An imine reductase mutant, its preparation method, and its application in the catalytic preparation of dextromethorphan intermediates.

Abstract

This invention provides an imine reductase mutant, its preparation method, and its application in the catalytic preparation of dextromethorphan intermediates. The imine reductase mutant is an imine reductase with an amino acid mutation, comprising the amino acid sequence shown in SEQ ID NO:1. The amino acid mutation type includes any one or a combination of at least two of I122T, D212A, or G228R. By introducing a mutation into the imine reductase, this invention significantly improves the enzyme's activity and stereoselectivity, enabling the high-yield synthesis of the dextromethorphan intermediate (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline under mild conditions, greatly reducing production costs and making it suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of protein modification technology, and relates to an imine reductase mutant, its preparation method and application, particularly to an imine reductase mutant, its preparation method and its application in the catalytic preparation of dextromethorphan intermediate. Background Technology

[0002] Dextromethorphan (DM), molecular formula C 18 H 25 NO is a centrally acting cough suppressant that works by inhibiting the cough center in the medulla oblongata of the brainstem and blocking the excitation of the vagus nerve. Its antitussive effect is comparable to, or even slightly stronger than, codeine. Because its central antitussive effect is non-anesthetic and non-addictive, it has been widely used in clinical treatment of cough for nearly 50 years.

[0003]

[0004] Dextromethorphan was originally developed by Roche in Switzerland. The U.S. Food and Drug Administration (FDA) listed it as an over-the-counter drug in 1956. Currently, dextromethorphan is an ingredient in over 100 over-the-counter drugs sold in the United States. It is primarily used in medications in the form of hydrobromide. In addition, common cold and cough medicines on the market, such as Miconazole, Pseudoephedrine, DayQuil, Pramipexole, Lizhu Kele, Parker, and Jianer Infant Cough Syrup, all contain dextromethorphan hydrobromide.

[0005] The asymmetric synthesis of dextromethorphan mainly involves the asymmetric synthesis of its intermediate (+)-p-methoxybenzyl-1,2,3,4,5,6,7,8-octahydroisoquinoline and its derivatives. This is primarily achieved through enantioselective hydrogenation catalyzed by the introduction of sterically hindered groups or metal complexes. Meyers et al. synthesized high-optical-purity dextromethorphan intermediates and homologues using the reduction product of isoquinoline as a raw material via asymmetric synthesis, achieving an ee value of over 98%. The final product, dextromethorphan, was obtained through a Grewe cyclization reaction. However, the cyclization reaction yield in this step was relatively low, only 45-50%. Furthermore, the reaction environment using butyllithium was quite harsh and the operation was complex. M. Kitamura et al. reported the selective hydrogenation reduction reaction of enamines and synthesized dextromethorphan using an asymmetric synthesis method. This type of method mainly synthesizes chiral intermediates and then dextromethorphan through corresponding selective hydrogenation catalyzed by BINAP-Ru(II) or other transition metal complexes. The catalysts used in this method for catalyzing enantioselective hydrogenation are relatively expensive, which increases the production cost.

[0006] Therefore, it is desirable in this field to develop a catalyst that can achieve the preparation of dextromethorphan intermediates under relatively mild reaction conditions. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an imine reductase mutant, its preparation method, and its application in the catalytic preparation of dextromethorphan intermediates. The imine reductase mutant of the present invention is obtained by random mutation based on the original sequence. Compared with the original imine reductase, changes in protein structure and function are observed, resulting in improved stereoselectivity and enzyme activity. It enables the high-yield synthesis of the dextromethorphan intermediate (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-2) under mild conditions, significantly reducing production costs and making it suitable for industrial production.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] On the one hand, the present invention provides an imine reductase mutant, wherein the imine reductase mutant is an imine reductase that has undergone amino acid mutation;

[0010] The imine reductase comprises the amino acid sequence shown in SEQ ID NO:1;

[0011] The types of amino acid mutations include any one or a combination of at least two of I122T, D212A, or G228R.

[0012] In this invention, the combination of mutation types includes the combination of I122T and D212A, the combination of D212A and G228R, the combination of I122T and G228R, and the combination of I122T, D212A and G228R.

