CN116083384A - Imine reductase mutant and encoding gene and application thereof - Google Patents
Imine reductase mutant and encoding gene and application thereof Download PDFInfo
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- CN116083384A CN116083384A CN202211462467.9A CN202211462467A CN116083384A CN 116083384 A CN116083384 A CN 116083384A CN 202211462467 A CN202211462467 A CN 202211462467A CN 116083384 A CN116083384 A CN 116083384A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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- C12N15/09—Recombinant DNA-technology
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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- C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
- C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
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Abstract
The invention provides an imine reductase mutant, and a coding gene and application thereof, wherein the 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 and 31; the 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 and 32; the recombinant vector comprises an imine reductase mutant gene; the genetically engineered bacteria used to produce the imine reductase mutants comprise recombinant vectors. The preparation method of the imine reductase mutant comprises the steps of culturing genetically engineered bacteria to obtain the recombinant imine reductase mutant. The invention provides a mutation mode of reverse imine reductase, which can simultaneously improve the activity of enzyme on a compound shown in a formula I and can be used for the synthesis reaction of cyclic imine with a structure shown in a formula b.
Description
Technical Field
The invention belongs to the field of bioconversion, and particularly relates to an imine reductase mutant, and a coding gene and application thereof.
Background
Atracurium besylate is a non-depolarizing muscle relaxant. The muscle relaxing non-depolarizing neuromuscular blocking agent is suitable for the muscle relaxing of the trachea cannula and the chest and abdomen operation, is used for assisting general anesthesia, enables the trachea cannula to be easy to carry out, enables skeletal muscles to relax during surgery or ventilation control, helps deep treatment patients to carry out artificial ventilation, and is used for various operations requiring muscle relaxing or respiratory control.
The cis-atracurium besylate is an isomer of atracurium besylate, has the same action as atracurium besylate, has the muscle relaxation action strength which is 3 times that of atracurium besylate, and does not release histamine when used in large dose, has smaller cardiovascular action and no accumulation effect. In addition, the metabolites of cis-atracurium are non-toxic and have no muscle relaxing effect. Thus, cis-atracurium has a more excellent clinical effect than atracurium.
Cis-atracurium has a more excellent clinical effect than atracurium, and thus it is necessary to control the content of enantiomer thereof during the production of atracurium. Formula a is a key intermediate in the synthesis process of cis-atracurium, and the production process also needs to control the content of enantiomer (shown as formula b). The existing chemical method for producing the compound shown in the formula a has complex reaction steps, high cost and harsh reaction conditions, and is easy to cause serious pollution to the environment. In addition, most of chemically synthesized intermediates are racemates, and multiple resolution steps are required to obtain cis-atracurium isomer compounds with high purity.
The imine reductase is NAD + Or NADP + Dependent enzymes, which are widely found in nature. Which is capable of reducing c=n to C-N to give chiral amines. However, imine reductases have difficulties in industrial applications, such as substrates or productsThe inhibition effect of the (B) and the application range of the substrate are narrow. For the wild enzyme in the invention, related researches are lack at present, and the enzyme has low activity against the substrate of the type shown in the formula a and the formula b and poor self stability. More inconvenient, the configuration of the product obtained by the catalysis of the wild enzyme is mainly R-type, and the chiral purity is only 84.8 percent. Therefore, the wild-type enzyme can be used for the production of cis-atracurium key intermediate shown in formula a, and can not be used for the production of S-configuration enantiomer shown in formula b.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide an imine reductase mutant with high catalytic activity and good thermal stability and a preparation method thereof, which are used for producing a compound shown in a formula b.
