CN114317472B - High stereoselectivity imine reductase and preparation method and application thereof - Google Patents

Abstract

The invention discloses a high stereoselectivity imine reductase and a preparation method and application thereof, and the imine reductase bacterial strain with high stereoselectivity is obtained by carrying out site-directed mutagenesis on wild type aeromonas. It can be used for preparing (S) -nornicotine by taking the myosmine as a substrate through biocatalysis; the invention further provides a cofactor circulating system containing glucose dehydrogenase and a process for producing (S) -nornicotine by utilizing the immobilized imine reductase, a high-concentration substrate can be converted in the reaction system, the conversion rate is not less than 99%, the optical purity of the product is not less than 99.5%, impurities such as cosolvent or free enzyme protein do not need to be introduced, the operation of the whole process is convenient, the separation of the product is simpler, and the repeated utilization rate of the immobilized enzyme is high.

Description

High stereoselectivity imine reductase and preparation method and application thereof

Technical Field

The invention relates to the field of biological enzyme catalysis, in particular to a high stereoselectivity imine reductase and a preparation method and application thereof.

Background

Nicotine, a common name, is nicotine, which is the main component of alkaloids in tobacco. It is an important chemical raw material for medicines, pesticides, spices and the like, and can be used for producing smoking cessation products, electronic cigarettes and pesticides. In recent years, the nicotine receptor acts on a central neurotransmitter system to promote the release of dopamine, so that the dopamine has a certain prevention effect on the onset of Parkinson's disease, and meanwhile, the nicotine is used as a raw material of medicaments for treating cardiovascular diseases, skin, snake and insect bite injuries and the like, so that the demand is increasing, and particularly, the high-purity nicotine is popular in the international market.

Nicotine contains one chiral center, and two enantiomers exist: the content, metabolic mechanism and physiological characteristics of (S) -nicotine and (R) -nicotine in tobacco are completely different, nicotine in natural tobacco is mainly S-configuration, and chemically synthesized nicotine is generally racemic during preparation process because chiral separation is not carried out. The presence of 50% of non-psychoactive (R) -nicotine and 50% of psychoactive (S) -nicotine, compared to natural tobacco extracts, divides the potential activity of tobacco tar into two parts.

(S) -nornicotine (compound of formula II) is an important intermediate for preparing (S) -nicotine (compound of formula III), and (S) -nicotine can be prepared by aminomethylation reaction of (S) -nornicotine, so that the optical purity of (S) -nornicotine determines the optical purity of (S) -nicotine. The biocatalysis method has the advantages of simple reaction operation, low cost, mild condition, low energy consumption, environmental friendliness, high optical purity of products and the like, and therefore, the biocatalysis method is receiving more and more attention. Enzymes commonly used in the bio-enzyme catalysis method of chiral amines mainly include transaminase, monoamine oxidase, dehydrogenase, imine reductase, and the like. Compared with other enzymes, the Imine Reductase (IRED) has obvious synthesis effect on the cyclic chiral amine, is obviously superior to resolution and deracemization, has wide applicable substrate range, mild reaction conditions, high catalytic efficiency and good stereoselectivity, and is an important way for synthesizing the chiral amine. An effective way to search for suitable S-type imine reductase is to synthesize (S) -nornicotine by imine reductase catalysis of Muscamine (formula I), and the process needs to consume the same amount of coenzyme NADPH (reduced nicotinamide adenine dinucleotide phosphate) for hydrogen supply and convert the coenzyme NADPH into NADP⁺Nicotinamide Adenine Dinucleotide Phosphate (NADPH), the coenzyme NADPH is expensive, glucose is generally used as an electron donor to oxidize glucose into corresponding gluconic acid under the catalysis of Glucose Dehydrogenase (GDH) in order to circulate the coenzyme NADPH, and electrons are transferred to NADP⁺Thereby circulating the coenzyme.