[0013] SEQ ID NO.1:

[0014] MTEHGKTPVTVLGLGAMGTALVEALLAAGHPVTAWNRTASRAEGVAAKGA

[0015] SVASTVSEALAANKTVIACLLDYDSVHEVLDPVASGLEGRQLINLTNGTPGQA

[0016] REMSAWAEELGAEYLDGGIMAVPPMIGTPGAFIFYSGSGTVFGQARTALDTFG

[0017] GVNYLGADPGLAPLHDIALLSGMYGNFIGVIQAFALVGSAGVKAREFAPLLR

[0018] GWMDAMSGFLERTAELIDDGDYERGVVSNIGMQAAAFPNLAKAAEEQGISAELLAPLQPLMDKRVAAGHGAEDLVGVIELLKK.

[0019] In this invention, by introducing the mutation described above into imine reductase, the activity and stereoselectivity of the enzyme are significantly improved, enabling the mild synthesis of the dextromethorphan intermediate (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-2) under low-temperature conditions, greatly reducing production costs and making it suitable for industrial production.

[0020] Preferably, the coding sequence of the imine reductase includes the nucleotide sequence shown in SEQ ID NO.2.

[0021] SEQ ID NO.2:

[0022] ATGACCGAACATGGTAAAACCCCGGTTACCGTTCTGGGCCTGGGCGCAAT

[0023] GGGCACCGCACTGGTGGAAGCCCTGCTGGCAGCAGGTCATCCGGTTACCG

[0024] CCTGGAATCGCACCGCAAGCCGTGCAGAAGGCGTGGCCGCCAAAGGCGC

[0025] AAGTGTGGCAAGTACCGTGAGTGAAGCCCTGGCAGCAAATAAAACCGTGA

[0026] TTGCCTGTCTGCTGGATTATGATAGTGTGCATGAAGTGCTGGGATCCGGTGG

[0027] CAAGCGGCCTGGAAGGTCGCCAGCTGATTAATCTGACCAATGGTACCCG

[0028] GGCCAGGCACGCGAAATGAGTGCATGGGCCGAAGAACTGGGTGCCGAATA

[0029] TCTGGATGGCGGTATTATGGCCGTTCCGCCGATGATTGGTACCCCGGGTGCA

[0030] TTTATTTTTTATAGTGGCAGCGGCACCGTGTTTGGCCAGGCACGTACCGCC

[0031] CTGGATAACCTTTGGTGGCGTGAATTATCTGGGTGCAGATCCGGGTCTGGCA

[0032] CCGCTGCATGATATTGCACTGCTGAGTGGTATGTATGGCAATTTTATTGGTG

[0033] TTATTCAGGCATTTGCACTGGTTGGTAGTGCCGGTGTTAAAGCACGCGAAT

[0034] TTGCACCGCTGCTGCGTGGCTGGATGGATGCAATGAGTGGCTTTCTGGAAC

[0035] GTACCGCCGAACTGATTGATGATGGTGATTATGAACGTGGCGTTGTTAGCA

[0036] ATATTGGTATGCAGGCCGCCGCATTTCCGAATCTGGCAAAAGCAGCCGAAG

[0037] AACAGGGCATTAGTGCCGAACTGCTGGCCCCGCTGCAGCCGTTAATGGATA

[0038] AACGGTTGCCGCCGGCCATGGCGCCGAAGATCTGGTTGGCGTGATTGAACTGCTGAAAAAAATAA.

[0039] Preferably, the imine reductase is derived from Amycolatopsis regifaucium.

[0040] In a second aspect, the present invention provides a nucleic acid molecule that encodes the imine reductase mutant described in the first aspect.

[0041] Thirdly, the present invention provides an expression vector containing at least one copy of the nucleic acid molecule described in the second aspect.

[0042] Fourthly, the present invention provides an imine reductase mutant transformant, wherein the imine reductase mutant transformant is a genetically engineered strain expressing the imine reductase mutant described in the first aspect.

[0043] Preferably, the imine reductase mutant transformant contains the nucleic acid molecule described in the second aspect.

[0044] Preferably, the imine reductase mutant transformant contains the expression vector described in the third aspect.

[0045] Preferably, the genetically engineered strain includes any one of Escherichia coli, Pichia pastoris, or Bacillus subtilis.

[0046] Fifthly, the present invention provides a method for preparing the imine reductase mutant described in the first aspect, the method comprising:

[0047] An expression vector was constructed and transformed into recipient cells to create an imine reductase mutant transformant.