In order to achieve the aim, the invention combines rational design, directed evolution and high-throughput screening technology of enzyme to modify enzyme protein. During the engineering process, the inventors have unexpectedly found that when the imine reductase from Actinomadura rifamycini is mutated from W to one of A, S, D, F at amino acid 175, the configuration of the enzymatic reaction product is reversed, and the enzyme activity is greatly improved. In addition, mutation of glutamine 168, leucine 171, arginine 219, asparagine 235, methionine 240, methionine 118 alone or in combination also resulted in inversion of the configuration of the enzymatic reaction product and a change in enzyme activity to varying degrees (Table 1). According to one aspect of the present invention there is provided a series of imine reductase mutants, the mutated amino acid sequence having at least 1 mutation site as follows: mutation of amino acid 175 from W to A, S, D, F; mutation of amino acid 168 from Q to A, D, F, S; mutation of amino acid 171 from L to A; the 219 th R mutation is S, F; asparagine at position 235 to alanine; methionine at position 240 is mutated to phenylalanine. The amino acid sequence of the imine reductase mutant has a mutation site in the mutated amino acid sequence, and has an amino acid sequence having a homology of 90% or more with the mutated amino acid sequence.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides an imine reductase mutant gene, which is derived from Actinomadura rifamycini wild type gene SEQ ID NO 2. "wild type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a natural source and is not intentionally modified by human manipulation. The enzyme obtained after the gene expression has low catalytic activity on certain substrates, and the chiral purity of the catalytic product is poor.
The present invention provides imine reductase mutants which are active on substrates of formula I. When the compounds of formula I are used as substrates, the enzyme activity of the imine reductase mutant is 1.6-31U/mg (Table 1), while the enzyme activity of the wild type enzyme is 6U/mg for these substrates. In addition, the chiral purity of the S-configuration product (shown as a formula b) obtained by catalysis of the enzyme mutants is 76.10% -100% (table 1), while the chiral purity of the R-configuration product (shown as a formula a) obtained by catalysis of the wild enzyme is 84.80%.
The invention mutates the protein molecule by rational design (changing individual amino acid in the protein molecule by site-directed mutagenesis or other methods on the basis of knowing the spatial structure of the protein), overlap extension PCR, recombinant PCR, large primer PCR, circular plasmid PCR and the like, thereby obtaining the target gene of the imine reductase mutant, and the 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 and 32.
The amino acid sequences of the imine reductase mutants coded by the imine reductase mutant genes are shown as SEQ ID NO 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31.
TABLE 1 catalytic performance of imine reductase and mutants thereof and chiral purity of dominant product
The invention provides a recombinant vector of an imine reductase mutant gene, which can be constructed by connecting a nucleotide sequence of the imine reductase gene of the invention to various prokaryotic expression vectors or eukaryotic expression vectors by a conventional method in the field. Prokaryotic expression vectors such as pGEX, pMAL, pET, pBAD, pBV220, pCold series and eukaryotic expression vectors, more preferably selected from pET series plasmids as starting vectors. The plasmid used in one embodiment of the invention is pET-24a.
The invention provides a genetic engineering bacterium for producing the imine reductase mutant, wherein the genetic engineering bacterium comprises the imine reductase mutant gene or the recombinant vector. The host cell of the genetically engineered bacterium is preferably Escherichia coli (Escherichia coli BL (DE 3)).
The imine reductase mutant gene, the recombinant vector and the genetically engineered bacterium can be used for preparing the imine reductase mutant.
The invention also provides a method for preparing the imine reductase mutant, which comprises the steps of fermenting and culturing the genetically engineered bacterium, and collecting and preparing the recombinant imine reductase mutant.
The method comprises the step of industrially preparing the recombinant imine reductase mutant under certain fermentation conditions of a production tank; the fermentation conditions of the production tank are preferably as follows: DO is more than 10 percent, and the air flow is 1:0.5-2 vvm.
In the oxidation-reduction reaction, the imine reductase mutant of the invention produces a compound shown in a formula b by reducing an imine reductase substrate shown in the formula I, wherein the formulas I and b are shown as follows:
the invention has the following beneficial effects:
the invention provides a mutation mode of reverse imine reductase, which can simultaneously improve the activity of the enzyme on a compound shown in a formula I and can be used for the synthesis reaction of cyclic imine with a structure shown in a formula b.
The enzyme has excellent catalytic activity, the catalyzed reaction is simple and mild, no waste is discharged, the reaction conversion rate is high, and the application prospect is good.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the present invention will be described in detail with reference to specific embodiments.