At present, in the field, preparation of high optical purity (S) -nicotine is mainly prepared by plant extraction, while chemical synthesis mainly comprises preparation of racemic nicotine and resolution of the racemic nicotine by using a chemical resolving agent to obtain (S) -nicotine, for example, in the Chinese patent application, "racemic nicotine is prepared by reacting ethyl nicotinate with N-vinyl pyrrolidone in the presence of alcoholate base and the subsequent processing steps" (publication No. CN111511726A, 2020, 08.07.s), mesmine is reduced to racemic nornicotine by using a reducing agent via a chemical method, and then the racemic nicotine is methylated to obtain levonicotine with chiral purity of 99% by using dibenzoyl tartaric acid for resolution. The chemical method needs a large amount of organic solvents and chemical resolving agents, has complex process, high cost and large pollution, and is contrary to the current green chemical theme. The chinese patent application "a method for preparing high optical purity nicotine" (publication No. CN112409327A, published 2021, 26.02) discloses the preparation of levonicotine using biological enzymes, but does not report the substrate concentration and enzyme dosage, and uses free enzymes to introduce more enzyme protein impurities into the enzyme conversion solution.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a high stereoselectivity S-type imine reductase and an expression sequence thereof, which can be used for reducing the Mesamine into (S) -nornicotine; it is also an object of the present invention to provide recombinant expression vectors and host cells based on the aforementioned expression sequences; it is still another object of the present invention to provide a process for preparing an S-type imine reductase comprising the above-mentioned S-type imine reductase; it is a further object of the present invention to provide the use of said S-imine reductase for the preparation of (S) -nornicotine.

The technical scheme is as follows: the S-type imine reductase disclosed by the invention comprises an amino acid sequence shown as SEQ ID No.1, and an encoding gene sequence of the S-type imine reductase comprises a nucleotide sequence shown as SEQ ID No. 2. The source of the sequence is obtained based on the wild type Aeromonas sp.fdaargos 1419, which is a model strain whose genome is disclosed in GenBank. A genome of Aeromonas sp.FDAARGOS 1419 is used as a template, a pair of primers is designed, and an imine reductase gene IRED I is obtained by amplification, wherein the nucleotide sequence of the imine reductase gene IRED I is shown as SEQ ID No.4, and the translated amino acid sequence IRED I is shown as SEQ ID No. 3. The invention obtains IRED II, namely an amino acid sequence shown in SEQ ID No.1, by carrying out site-directed mutagenesis on IRED I. IRED II is codon optimized to obtain the corresponding nucleotide sequence, namely SEQ ID No. 2.

Compared with IRED I, IRED II obtained in the above way has higher stereoselectivity to a substrate, namely, Mesamine (CAS:532-12-7), the enzyme can reduce Mesamine into (S) -nornicotine, the enantiomeric excess e.e.% >99 percent, especially, the enzyme can tolerate the Mesamine substrate with high concentration after being prepared into immobilized enzyme, and the enzyme can be recycled, so that the enzyme has higher industrial application value.

The invention can design a recombinant expression vector based on IRED II after site-directed mutagenesis and transfect to competent host cells, and obtain the S-type imine reductase with high stereoselectivity through fermentation engineering.

The preparation method of the S-type imine reductase comprises the following steps:

(1) carrying out site-directed mutagenesis on an imine reductase amino acid sequence IRED I of an Aeromonas sp.FDAARGOS 1419 strain, mutating A at the 57 th position to R, D at the 122 th position to A, A at the 176 th position to G, and S at the 241 th position to G, obtaining an amino acid sequence shown as SEQ ID No.1 after mutagenesis, and obtaining a nucleotide sequence shown as SEQ ID No.2 through codon optimization;

(2) constructing a recombinant expression vector containing the nucleotide sequence of SEQ ID No.2, and adding competent host cells to obtain a recombinant strain expressing the IRED protein;

(3) culturing the recombinant strain, and ultrasonically crushing to obtain crude enzyme solution for microorganism immobilization and/or separation and purification and/or biocatalysis reaction.

The nucleotide sequence shown in SEQ ID No.2 is utilized, an expression vector is constructed through the existing genetic engineering technology, and then competent host cells are added to obtain the corresponding strain for expressing the IRED protein.

The expression vector is selected from any one of pET, pGEX and pMAL, preferably pET-28 plasmid; the host cell is selected from any one of but not limited to escherichia coli, bacillus subtilis and pichia pastoris, the growth and propagation speed of the escherichia coli cell is high, and BL21(DE3) is suitable for high-efficiency expression of exogenous genes, so that the escherichia coli is preferably used as the host cell.

The S-type imine reductase prepared by the method can be directly used for biocatalysis and can also be used for industrially preparing (S) -nornicotine. The (S) -nornicotine is prepared by taking the myosmine as a substrate and adding the crude enzyme solution or the product thereof to construct a biocatalytic reaction system so as to obtain the (S) -nornicotine with high yield and high stereoselectivity. Wherein, the product of the crude enzyme liquid of the S-type imine reductase is a refined biological enzyme product obtained by separating and purifying the crude enzyme liquid or immobilized imine reductase obtained by immobilizing the crude enzyme liquid; the reaction system comprises glucose dehydrogenase, glucose and a hydrogen donor.

