Imine reductase mutant, encoding gene and application thereof

Abstract

The application provides an imine reductase mutant, a coding gene and application thereof, an amino acid sequence of the imine reductase mutant is shown as SEQ ID NO 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31; a nucleotide sequence of the imine reductase mutant gene is shown as SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32; a recombinant carrier comprises the imine reductase mutant gene; a genetically engineered bacterium for producing the imine reductase mutant comprises the recombinant carrier. A preparation method of the imine reductase mutant comprises culturing the genetically engineered bacterium to obtain the recombinant imine reductase mutant. The application provides a mutation mode of reversing the imine reductase, can simultaneously improve the activity of the enzyme to the compound shown as formula I, and can be used for synthesizing the cyclic imine with the structure of formula b.

Description

Technical Field

[0001] This invention belongs to the field of biotransformation, and specifically relates to an imine reductase mutant, its encoding gene, and its applications. Background Technology

[0002] Atracurium besylate is a non-depolarizing muscle relaxant. It is indicated for muscle relaxation during endotracheal intubation and for muscle relaxation required in thoracic and abdominal surgeries. It is a non-depolarizing neuromuscular blocker used as an adjunct to general anesthesia to facilitate endotracheal intubation and to relax skeletal muscles during surgery or controlled ventilation. It assists in artificial ventilation for patients in intensive care units and is used in various surgeries requiring muscle relaxation or respiratory control.

[0003] Atracurium cisasulfonate is an isomer of atracurium and has the same effects as atracurium, but its muscle relaxant effect is three times stronger. At high doses, cisatracurium does not release histamine, has less cardiovascular effect, and does not accumulate. Furthermore, the metabolites of cisatracurium are non-toxic and do not have a muscle relaxant effect. Therefore, cisatracurium has superior clinical efficacy compared to atracurium.

[0004] Compared to atracurium, cisatracurium has superior clinical efficacy, therefore, it is necessary to control the content of its enantiomers during the production of atracurium. Formula a is a key intermediate in the synthesis of cisatracurium, and its production process also requires control of the content of its enantiomers (as shown in Formula b). Existing chemical methods for producing the compound shown in Formula a involve complex reaction steps, high costs, and harsh reaction conditions, and also easily cause serious environmental pollution. In addition, the intermediates synthesized by chemical methods are mostly racemic, requiring multiple resolution steps to obtain high-purity cisatracurium isomer compounds.

[0005]

[0006] Imine reductase is NAD + or NADP + Imine reductases are widely found in nature. They can reduce C=N to CN to obtain chiral amines. However, imine reductases also face some difficulties in industrial applications, such as substrate or product inhibition and a narrow substrate applicability range. Regarding the wild-type enzyme in this invention, there are currently few research reports, and the enzyme exhibits low activity against substrates of the types shown in formulas a and b, as well as poor stability. More significantly, the product catalyzed by this wild-type enzyme is primarily in the R-configuration, and its chiral purity is only 84.8%. Therefore, this wild-type enzyme cannot be used for the production of the key intermediate of cisatracurium shown in formula a, nor for the production of the S-configuration enantiomer shown in formula b. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide an imine reductase mutant with high catalytic activity and good thermal stability, as well as a preparation method thereof, for the production of the compound shown in formula b.

[0008] To achieve the above objectives, this invention combines rational enzyme design, directed evolution, and high-throughput screening technology to modify enzyme proteins. During the modification process, the inventors unexpectedly discovered that when the imine reductase derived from *Actinomadura rifamycini* mutates from amino acid W at position 175 to one of A, S, D, or F, the conformation of the enzymatic reaction product is reversed, and the enzyme activity is greatly enhanced. Furthermore, mutations at positions 168 (glutamine), 171 (leucine), 219 (arginine), 235 (asparagine), 240 (methionine), and 118 (methionine), either individually or in combination, also lead to conformation reversal of the enzymatic reaction product and varying degrees of alteration in enzyme activity (Table 1). According to one aspect of the invention, a series of imine reductase mutants are provided, wherein the mutated amino acid sequence has at least one mutation site as follows: amino acid position 175 is mutated from W to A, S, D, or F; amino acid position 168 is mutated from Q to A, D, F, or S; amino acid position 171 is mutated from L to A; amino acid position 219 is mutated from R to S or F; asparagine position 235 is mutated to alanine; and methionine position 240 is mutated to phenylalanine. The amino acid sequence of the imine reductase mutant has the mutation sites in the mutated amino acid sequence and has more than 90% homology with the mutated amino acid sequence.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides an imine reductase mutant gene derived from the wild-type gene *Actinomadura rifamycini*, SEQ ID NO 2. "Wild-type" refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences present in organisms, which can be isolated from natural sources and have not been intentionally modified by human intervention. Enzymes obtained after expression of these genes exhibit low catalytic activity for certain substrates, and the catalytic products have poor chiral purity.

