An enantioselective imine reductase mutant and its application in the production of S-arecoline
By combining specific site mutations of Pseudomonas imine reductase with a coenzyme cycle system, the problems of cumbersome acquisition methods and low purity of S-arecoline in existing technologies have been solved, achieving efficient and low-cost preparation of high-purity S-arecoline, which is suitable for the supply of high-purity raw materials for fiberless areca chewing gum.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HAINAN SPECIAL ECONOMIC ZONE LANGJI ARECA HEALTH BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies for obtaining S-arecoline involve cumbersome processes, long cycles, limited purity, high costs, significant pollution, and low conversion rates. Furthermore, the stereoselectivity of natural imine reductase does not meet the requirements for industrial applications.
By mutating specific amino acid sites of the imine reductase in Pseudomonas sp. RC4D1, an enantioselective imine reductase mutant was developed, and combined with a coenzyme cycle system, it was used to catalyze the preparation of S-arecoline from pseudooxyarecoline.
The preparation of high optical purity (99.9%) S-arecoline has been achieved, which simplifies the production process, reduces costs, and improves catalytic selectivity, making it suitable for the supply of high-purity raw materials for fiberless areca chewing gum.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to an enantioselective imine reductase mutant and its application in the production of S-arecoline, and particularly to the application of this technology in the preparation of fiberless areca chewing gum raw materials. Background Technology
[0002] S-arecoline, chemically known as 1-methyl-3-methoxycarbonyl-1,2,5,6-tetrahydropyridine, is the core active ingredient in areca nut. It possesses physiological activities such as stimulating mental alertness and promoting digestion, and is also an important pharmaceutical intermediate. Currently, there are two main methods for obtaining arecoline: one is extraction from areca nut fruit, which is a complex and time-consuming process, susceptible to impurities, resulting in limited purity, and is also limited by the supply of areca nut raw materials; the other is chemical synthesis, which requires resolution to obtain S-arecoline, is costly, and suffers from high consumption, high pollution, and low conversion rates, thus having many limitations in actual production.
[0003] Enzymatic synthesis of chiral compounds has attracted much attention due to its low cost and environmental friendliness. Imine reductase (IRED) is an effective catalyst for the preparation of various chiral amines, catalyzing the asymmetric reduction of imines to chiral amines. However, the industrial application of natural imine reductases is hindered by their stereoselectivity not meeting production requirements or insufficient catalytic activity for arecoline precursor substrates. Therefore, further exploration of imine reductase mutants with high catalytic efficiency and good enantioselectivity for the catalytic synthesis of S-arecoline is of great significance. Summary of the Invention
[0004] To address the shortcomings of existing S-arecoline synthesis processes, this invention provides an enantioselective imine reductase mutant and its application in S-arecoline production. The specific technical solution is as follows:
[0005] In a first aspect, the present invention provides an imine reductase mutant obtained by single-point or multi-point mutation at positions 118, 168, 169, 172, 234, and 238 of the imine reductase as shown in SEQ ID NO.2.
[0006] Furthermore, the imine reductase mutant is mutated in one of the following ways:
[0007] • (1) Alanine at position 118 is mutated to serine;
[0008] (2) Serine at position 168 is mutated to arginine;
[0009] (3) Alanine at position 169 is mutated to threonine;
[0010] (4) Cysteine at position 172 is mutated to tyrosine;
[0011] (5) Alanine at position 234 is mutated to serine;
[0012] (6) Threonine at position 238 is mutated to methionine;
[0013] (7) The alanine at position 118 is mutated to serine, and the serine at position 168 is mutated to arginine.
[0014] (8) Alanine at position 118 is mutated to serine, and alanine at position 169 is mutated to threonine;
[0015] (9) Alanine at position 169 is mutated to threonine, and cysteine at position 172 is mutated to tyrosine.
[0016] •(10) Alanine at position 118 is mutated to serine, alanine at position 169 is mutated to threonine, and threonine at position 238 is mutated to methionine.
[0017] (11) Alanine at position 118 is mutated to serine, alanine at position 169 is mutated to threonine, alanine at position 234 is mutated to serine, and threonine at position 238 is mutated to methionine.
[0018] This invention is based on the imine reductase of Pseudomonas sp. RC4D1 (NCBI accession number WP_213665450.1), which exhibits high enzyme activity and low enantioselectivity among wild-type enzymes. By mutating the residues surrounding the substrate of the imine reductase, this invention alters the interaction between the enzyme molecule and the substrate. The resulting imine reductase mutant can achieve good, even 99.9% optical purity, in the reaction catalyzing the preparation of S-arecoline from pseudooxyarecoline.