The glucose dehydrogenase is a crude enzyme solution or a product thereof prepared by culturing and ultrasonically crushing a glucose dehydrogenase-producing strain; wherein the crude enzyme liquid product prepared by the strain for producing the glucose dehydrogenase is a refined biological enzyme product obtained by separating and purifying crude enzyme liquid or immobilized glucose dehydrogenase obtained by immobilizing the crude enzyme liquid.

The amino acid Sequence of the glucose dehydrogenase GDH is shown in SEQ ID No.5, NCBI Reference Sequence: WP _ 214850512.1. The nucleotide sequence is shown in SEQ ID No. 6. As shown in formula IV, the substrate is dissolved in a buffer solution with proper pH, imine reductase and NADPH are used as hydrogen donors, imine in the substrate is reduced to amine with S-configuration, and NADP is generated⁺Glucose in the system is used as a hydrogen donor and is oxidized into gluconic acid under the catalysis of glucose dehydrogenase, and NADPH is generated, so that the regeneration cycle of the cofactor is realized.

Further, the crude enzyme solution product prepared by the glucose dehydrogenase expression strain is a refined biological enzyme product obtained by separating and purifying the crude enzyme solution or immobilized glucose dehydrogenase obtained by immobilizing the crude enzyme solution.

The crude enzyme solution prepared by the S-type imine reductase expression strain and the crude enzyme solution prepared by the glucose dehydrogenase expression strain can be selectively and directly used in a biocatalysis reaction system; optionally immobilizing the immobilized chitosan and adding the immobilized chitosan into a reaction system; one of them can be selected to be added as crude enzyme solution, and the other can be added after immobilization.

The preparation method of the immobilized enzyme comprises the following steps: adding the crude enzyme solution into an ammonium sulfate solution, then adding polyethyleneimine, and centrifuging to obtain a supernatant; and adding the activated amino resin or the activated epoxy resin into the supernatant, oscillating, crosslinking, washing with water, repeating the steps for a plurality of times, and performing suction filtration to obtain the immobilized enzyme.

More specifically, the preparation method of the immobilized enzyme comprises the following steps:

(IN-1) adding 1-10g/L ammonium sulfate solution into the crude enzyme solution, stirring, adding 0.05-2% (m/v) polyethyleneimine solution, stirring, centrifuging, and collecting the flocculated enzyme clear solution;

(IN-2A) amino resin Carrier activation

a. Adding PBS buffer solution (pH 4.2-4.5) into amino resin carrier, stirring or oscillating at 20-25 deg.C, adding alkaline solution to adjust pH to 8.0, and discarding liquid;

b. adding diluted PBS buffer (pH 8.0), stirring or shaking at 20-25 deg.C, and removing liquid;

c. adding diluted PBS buffer solution (pH 8.0), adding glutaraldehyde, stirring or oscillating at 20-25 deg.C, discarding liquid, washing with diluted PBS repeatedly for several times, and draining to obtain activated amino resin immobilized carrier;

(IN-2B) epoxy Carrier activation

Adding PBS buffer solution (pH 8.0) into the epoxy carrier, stirring or oscillating at the temperature of 20-25 ℃, performing suction filtration, washing twice with diluted PBS buffer solution (pH 8.0), and performing suction drying to obtain the activated epoxy resin immobilized carrier.

(IN-3) immobilization of imine reductase or glucose dehydrogenase

And (2) taking any one of the activated carriers IN the step (IN-2A) or the step (IN-2B), adding the flocculated enzyme clear liquid obtained IN the step (IN-1) into the activated carrier, wherein the mass ratio of the flocculated enzyme clear liquid to the activated carrier is 1:5-15, oscillating and crosslinking the enzyme clear liquid for 18-30h, carrying out suction filtration and water washing for a plurality of times, adding diluted PBS (pH 8.0) into the enzyme clear liquid, stirring or oscillating for a plurality of times, and carrying out suction filtration to obtain the corresponding immobilized enzyme.

The charging concentration of the substrate myosmine in the catalytic reaction system is at least 100g/L, and the buffer solution is selected from any one or a combination of more of but not limited to phosphate buffer solution, triethanolamine and Tris-HCl buffer solution. The conversion rate after 20 hours is not less than 80 percent, and the optical purity of the (S) -nornicotine product is not less than 98 percent.