[0011] This invention provides several imine reductase mutants that are active against the substrates shown in Formula I. When the compounds of Formula I are used as substrates, the imine reductase mutants exhibit enzyme activity of 1.6–31 U / mg (Table 1), while the wild-type enzyme shows an activity of 6 U / mg against these substrates. Furthermore, the chiral purity of the S-configuration products (as shown in Formula b) obtained by these enzyme mutants is 76.10%–100% (Table 1), while the chiral purity of the R-configuration products (as shown in Formula a) obtained by the wild-type enzyme is 84.80%.

[0012]

[0013] This invention obtains the target gene of the imine reductase mutant by rational design (changing individual amino acids in the protein molecule through site-directed mutagenesis or other methods based on understanding the spatial structure of the protein) and by methods such as overlap extension PCR, recombinant PCR, large primer PCR and circular plasmid PCR. The nucleotide sequence of the imine reductase mutant gene is shown in SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32.

[0014] The amino acid sequences of the imine reductase mutants encoded by the above imine reductase mutant genes are shown in SEQ ID NO3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31.

[0015] Table 1 Catalytic properties of imine reductase and its mutants, and chiral purity of dominant products.

[0016]

[0017]

[0018] This invention provides a recombinant vector for an imine reductase mutant gene, which can be constructed by linking the nucleotide sequence of the imine reductase gene of this invention to various prokaryotic or eukaryotic expression vectors using conventional methods in the art. Examples include prokaryotic and eukaryotic expression vectors such as pGEX, pMAL, pET, pBAD, pBV220, and pCold series, with pET series plasmids being more preferred as the starting vector. In one embodiment of this invention, the plasmid used is pET-24a.

[0019] This invention provides a genetically engineered bacterium that produces the imine reductase mutant described above, wherein the genetically engineered bacterium contains the imine reductase mutant gene or the recombinant vector described above. The host cell of the above-mentioned genetically engineered bacterium is preferably *Escherichia coli* BL21(DE3).

[0020] The imine reductase mutant gene, recombinant vector, and genetically engineered bacteria described above can be used to prepare the imine reductase mutant of this invention.

[0021] The present invention also provides a method for preparing the imine reductase mutant, comprising fermenting and culturing the genetically engineered bacteria, and collecting and preparing the recombinant imine reductase mutant.

[0022] The above method includes the step of industrially preparing the recombinant imine reductase mutant under certain fermentation conditions in a production tank; the preferred fermentation conditions in the production tank are: DO ≥ 10% and air flow rate 1:0.5~2vvm.

[0023] In a redox reaction, the imine reductase mutant of this invention reduces the imine reductase substrate shown in Formula I to produce the compound shown in Formula b, as shown below:

[0024]

[0025] The present invention has the following beneficial effects:

[0026] This invention provides a mutation method for reversing imine reductase, which can simultaneously improve the enzyme's activity for compounds shown in Formula I and can be used for the synthesis of cyclic imines with the structure of Formula b.

[0027] The enzyme involved in this invention has excellent catalytic activity, and the reaction it catalyzes is simple and mild, with no waste discharge and high reaction conversion rate, showing good application prospects. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1: Establishment of a wild-type imine reductase genetically engineered bacterium

[0030] The sequence of the wild-type Actinomadura rifamycini imine reductase gene (GenBank: WP_026403156.1) was optimized based on the NCBI database. The whole gene fragment was then artificially synthesized by a gene synthesis company and inserted into the NdeI and BamHI sites of the pET-24a plasmid. The ligated vector was then transformed into Escherichia coli BL21(DE3) to establish an engineered imine reductase bacterium.