[0019] Secondly, the present invention provides a gene encoding the above-mentioned imine reductase mutant.
[0020] Thirdly, the present invention provides a recombinant vector comprising the aforementioned gene.
[0021] Fourthly, the present invention provides a genetically engineered bacterium containing the aforementioned genes.
[0022] Fifthly, the present invention provides the application of the above-mentioned imine reductase mutant, or the above-mentioned recombinant vector, or the above-mentioned genetically engineered bacteria in the preparation of S-arecoline.
[0023] In a sixth aspect, the present invention provides a method for preparing S-arecoline, wherein S-arecoline is prepared by reacting with pseudooxyarecoline as a substrate under a coenzyme cycle system, using the crude enzyme solution, pure enzyme or immobilized enzyme of the above-mentioned imine reductase mutant as a catalyst, or the wet cell or whole cell of the above-mentioned genetically engineered bacteria as a catalyst.
[0024] Furthermore, the coenzyme cycle system includes a coenzyme, glucose, and glucose dehydrogenase, wherein the coenzyme is NADP+ or NAD+.
[0025] Furthermore, the glucose dehydrogenase is one of BcGDH derived from Bacillus cereus, BmGDH derived from Bacillus megaterium, or BaGDH derived from Bacillus amyloliquefaciens.
[0026] Furthermore, in the reaction, the reaction temperature is 15~35℃, the pH value is 5.5~8.5, and the substrate concentration is 50~500mM.
[0027] Furthermore, the catalytic reduction reaction is carried out in 100 mM phosphate buffer with a coenzyme concentration of 0.01-0.1 mM.
[0028] Compared with existing technologies, this invention has the following advantages: This invention mutates an imine reductase derived from Pseudomonas to obtain a series of mutants, which, while maintaining the high enzyme activity of the wild-type imine reductase, improve its enantioselectivity in the catalytic preparation of S-arecoline. Compared with existing technologies, this invention has a simple production process, mild reaction conditions, high selectivity for imine reductase, and high optical purity of the product, showing good prospects for industrial application and can be used for the supply of high-purity arecoline raw materials for fiberless areca chewing gum. Attached Figure Description
[0029] Figure 1 The reaction equation for the formation of S-arecoline from pseudooxyarecoline by imine reductase catalysis. Detailed Implementation
[0030] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.
[0031] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the supplier's recommended operating instructions. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.
[0033] Example 1 Construction of wild-type enzyme engineered bacteria
[0034] Imine reductase was retrieved from the NCBI database and selected from *Pseudomonas* sp. RC4D1 (Ps IRED, NCBI accession number WP_213665450.1, base sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2). The amino acid sequence was converted to a nucleotide sequence through codon optimization. The nucleotide sequence was then totalized chemically and integrated into the multiple cloning site of the expression vector pET-28a(+). Finally, the constructed plasmid was introduced into *Escherichia coli* BL21(DE3) host cells to construct a wild-type imine reductase engineered bacterium.
[0035] Example 2 Construction of imine reductase mutant
[0036] I. Activation of engineered bacteria and plasmid extraction
[0037] The engineered bacteria obtained in Example 1 were activated and cultured using LB medium. The LB medium formulation was: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, dissolved in deionized water and brought to a final volume. The medium was then sterilized at 121°C for 20 min and set aside for use. The solid medium was LB medium with 2% (w / w) agar added.
[0038] The preserved engineered bacteria glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured at 37°C and 200 rpm for 12 h. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored at -80°C for long-term storage.
[0039] II. Site-directed gene mutation
[0040] Gene mutations are performed using whole plasmid PCR. When only a small-scale mutation is needed, the mutation can be designed in the upstream and downstream primers, and PCR can be performed using the plasmid as a template to obtain the target plasmid.
[0041] PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55-58℃ annealing for 15 s (adjusted according to primer Tm value), 72℃ extension for 75 s (adjusted according to gene length, 10 s / 1 kb), for a total of 30 cycles; 72℃ extension for 10 min; store at 4℃.
[0042] After PCR amplification, the amplification products were detected by 0.9% (w / v) agarose gel electrophoresis. The results showed that the amplification products were a single band. The amplification products were purified and recovered using a DNA recovery kit. The specific steps were as described in the instructions of the purification kit.