As a preferable biocatalytic reaction system of the invention, the feeding concentration of the substrate myosmine can reach more than 300g/L, and the substrate of 200-300g/L can be efficiently catalyzed generally. The mass ratio of the immobilized imine reductase to the substrate, namely the masamine, is 0.5-1: 1, the mass ratio of the glucose to the substrate is 1.4-1.8: 1, the mass ratio of the immobilized glucose dehydrogenase to the substrate is 0.1-0.5: 1, and the hydrogen donor NADP⁺Is 0.1 to 0.5 percent of the mass of the substrate; catalytic reaction is carried out at 25-35 ℃ and pH 6.2-6.8, and the buffer solution is 0.1M triethanolamine hydrochloride buffer solution. The conversion rate after 20 hours of biocatalysis is not less than 99.5 percent, and the optical purity of the (S) -nornicotine product is not less than 99.5 percent. Wherein the imine reductase still retains more than 90% of the original enzyme activity after being used for 20 times.

The crude enzyme solution of the S-type imine reductase obtained by the invention can be separated and purified to obtain a biological enzyme refining agent sold in the market, and the separation and purification methods include but are not limited to salting-out precipitation, organic solvent precipitation, semi-permeable membrane dialysis, gel chromatography, PAGE, ion exchange chromatography, affinity chromatography and other methods.

The invention utilizes the strain obtained by site-directed mutagenesis to ferment and obtain biological enzyme, the biological enzyme is prepared into immobilized enzyme which is used for industrially catalyzing high-concentration myosmin substrate, the immobilized enzyme can be directly separated from conversion liquid by suction filtration after the conversion is finished, the immobilized enzyme can be repeatedly used for the next conversion, and the conversion liquid can be used for preparing S-nicotine. The method does not need to introduce impurities such as cosolvent or free enzyme protein, the whole process is convenient to operate, the product separation is simpler, and the reuse rate of the immobilized enzyme is high.

Drawings

FIG. 1 is a comparison of the resistance of example 6 free enzyme to a Mysmine substrate versus immobilized enzyme;

FIG. 2 is a graph showing the relative enzyme activity of example 7 after 20 times of repeated use of immobilized enzyme.

Detailed Description

The invention is further described with reference to the following figures and specific examples.

Example 1

1. Construction of Iminireductase Strain

Using genome of Aeromonas sp.FDAARGOS 1419 as template, and searching and designing the following primers according to NCBI:

forward primer (IRED-P-Nde I): 5'-GGAATTCCATATGCGCCATCTCTCTGTC-3'

Reverse primer (IRED-R-Xho I): 5'-CCGCTCGAGTCATTGTGCCGCTCCATTG-3'

PCR amplification, with the reaction conditions of 94 ℃ pre-denaturation for 5min, 30 cycles (94 ℃ 30s, 59 ℃ 30s, 72 ℃ 1.5min), and 72 ℃ re-extension for 10 min.

And performing gel validation and product recovery after the PCR is finished. And then carrying out enzyme digestion on the target fragment and pET-26b (+) plasmid respectively by a double enzyme digestion system consisting of Nde I and Xho I, recovering the enzyme digestion product after the reaction is finished, mixing the enzyme digested target fragment and pET-26b (+) vector according to a certain proportion, adding a solution containing DNA ligase, and carrying out ligation reaction at 16 ℃.

And adding the ligation product into escherichia coli DH5 alpha competent cells for transformation, quickly transferring the cells into a water bath kettle after ice bath for 30min, carrying out heat shock at 42 ℃ for 60s, and adding 500 mu L of sterile and non-resistant LB liquid culture medium into an ultra-clean bench after ice bath for 2min for incubation. After incubation, a proper amount of the bacterial liquid is uniformly spread on a flat plate containing a kanamycin-resistant LB solid culture medium for culture, a monoclonal colony is picked up to 4mL of kanamycin-resistant LB liquid culture medium, the colony is cultured at 37 ℃ and 220rpm for 14h, and PCR verification is carried out by taking the bacterial liquid as a template. And (3) extracting recombinant plasmids after the PCR verification succeeds, carrying out enzyme digestion and sequencing on the extracted plasmids respectively, and verifying to obtain a recombinant imine reductase clone strain, wherein the gene IRED I contains a nucleic acid sequence shown as SEQ ID NO.4, and the translation amino acid sequence IRED I is shown as SEQ ID NO. 3.

