.5 is 94.9%; and when the pH range is gradually increased to 9.0, the conversion rate of the enzyme is reduced to about 85%. The e.e.% value of the product is not affected by the change of the pH value, and reaches more than 99.0 percent.

Experimental results show that the conversion efficiency of enzyme catalysis can still be kept high after the pH value of the buffer solution is increased to 8.0. This suggests that the pH tolerance of the enzyme is enhanced after mutation, and the enzyme activity of the enzyme becomes more stable, improving its usability in synthetic chemistry to some extent.

EXAMPLE 7 Whole-cell catalytic Synthesis of (S) -nornicotine by mutant A126N/M181L

50G of myosmine, 0.3 wt% of the mutant crude enzyme solution, 0.5L of phosphate buffer (100 mM, pH 7.2), 0.4g of NADP ⁺, 40g of glucose and 0.08L of coenzyme (glucose dehydrogenase, GDH) crude enzyme solution are added into a 1L reaction bottle, the pH of the reaction solution is controlled to be 7.2, the reaction solution is respectively placed at 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ for 12 hours, then sodium hydroxide saturated solution is added for regulating the pH value to be more than 10, denatured proteins are removed by centrifugation, the supernatant is extracted by methylene dichloride, the product is dried and collected by spin drying, the imine reductase mutant has the highest reduction reaction activity on the myosmine in the range of 25-35 ℃ through HPLC detection, the conversion rate can reach 99.9%, and the catalytic efficiency at other temperatures is reduced to be less than 90.0%. The e.e.% value of the product is not affected by temperature changes.

The results demonstrate that temperature has a very important effect on the enzyme-catalyzed reaction. The reactivity of the enzyme increases with increasing temperature over a range of temperatures, but too high a temperature reduces or even deactivates the reactivity of the enzyme. The mutant has the best catalytic efficiency at 25-35℃and thus 30℃is considered the optimal reaction temperature for the enzyme, in combination with the conversion, conversion rate and product optics.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. An imine reductase mutant is characterized in that the amino acid sequence of the imine reductase mutant is shown as any one of SEQ ID NO. 2-7.

2. The imine reductase mutant coding gene as set forth in claim 1, characterized in that the nucleotide sequence is any one of SEQ ID NO 9-14.

3. A recombinant plasmid comprising a gene encoding the imine reductase mutant according to claim 2.

4. The recombinant plasmid according to claim 3, wherein the plasmid is pET-28a (+).

5. A host cell comprising the recombinant plasmid according to claim 3 or 4.

6. The host cell of claim 5, wherein the host cell is an E.coli BL21 (DE 3) cell.

7. Use of the imine reductase mutant according to claim 1 for the preparation of chiral (S) -nornicotine.

8. The use according to claim 7, characterized in that (S) -nornicotine is synthesized by catalytic reaction with a substrate of myosmine, a coenzyme of NAD (P ⁺), a regenerating system of glucose dehydrogenase/glucose, at 25-35 ℃ and with stirring at ph6.0-9.0, 150-250 rpm for 12±4 hours.

9. The use according to claim 8, wherein in the catalytic reaction, the concentration of the catalytic substrate is 50±10 g/L, the cell content is 30-100 g/L, the pH of the reaction system is 7.2±0.8, and the reaction temperature is 30 ℃; the bacterial strain is Escherichia coli BL21 (DE 3) according to claim 6.

10. The use according to claim 9, characterized in that the concentration of NAD (P ⁺) is 0.1-1.0 g/L, the concentration of glucose is 30-100 g/L and the concentration of glucose dehydrogenase crude enzyme is 50-500 mL/L.