[0043] III. Construction of mutant engineered bacteria
[0044] The purified gene fragment was digested with DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into E. coil BL21(DE3) competent cells, plated, and single colonies were picked and cultured in LB liquid. Positive transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, sterile glycerol was added to a final concentration of 20% (v / v), labeled, and stored at -80℃ for later use.
[0045] Example 3: Bacterial culture and enzyme powder preparation
[0046] Engineered bacteria containing the imine reductase gene were activated by streak plating. Single colonies were inoculated into 5 mL LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 10 h. A 2% inoculum was then transferred to 50 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking until the OD600 reached approximately 0.6-0.8. IPTG was then added to a final concentration of 0.25 mM, and the culture was induced at 18°C for 18 h. After culturing, the culture was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were resuspended in twice the volume of 100 mM pH 7.5 Tris-HCl buffer, sonicated, and centrifuged at 12000 rpm at 4°C for 10 min to remove the precipitate. The resulting supernatant was freeze-dried to obtain imine reductase powder.
[0047] The same process was used to prepare glucose dehydrogenase powder, ultimately yielding glucose dehydrogenase powder that can be used in the coenzyme cycle.
[0048] Example 4 Catalytic Synthesis of S-Arecoline
[0049] Prepare a 100 mM pH 7.5 phosphate buffer solution, add pseudo-arecoline substrate to a final concentration of 200 mM, add 220 mM glucose, add NADP+ to a final concentration of 0.05 mM, add 0.1 g / L of the imine reductase mutant enzyme powder prepared above, add 0.05 g / L of glucose dehydrogenase enzyme powder, and react at 30℃ for 24 h.
[0050] After the reaction was completed, the product was detected by chiral high-performance liquid chromatography. The results showed that the substrate conversion rate reached 98.2% and the optical purity of the product S-arecoline reached 99.9%, which fully meets the requirements for industrial production.
Claims
1. An imine reductase mutant, characterized in that, It is obtained by single-point or multi-point mutation of the amino acid sequence of imine reductase as shown in SEQ ID NO.2 at positions 118, 168, 169, 172, 234 and 238.
2. The imine reductase mutant according to claim 1, characterized in that, The imine reductase mutant is mutated in one of the following ways: (1) The alanine at position 118 is mutated to serine; (2) Serine at position 168 is mutated to arginine; (3) Alanine at position 169 is mutated to threonine; (4) Cysteine at position 172 is mutated to tyrosine; (5) Alanine at position 234 is mutated to serine; (6) Threonine at position 238 is mutated to methionine; (7) The alanine at position 118 is mutated to serine, and the serine at position 168 is mutated to arginine. (8) Alanine at position 118 is mutated to serine, and alanine at position 169 is mutated to threonine; (9) Alanine at position 169 is mutated to threonine, and cysteine at position 172 is mutated to tyrosine. (10) Alanine at position 118 is mutated to serine, alanine at position 169 is mutated to threonine, and threonine at position 238 is mutated to methionine. (11) Alanine at position 118 is mutated to serine, alanine at position 169 is mutated to threonine, alanine at position 234 is mutated to serine, and threonine at position 238 is mutated to methionine.
3. The gene encoding the imine reductase mutant as described in claim 1.
4. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 3.
5. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contains the gene as described in claim 3.
6. The application of the imine reductase mutant as described in claim 1, or the recombinant vector as described in claim 4, or the genetically engineered bacteria as described in claim 5 in the preparation of S-arecoline.
7. A method for preparing S-arecoline, characterized in that, S-arecoline is prepared by reacting pseudooxyarecoline with crude enzyme solution, pure enzyme or immobilized enzyme of the imine reductase mutant as described in claim 1, or wet bacterial cells or whole cells of the genetically engineered bacteria as described in claim 5, under a coenzyme cycle system.
8. The method according to claim 7, characterized in that, The coenzyme cycle system includes a coenzyme, glucose, and glucose dehydrogenase, wherein the coenzyme is NADP+ or NAD+.
9. The method according to claim 7, characterized in that, In the reaction, the reaction temperature is 15~35℃, the pH value is 5.5~8.5, and the substrate concentration is 50~500 mM.
10. The method according to claim 7, characterized in that, The catalytic reduction reaction was carried out in 100 mM phosphate buffer with a coenzyme concentration of 0.01-0.1 mM.