[0031] Example 2: Obtaining the imine reductase mutant gene

[0032] The three-dimensional structure of the wild-type gene for this enzyme has not yet been revealed. However, this study used SWISS-MODEL to construct a three-dimensional model of the wild-type gene sequence and found that it is highly homologous to the imine reductase (PDB:6JIZ) derived from Stackebrandtia nassauensis (strain DSM44728 / CIP108903 / NRRL B-16338 / NBRC 102104 / LLR-40K-21). Therefore, based on the three-dimensional structure of this enzyme, we performed substrate-protein binding simulations using Docking software, and finally used Pymol analysis to select NAD(P) enzymes that are likely to bind to the substrate. + Amino acids involved in binding, proton transfer, etc., are mutated amino acids.

[0033] In addition to the rational design described above, this study utilized error-prone PCR random mutagenesis to engineer imine reductase. Generally, error-prone PCR can alter the mutation frequency during DNA polymerase amplification by adjusting reaction conditions (such as increasing magnesium ion concentration, adding manganese ions, changing the concentration of the four dNTPs in the system, or using low-fidelity DNA polymerase). This allows for the random introduction of mutations into the target gene at a specific frequency, resulting in random mutants of the protein molecule.

[0034] This study used a low-fidelity Taq polymerase, while utilizing Mn... 2+ Alternative natural cofactor Mg 2+ Increase the probability of error.

[0035] The 50μL PCR system is as follows:

[0036]

[0037] Add sterile double-distilled water to a final volume of 50 μL.

[0038] Among them: the imine reductase template gene was constructed by PCR amplification of the imine reductase gene according to the method of Example 1 and insertion of the gene into the pET-24a plasmid; the primer design was based on the upstream and downstream sequences of the target gene in the recombinant plasmid constructed in Example 1.

[0039] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 2.5 min; 94℃ denaturation for 15 s, 53℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 10 min, and cooling to 4℃.

[0040] The obtained PCR amplification product was ligated into the pET-24a vector and transformed into Escherichia coli BL21(DE3) to construct an imine reductase gene mutant library.

[0041] Using *E. coli* BL21(DE3) as the host and pET-24a plasmid as the vector, extended imine reductase was expressed. High-activity mutant strains were screened using the enzyme activity detection method described in Example 5. The mutant high-activity imine reductase gene was identified. The nucleotide sequence of the screened high-activity imine reductase mutant gene is shown in SEQ ID NO 4.

[0042] Example 3: Small-scale production of imine reductase in shake flasks

[0043] E. coli containing the recombinant plasmids constructed in Examples 1 and 2 were inoculated into 50 mL of LB medium containing kanamycin (50 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.2). The culture was incubated at 37°C with shaking at 210 rpm for 16 hours. Then, the culture was transferred at a 1:100 ratio to 100 mL of LB medium containing kanamycin and incubated at 37°C with shaking at 210 rpm. The absorbance (OD) of the bacterial culture at 600 nm was measured periodically. 600 ), to monitor bacterial cell growth density. When the OD of the culture... 600 When the concentration of the enzyme is 0.6–0.8, add isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.8 mM to induce the expression of the target imine reductase gene, and culture overnight (≥16 hours). Centrifuge at 10,000 rpm and 4°C for 10 min, discard the supernatant, resuspend the cell pellet in pre-cooled 50 mM Tris-HCl buffer (pH 7.5) at 200 g / L, sonicate to disrupt the cell structure, and then centrifuge at 13,000 rpm and 4°C for 30 min. Collect the supernatant, which is the crude enzyme solution, and store it at -20°C.