2. Construction of an imine reductase expressing Strain

The method comprises the steps of performing site-directed mutagenesis on SEQ ID No.3, wherein A at the 57 th position is mutated into R, D at the 122 th position is mutated into A, A at the 176 th position is mutated into G, S at the 241 th position is mutated into G, and an amino acid sequence IRED II after mutation is represented by SEQ ID No.1, entrusting Shanghai Jieli bioengineering limited company to synthesize a nucleic acid sequence SEQ ID No.2 containing a vector pET-26b (+) gene after codon optimization according to the sequence SEQ ID No.3, introducing the nucleic acid sequence into escherichia coli BL21 star (DE3) competent cells for transformation, performing ice bath for 30min, performing heat shock water bath at 45 ℃ for 90S, uniformly coating a bacterial liquid on a flat plate containing a kanamycin-resistant LB solid culture medium after ice bath for 2min, selecting a monoclonal bacterial colony to 4mL kanamycin-resistant LB liquid culture medium after culture at 37 ℃ for 18h, and performing culture at 37 ℃ at 220rpm for 14 h. Thus obtaining the imine reductase expression strain BL21 star (DE3)/pET-26b (+) -IRED.

3. Construction of glucose dehydrogenase-expressing Strain

A pET-26b (+) vector gene was synthesized by Shanghai Czeri bioengineering GmbH based on the published glucose dehydrogenase GDH Sequence (NCBI Reference Sequence: WP-214850512.1), the amino acid Sequence of which is shown in SEQ ID No.5 and the codon-optimized nucleic acid Sequence of which is shown in SEQ ID No. 6. And adding the connected plasmid into competent cells of escherichia coli BL21 star (DE3) for transformation, carrying out ice bath for 30min, carrying out heat shock on the escherichia coli in the water bath at 45 ℃ for 90s, carrying out ice bath for 2min, uniformly coating the bacterial liquid on a plate containing a kanamycin-resistant LB solid culture medium, culturing at 37 ℃ for 18h, picking out a monoclonal bacterial colony to 4mL of kanamycin-resistant LB liquid culture medium, and culturing at 37 ℃ and 220rpm for 14 h. Thus obtaining the imine reductase expression strain BL21 star (DE3)/pET-26b (+) -GDH.

4. Preparation of crude enzyme solution of imine reductase and glucose dehydrogenase

The above strains were inoculated into 4mL of each LB liquid medium containing 50mg/L kanamycin, cultured at 37 ℃ and 180rpm for 12 hours, then the whole of the strain was inoculated into 600mL of TB liquid medium, cultured at 37 ℃ and 180rpm to an OD600 of about 2, induced by adding IPTG having a final concentration of 0.3mM, and induced at 25 ℃ for 16 hours. Then, the bacterial cells containing the carbonyl reductase are collected by centrifugation at 5000rpm for 20 minutes, 30g of the bacterial cells are resuspended by 70g of phosphate buffer (0.1mol/L, pH 7.0), and the crude enzyme liquid of the imine reductase and the crude enzyme liquid of the glucose dehydrogenase are obtained after ultrasonic disruption.

Example 2

In this example, amino resin and epoxy resin were used as carriers to prepare immobilized imine reductase and glucose dehydrogenase by covalent bonding.

1. Activation of amino resin immobilized carrier

a. Adding 40ml of 0.1M PBS buffer (pH 4.2-4.5) into 10g of amino resin carrier, stirring or oscillating for 15min at the temperature of 20-25 ℃ and the rotation speed of 150rpm, adjusting the pH to 8.0 by using 4M NaOH solution after the stirring or oscillation is finished, and discarding the liquid;

b. adding 40mL of 0.02M PBS buffer solution (pH 8.0), stirring or oscillating at 20-25 deg.C and 150rpm for 5min, and discarding the liquid;

c. adding 40mL of 0.02M PBS buffer solution (pH 8.0), adding 1.6mL of 50% glutaraldehyde (the use concentration of the glutaraldehyde is 2%), stirring or oscillating at the temperature of 20-25 ℃ and the rotation speed of 150rpm for 60min, discarding the liquid, washing twice with 0.02M PBS, and draining the liquid to obtain the activated amino immobilized carrier for later use.

2. Activation of epoxy resin immobilized carrier

Adding 10g of epoxy carrier into 40mL of 0.1M PBS buffer solution (pH 8.0), stirring or oscillating at the temperature of 20-25 ℃ and the rotation speed of 150rpm for 15min, carrying out suction filtration, washing twice with 0.02M PBS buffer solution (pH 8.0), and carrying out suction drying to obtain the activated epoxy resin immobilized carrier.

3. Flocculation treatment of crude enzyme liquid

The crude enzyme solution of imine reductase and the crude enzyme solution of glucose dehydrogenase obtained in example 1 are respectively added with 5g/L ammonium sulfate and stirred for 5min, then 0.1% polyethyleneimine is added and stirred for 10min, and centrifuged for 10min at 5000rpm, and the supernatant is taken to obtain the clear solution of flocculated imine reductase and the clear solution of flocculated glucose dehydrogenase.