Example 1: establishment of wild imine reductase enzyme genetic engineering bacteria
Sequence optimization is carried out according to the wild type gene sequence (GenBank: WP_ 026403156.1) of the Actinomadura rifamycini imine reductase recorded by NCBI, then a whole gene fragment is artificially synthesized by a gene synthesis company, genes are inserted into NdeI and BamHI sites of a pET-24a plasmid, and the linked vector is transferred into escherichia coli BL21 (DE 3) to establish the imine reductase genetic engineering bacterium.
Example 2: obtaining of imine reductase mutant Gene
The three-dimensional structure of the wild-type gene of the enzyme has not been revealed yet. However, the three-dimensional MODEL construction of the wild-type gene sequence using SWISS-MODEL in this study revealed that it was highly homologous to Stackebrandtia nassauensis (strain DSM44728/CIP 108903/NRRL B-16338/NBRC 102104/LLR-40K-21) derived imine reductase (PDB: 6 JIZ). Thus, by reference to the three-dimensional structure of the enzyme, the binding of the substrate of formula I to the protein is simulated by the Docking software, and finally by Pymol analysis, NAD (P) is selected, possibly bound to the substrate + Amino acids involved in binding, proton transfer, etc., are used as mutant amino acids.
In addition to the rational design described above, the present study utilized error-prone PCR random mutagenesis to protein engineer imine reductases. In general, error-prone PCR can be used to randomly introduce mutations into a target gene at a certain frequency by adjusting reaction conditions (e.g., increasing magnesium ion concentration, adding manganese ions, changing dNTP concentrations of four species in a system, or using low-fidelity DNA polymerase) to change the mutation frequency during amplification when the target gene is amplified by DNA polymerase, thereby obtaining random mutants of protein molecules.
The study uses Taq polymerase with lower fidelity while using Mn 2+ Substitution of the natural cofactor Mg 2+ Increasing the probability of error.
The 50. Mu.L PCR system was as follows:
sterilized double distilled water was added to 50. Mu.L.
Wherein: an imine reductase template gene constructed by PCR amplifying an imine reductase gene and inserting the gene into pET-24a plasmid according to the method of example 1; primer design, this study was based on the design of the upstream and downstream sequences of the gene of interest in the construction of the recombinant plasmid in example 1.
The PCR reaction conditions were: pre-denaturation at 95℃for 2.5min; denaturation at 94℃for 15s, annealing at 53℃for 30s, extension at 72℃for 30s for 35 cycles; the extension was continued at 72℃for 10min and cooled to 4 ℃.
The PCR amplified product was ligated to pET-24a vector and transferred into E.coli BL21 (DE 3) to create an imine reductase gene mutation library.
The E.coli BL21 (DE 3) was used as host, pET-24a plasmid was used as vector, and extended imine reductase was expressed, and the high activity mutant was screened with high throughput using the enzyme activity detection method described in example 5. And identifying the mutated high-activity imine reductase gene. The nucleotide sequence of the screened high-activity imine reductase mutant gene is shown as SEQ ID NO 4.
Example 3: small scale production of imine reductase in shake flasks
Will include implementation ofExamples 1 and 2 E.coli, which constructed recombinant plasmids, was inoculated into 50mL of LB medium (peptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH 7.2) containing kanamycin (50. Mu.g/mL). Shake-culturing at 37℃and 210rpm for 16 hours. Then transferring at a ratio of 1:100, shake culturing at 37deg.C and 210rpm in 100mL LB medium containing kanamycin, and measuring absorbance (OD) of bacterial liquid at 600nm at regular time 600 ) To monitor the cell growth density. When the OD of the culture 600 When the target imine reductase gene expression is carried out in the range of 0.6 to 0.8mM, isopropyl beta-D-thiogalactoside (IPTG) with the final concentration of 0.8mM is added to induce the target imine reductase gene expression, and the target imine reductase gene expression is induced and cultured overnight (more than or equal to 16 hours). Centrifugation at 10000rpm at 4℃for 10min, discarding the supernatant, and after re-suspending the cell pellet at 200g/L with pre-chilled 50mM Tris-HCl buffer (pH 7.5), ultrasonication, centrifugation at 13000rpm at 4℃for 30min, collecting the supernatant, i.e., crude enzyme solution, and storing at-20 ℃.