[0044] Example 4: Fermentation production of imine reductase

[0045] Fermentation protocol: The recombinant *E. coli* (containing a mutated imine reductase gene) constructed in Examples 1 and 2 were inoculated as single colonies in 120 mL of LB medium (containing 50 μg / mL kanamycin) and cultured overnight (≥5 hours) at 37°C and 210 rpm with shaking. Fermentation was then carried out in a 15 L fermenter: 2% of the seed culture was inoculated into 6 L of fermentation medium. The pH of the fermentation broth was maintained at 7.0-7.2 by adding ammonia. The fermenter temperature was 37°C, the stirring speed was 300-900 rpm, dissolved oxygen was controlled at approximately 30%, and the air flow rate was 1:1-2 vvm. After 8 hours of culture, IPTG (final concentration 0.8 mmol / L) was added, the fermenter temperature was adjusted to 22°C, and fermentation continued for 12-16 hours. During fermentation, a feed solution (200 g / L glucose, 100 g / L yeast extract, pH 7.2) was added to maintain the growth of the culture. After fermentation, the culture was directly homogenized using a high-pressure homogenizer. After crushing, the fermentation broth was mixed with polyethyleneimine to a final concentration of 2 g / L and diatomaceous earth to a final concentration of 150 g / L, and stirred for 30 minutes. After flocculation and sedimentation, the mixture was filtered through a filter cloth lined with diatomaceous earth. The filtered enzyme solution was then concentrated using an ultrafiltration membrane to prepare crude imine reductase enzyme solution, which was stored at -20°C.

[0046] Example 5: Determination of imine reductase activity

[0047] The assay system for imine reductase activity is as follows:

[0048] A 50 mM Tris-HCl buffer solution containing 10 g / L of the substrate shown in Formula I, 1 mM NADPH, and 10% methanol was added to adjust the pH to 8.0 and brought to a final volume of 270 μL. The mixture was then added to a 96-well plate. 30 μL of crude imine reductase solution or its dilution was added and mixed. The plate was then incubated at 40 °C and the absorbance was measured at 345 nm. NADPH, acting as a reducing agent, will generate NADP during the reaction. + Its light absorption at 345nm will gradually decrease.

[0049] Enzyme activity is defined as the amount of enzyme required to catalyze the consumption of 1 μmol of NADPH per minute under the above conditions.

[0050] The specific activity of the recombinant imine reductase mutant was 1.6–31 U / mg (Table 1). Before the mutation, the specific activity of wild-type imine reductase was 6 U / mg (Table 1).

[0051] The embodiments of the present invention have been described in detail above through examples, but the content described is only an exemplary embodiment of the present invention and should not be considered as limiting the scope of implementation of the present invention. The protection scope of the present invention is defined by the claims. Any technical solutions designed by those skilled in the art using the technical solutions described in the embodiments of the present invention, or designed by those skilled in the art under the inspiration of the technical solutions of the embodiments of the present invention, within the substance and protection scope of the present invention, to achieve the above-mentioned technical effects, or any equivalent changes and improvements made to the scope of the application, should still fall within the patent protection scope of the present invention.

Claims

1. An imine reductase mutant, characterized in that, The amino acid sequences are shown in SEQ ID NO: 3, 5, 21, 25, 29, 31.

2. An imine reductase mutant gene, characterized in that, The imine reductase mutant gene is used to encode the imine reductase mutant of claim 1, and its nucleotide sequence is shown in SEQ ID NO: 4, 6, 22, 26, 30, 32.

3. A recombinant vector, characterized in that, The recombinant vector contains the imine reductase mutant gene as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector uses pET series plasmids as the starting vector.

5. A genetically engineered bacterium for producing the imine reductase mutant of claim 1, characterized in that, The genetically engineered bacteria comprises the recombinant vector of claim 3, and the host cell of the genetically engineered bacteria is Escherichia coli.

6. The use of the imine reductase mutant gene of claim 2, the recombinant vector of claim 3, and the genetically engineered bacteria of claim 5 in the preparation of the imine reductase mutant of claim 1.

7. A method for preparing the imine reductase mutant according to claim 1, characterized in that, The method includes the following steps: culturing the genetically engineered bacteria as described in claim 5 to obtain a recombinant imine reductase mutant.

8. The preparation method according to claim 7, characterized in that, The process includes the step of preparing the imine reductase mutant by fermentation.

9. The application of the imine reductase mutant according to claim 1 in redox reactions, characterized in that, The imine reductase mutant produces the compound shown in formula b by reducing the imine reductase substrate shown in formula I, as shown below: （Ⅰ）， （b）。

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