4. Imine reductase and glucose dehydrogenase immobilization

And (2) taking 1g of the activated amino resin carrier or epoxy resin carrier, adding 10g of flocculated imine reductase clear liquid or flocculated glucose dehydrogenase clear liquid, carrying out vibration crosslinking at 25 ℃ and 180rpm for 20 hours, carrying out suction filtration and washing for three times, then using 0.02M PBS (phosphate buffer solution) with pH8.0, 0.5M NaCl and 150rpm, stirring or vibrating for 45min, then using 0.02M PBS (phosphate buffer solution) with pH8.0 to wash for 2 times, and carrying out suction filtration respectively to obtain the immobilized imine reductase and the immobilized glucose dehydrogenase.

Example 3 specific enzyme Activity and enzyme Activity recovery assay

1. Method for measuring enzyme activity of free imine reductase or immobilized imine reductase

10mL of triethanolamine buffer containing 0.5M myosmine pH 6.5 was added with 1g of glucose and 3mg of NADP⁺2mL of flocculated glucose dehydrogenase clear liquid, 0.1mL of flocculated imine reductase clear liquid or 0.5g of immobilized imine reductase, reacting at 30 ℃ for 10min, detecting the generation amount of (S) -nornicotine by a liquid phase, and defining the enzyme activity: 1min produces 1 mu mol of (S) -nornicotine as an enzyme active unit.

2. Method for measuring enzyme activity of free glucose dehydrogenase or immobilized glucose dehydrogenase

Adding 0.1M PBS buffer solution with pH7.0, NADP into the cuvette⁺(10g/L)300 mu L, flocculating the supernatant of the glucose dehydrogenase to 100 mu L or immobilizing the glucose dehydrogenase to 0.1g, putting the cuvette into a spectrophotometer, adding 400 mu L of 0.1M glucose solution into the cuvette, reading after blank, recording an absorbance value every 5 seconds, drawing by taking time as an abscissa and a corresponding absorbance value as an ordinate to calculate the slope, and detecting the wavelength to be 340 nm. The measurement temperature was 30 ℃.

Enzyme activity (U/g) slope x dilution factor x 7.1 x 60 x 10

3. Results of enzyme activity measurements on different resins

Eight kinds of resins, namely ESR-1, ES-101 and ES-103, synthesized by Sedan Xiaojie LX-1000HA, LX-1000NH, LX-1000EP, LX-1000HFA and Tianjin Nankai, were immobilized according to the procedure of example 2, and then enzyme activity was measured according to the above test method, the results of immobilized imine reductase are shown in Table 1, and the results of immobilized glucose dehydrogenase are shown in Table 2:

TABLE 1 results for different resin immobilized imine reductases

TABLE 2 results of different resin-immobilized glucose dehydrogenases

As can be seen from the table above, the most suitable resin type of the imine reductase is LX-1000NH, the specific enzyme activity of the prepared immobilized enzyme reaches 425U/g, the recovery rate of the enzyme activity is 40.8%, and compared with free enzyme, the immobilized enzyme hardly has influence on the stereoselectivity of the enzyme; the most suitable resin type of the glucose dehydrogenase is ESR-3, the specific enzyme activity of the prepared immobilized enzyme reaches 1585U/g, the recovery rate of the enzyme activity is 36.5 percent, and the most suitable resin carriers of the two enzymes are amino resin.

EXAMPLE 4 preparation of (S) -nornicotine with biocatalytic System

In this example, immobilized imine reductase and immobilized glucose dehydrogenase prepared in example 2 were used to biocatalytic myosmine for the industrial preparation of (S) -nornicotine.

Adding 200g of Mycosamine, 295g of glucose and 600g of triethanolamine hydrochloric acid buffer (0.1M, pH 6.5) into 1L of catalytic system, stirring, adding 300mg of NADP⁺50g of immobilized imine reductase and 50g of immobilized glucose dehydrogenase are mechanically stirred at 30 ℃, the conversion rate is 99.9 percent after the reaction is carried out for 20 hours, and the optical purity of (S) -nornicotine is more than 99.9 percent.

Example 5300L preparation of (S) -nornicotine by biocatalytic System

Adding 90kg of Mycosamine, 133kg of glucose and 180kg of triethanolamine hydrochloric acid buffer (0.1M, pH 6.5) into 300L of catalytic system, stirring, adding 90g of NADP⁺15kg of immobilized imine reductase and 15kg of immobilized glucose dehydrogenase are mechanically stirred at 30 ℃, after the reaction is carried out for 20 hours, the conversion rate is 99.9 percent, the optical purity of (S) -nornicotine is more than 99.5 percent, and the intermediate test is carried out for growingThe production-level immobilized enzyme can be used for substrate conversion of about 300g/L, and the conversion efficiency is improved probably due to the good stirring condition and the high speed of the Chinese-style production tank.