Example 4: fermentation production of imine reductase
Fermentation scheme: the recombinant E.coli constructed in examples 1 and 2 (containing mutated imine reductase gene) was inoculated into 120mL LB medium (containing 50. Mu.g/mL kanamycin), shake-cultured overnight (5 hours or more) at 37℃at 210rpm, and then fermented in a 15L fermenter: the seed liquid is inoculated into 6L fermentation culture medium according to the inoculation amount of 2 percent, the pH value of the fermentation liquid is maintained to be 7.0-7.2 by adding ammonia water, the temperature of the fermentation liquid is 37 ℃, the stirring rotation speed is 300-900rpm, the dissolved oxygen is controlled to be about 30 percent in the process, and the air flow is 1:1-2 vvm. After 8 hours of cultivation, IPTG (final concentration 0.8 mmol/L) was added, the pot temperature was adjusted to 22℃and fermentation was continued for 12-16 hours. During the fermentation process, a feed solution (200 g/L glucose, 100g/L yeast extract, pH 7.2) is added to maintain the growth of the culture. After fermentation, the culture is directly homogenized and crushed by a high-pressure homogenizer. The crushed fermentation broth is added with polyethyleneimine with the final concentration of 2g/L and diatomite with the final concentration of 150g/L, and stirred for 30 minutes. After the flocculation sedimentation is finished, the mixture is filtered by filter cloth paved with diatomite. Filtering and concentrating the filtered enzyme solution by using an ultrafiltration membrane to prepare an imine reductase crude enzyme solution, and preserving the imine reductase crude enzyme solution at the temperature of minus 20 ℃.
Example 5: determination of the enzyme Activity of imine reductase
The enzyme activity measuring system of the imine reductase is as follows:
50mM Tris-HCl buffer containing 10g/L of the substrate of formula I, 1mM NADPH,10% methanol, pH 8.0, constant volume to 270. Mu.L, and adding to a 96-well plate after mixing. Adding 30 mu L of crude enzyme solution of imine reductase or diluted solution thereof, uniformly mixing, then placing into an enzyme labeling instrument, reacting at 40 ℃ and detecting 345nm light absorption. NADPH as a reducing agent generates NADP as the reaction proceeds + Its light absorption at 345nm gradually decreases.
Definition of enzyme activity: under the above conditions, the amount of enzyme required to catalyze the consumption of 1. Mu. Mol of NADPH per minute is defined as 1 enzyme activity unit.
The specific activity of the recombinant imine reductase mutant is 1.6-31U/mg (Table 1). The specific activity of the wild imine reductase before mutation was 6U/mg (Table 1).
The embodiments of the present invention have been described in detail by way of examples, but the descriptions are merely exemplary of the embodiments of the present invention and are not to be construed as limiting the scope of the embodiments of the present invention. The protection scope of the embodiments of the invention is defined by the claims. In the technical scheme of the embodiment of the invention, or under the inspired by those skilled in the art, similar technical schemes are designed within the spirit and the protection scope of the embodiment of the invention, or equivalent changes and improvements made to the application scope are still included in the patent coverage protection scope of the embodiment of the invention.
Claims (9)
1. An imine reductase mutant is characterized in that the amino acid sequence is shown as SEQ ID NO 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31.
2. An imine reductase mutant gene, which is used for encoding the imine reductase mutant of claim 1, and has nucleotide sequences shown in SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32.
3. A recombinant vector comprising the imine reductase mutant gene of claim 2.
4. The recombinant vector according to claim 3, wherein the recombinant vector uses pET series plasmid as a starting vector.
5. A genetically engineered bacterium for producing the imine reductase mutant according to claim 1, characterized in that the genetically engineered bacterium comprises the recombinant vector according to claim 3, and the host cell of the genetically engineered bacterium 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 bacterium 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 by comprising the steps of: culturing the genetically engineered bacterium of claim 5 to obtain a recombinant imine reductase mutant.
8. The method according to claim 7, comprising a step of preparing the imine reductase mutant by fermentation.
9. Use of an imine reductase mutant according to claim 1 in a redox reaction to produce a compound of formula b by reduction of an imine reductase substrate of formula i, formula b being as follows:
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