Example 6 tolerance comparison of free enzyme with immobilized enzyme substrate

Imine reductase free enzyme with the same enzyme activity and immobilized imine reductase are respectively selected to carry out catalytic reaction under different concentrations of Mesamine, the inhibition condition of substrate concentration on the enzyme activity is verified, the conversion rate is detected by reacting for 30h in a liquid phase, the result is shown in figure 1, the conversion rate is not less than 99% when the immobilized imine reductase is within 200g/L of substrate concentration, the addition amount of the free enzyme substrate is 90g/L, and the conversion rate has a remarkable reduction trend.

Example 7

Continuous conversion is carried out according to the 1L reaction system in the embodiment 4, namely after each conversion is finished, the immobilized enzyme obtained by suction filtration is continuously used for the next catalytic reaction, the relative enzyme activity stability is determined as shown in figure 2, the enzyme activity of the immobilized enzyme used for the first time is determined as 100%, and more than 90% of the initial enzyme activity is still kept after 20 times of use, which indicates that the immobilized carbonyl reductase has good operation stability.

Sequence listing

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Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Asp Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 4

<211> 879

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgcgccatc tctctgtcat cggcctcggg gccatgggct ccgccctcgc caccaccttg 60

ctcaaggcgg gccatccggt caccgtctgg aatcgcagcg ccgccaaggc cgccccgctg 120

caagcgctgg gagcgactct cgccccctcg gttggcgcgg ctattgccgc aagcgatatc 180

acccttgtct gcgtcgataa ctacgccgtc agccagcagt tgctggacga ggcaagcgat 240

gcggtggcgg gcaagctgct ggtgcaactc tccaccggca gcccgcaggg ggccagagcg 300

ctggagagct ggagccacgc ccgcggcgcc cgttatctgg atggcgccat cctctgcttc 360

ccggatcaga tcggcaccag cgatgccagc atcatctgct cgggggccag cgctgccttt 420

agcgaggccg agccggtact gcgcctgctc gcccccaccc tcgaccatgt ggccgaggcg 480

gtgggcgccg ccgcggcgca agattgtgcc gtcgccgcct actttgccgg tggtctgctc 540

ggtgctctgc acggcgcgct gatctgcgag gcggaagggt tgccggtagc caaggtgtgc 600

gcccagttca gcgagctctc gcccattctc ggtggcgatg tggcccacct tggcaagacg 660

ttggcgagcg gtgatttcga tcacccctac gccagcctga aaacctggag cgccgccatc 720

agccgcctcg ccggtcacgc caccgacgcc gggatcgaca gccgcttccc gcgttttgcc 780

gccgatctgt ttgaggaagg ggtggcgcaa gggtttggtc agcaggaggt gtctgccctc 840

atcaaggtgc tgcgggcacg caatggagcg gcacaatga 879

<210> 5

<211> 261

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Tyr Asn Ser Leu Lys Gly Lys Val Ala Val Val Thr Gly Gly Ser

1 5 10 15

Met Gly Ile Gly Glu Ala Ile Ile Arg Arg Tyr Ala Glu Glu Gly Met

20 25 30

Arg Val Val Ile Asn Tyr Arg Ser His Pro Glu Glu Ala Lys Lys Ile

35 40 45

Ala Glu Asp Ile Lys Gln Ala Gly Gly Glu Ala Leu Thr Val Gln Gly

50 55 60

Asp Val Ser Lys Glu Glu Asp Met Ile Asn Leu Val Lys Gln Ala Val

65 70 75 80

Asp His Phe Gly Gln Leu Asp Val Phe Val Asn Asn Ala Gly Val Glu

85 90 95

Met Pro Ser Pro Ser His Glu Met Ser Leu Glu Asp Trp Gln Lys Val

100 105 110

Ile Asp Val Asn Leu Thr Gly Ala Phe Leu Gly Ala Arg Glu Ala Leu

115 120 125

Lys Tyr Phe Val Glu His Asn Val Lys Gly Asn Ile Ile Asn Met Ser

130 135 140

Ser Val His Glu Ile Ile Pro Trp Pro Thr Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Val Lys Leu Met Thr Gln Thr Leu Ala Met Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Ile Asn Ala Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Glu Asp Pro Lys Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Asn Ile Gly Lys Pro Glu Glu Ile

210 215 220

Ser Ala Val Ala Ala Trp Leu Ala Ser Asp Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

<210> 6

<211> 786

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgtataata gcttaaaagg taaagtggcc attgttaccg gcggtagtat gggcattggc 60

gaagccatta ttcgtcgcta tgcagaagaa ggtatgcgcg ttgtgattaa ttatcgctca 120

catccggaag aagccaaaaa aattgccgaa gatattaaac aggcaggtgg tgaagcactg 180

accgttcagg gcgatgtgtc taaagaagaa gatatgatta atctggttaa acaggccgtg 240

gatcattttg gtcagctgga tgtgtttgtg aataatgcag gcgttgaaat gccctcgccg 300

tcacatgaaa tgagtttaga agattggcag aaagtgattg atgtgaatct gacaggggcc 360

tttctgggtg cacgcgaagc actgaaatat tttgtggaac ataatgttaa aggcaatatt 420

attaatatgt ctagtgttca tgaaattatt ccgtggccga cctttgttca ttatgccgcc 480

tctaaaggcg gcgttaaact gatgacccag accttagcaa tggaatatgc cccgaaaggt 540

attcgtatta atgcaattgg tccgggtgca attaataccc cgattaatgc cgaaaaattt 600

gaagatccga aacagcgcgc agatgtggaa tcaatgattc cgatgggtaa tattggcaaa 660

ccggaagaaa tttcagcagt tgccgcctgg ttagccagcg atgaagcaag ctatgttacc 720

ggcattaccc tgtttgcaga tggcggcatg accctgtatc cgagctttca ggccggtcgc 780

ggctaa 786

Claims (10)

1. The high stereoselectivity S type imine reductase has an amino acid sequence shown as SEQ ID No. 1.

2. The gene for coding the high stereoselectivity S-type imine reductase of claim 1, the nucleotide sequence of which is shown as SEQ ID No. 2.

3. A recombinant expression vector comprising the gene of claim 2.

4. A host cell comprising the recombinant expression vector of claim 3.

5. A preparation method of S-type imine reductase is characterized by comprising the following steps:

(1) for is toAeromonas sp. Performing site-directed mutagenesis on an imine reductase amino acid sequence IRED I of the FDAARGOS 1419 strain, mutating A at the 57 th position to R, D at the 122 th position to A, A at the 176 th position to G, and S at the 241 th position to G, and obtaining the amino acid shown as SEQ ID No.1 after mutationIRED II, a nucleotide sequence shown as SEQ ID No.2 is obtained through codon optimization;

(2) constructing a recombinant expression vector containing SEQ ID No.2, and adding competent host cells to obtain a recombinant strain expressing IRED II;

(3) culturing the recombinant strain, and ultrasonically crushing to obtain a crude enzyme solution.

6. The method of claim 5, wherein the step of preparing the S-imine reductase comprises: the initial vector of the recombinant expression vector is selected from any one of pET, pGEX and pMAL.

7. The method of claim 6, wherein the step of preparing the S-imine reductase comprises: the host cell is selected from any one of escherichia coli, bacillus subtilis and pichia pastoris.

The application of S-type imine reductase in preparing (S) -nornicotine, which takes myosmine as a substrate, and adds crude enzyme solution of S-type imine reductase obtained by the method of claim 5 or products thereof into a reaction system for biocatalysis to obtain (S) -nornicotine;

wherein, the product of the crude enzyme liquid of the S-type imine reductase is a refined biological enzyme product obtained by separating and purifying the crude enzyme liquid or an immobilized imine reductase obtained by immobilizing the crude enzyme liquid; the reaction system comprises glucose dehydrogenase, glucose and a hydrogen donor.

9. Use of an S-imine reductase according to claim 8, for the preparation of (S) -nornicotine, characterized in that: the glucose dehydrogenase is a crude enzyme solution or a product thereof prepared by culturing and ultrasonically crushing a glucose dehydrogenase-producing strain; wherein the crude enzyme liquid product prepared by the strain for producing the glucose dehydrogenase is a refined biological enzyme product obtained by separating and purifying crude enzyme liquid or immobilized glucose dehydrogenase obtained by immobilizing the crude enzyme liquid.

10. The use of an S-type imine reductase enzyme according to claim 9, for the preparation of (S) -nornicotine, wherein said immobilized imine reductase and immobilized glucose dehydrogenase are prepared by a process comprising: adding the crude enzyme solution into an ammonium sulfate solution, then adding polyethyleneimine, and centrifuging to obtain a supernatant; and adding the activated amino resin carrier or the activated epoxy resin carrier into the supernatant, oscillating, crosslinking, washing with water, repeating the steps for several times, and performing suction filtration.

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