[0048] The imine reductase mutant transformant was cultured, and the culture was collected to obtain the imine reductase mutant.

[0049] As a preferred technical solution, the method for preparing the imine reductase mutant of the present invention includes the following steps:

[0050] Random mutations were performed using nucleotide sequence SEQ ID NO: 2 as a template, and the resulting material was digested and recombined into the expression vector pET28a(+), which was then transformed into host cells BL21(DE3) to obtain a random mutant library.

[0051] High-throughput screening of a random mutant library was performed to obtain mutant strains that caused significant changes in enzyme activity. The mutant strains were then cultured in LB liquid medium to obtain imine reductase mutant enzyme solution.

[0052] In a sixth aspect, the present invention provides a method for synthesizing (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-2) using an imine reductase mutant as described above, comprising the following steps:

[0053] 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline was mixed with an imine reductase mutant enzyme solution and reacted to obtain (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline.

[0054] The imine reductase mutant described in this invention is used to prepare (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline, which exhibits high stereoselectivity and an optical purity of over 99%.

[0055] Preferably, the concentration of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline in the reaction system is 10mM-100mM, for example 10mM, 20mM, 30mM, 50mM, 80mM, 90mM or 100mM.

[0056] Preferably, the amount of imine reductase mutant used in the reaction system is 5-10 U / mL, for example, 5 U / mL, 6 U / mL, 7 U / mL, 8 U / mL, 9 U / mL or 10 U / mL.

[0057] Preferably, the reaction system further contains glucose dehydrogenase, coenzyme, and glucose;

[0058] Preferably, the amount of glucose dehydrogenase used in the reaction system is 10-25 U / mL, for example, 10 U / mL, 12 U / mL, 15 U / mL, 18 U / mL, 20 U / mL, 22 U / mL or 25 U / mL.

[0059] Preferably, the coenzyme is NADP+;

[0060] Preferably, the concentration of the coenzyme in the reaction system is 0.05mM-0.5mM, for example 0.05mM, 0.08mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM or 0.5mM.

[0061] Preferably, the concentration of glucose in the reaction system is 50mM-500mM, for example 50mM, 80mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM or 500mM.

[0062] Preferably, the solvent for the reaction is a Tris-HCl buffer solution with a concentration of 10-100 mM (e.g., 10 mM, 20 mM, 30 mM, 50 mM, 80 mM, 90 mM or 100 mM) and a pH of 8.0-8.5, e.g., 8.0, 8.2, 8.4 or 8.5.

[0063] Preferably, the reaction is carried out at pH 8.0-8.5 (e.g., 8.0, 8.2, 8.4 or 8.5).

[0064] Preferably, the reaction temperature is 20-25°C (e.g., 20°C, 22°C, 24°C or 25°C), and the reaction time is 12-24 hours (e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours).

[0065] In a seventh aspect, the present invention provides the use of the imine reductase mutant described in the first aspect, or the imine reductase mutant transformant described in the fourth aspect, or the method described in the sixth aspect in the preparation of dextromethorphan.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] The imine reductase mutant of the present invention exhibits changes in protein structure and function compared to the original imine reductase, resulting in improved stereoselectivity and enzyme activity. It can synthesize the dextromethorphan intermediate (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-2) in high yield under mild conditions. The product optical purity can reach over 90%, and the yield can reach over 53%, or even over 80%. Moreover, it greatly reduces production costs and is suitable for industrial production. Attached Figure Description

[0068] Figure 1 A schematic diagram illustrating the reaction principle for preparing (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline. Detailed Implementation

[0069] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0070] Example 1 Construction of wild-type imine reductase expression strain

[0071] The fully synthesized imine reductase IRED fragment (sequence shown in SEQ ID NO: 2, synthesized by Changzhou Jiyu Biotechnology Co., Ltd.) was digested with restriction endonucleases NdeⅠ and XhoⅠ (purchased from New England Biolabs, operated according to the instructions) and recombined into the vector pET 28a(+). The vector was then transformed into Tran5α competent cells (purchased from TransGen Biotech). E. coli Tran5α cells were placed in LB liquid medium and cultured overnight at 37°C and 160 rpm with shaking. The recombinant plasmid IRED-pET28a(+) was extracted and transformed into chemocompetent cells of Escherichia coli expressing wild-type imine reductase (TransGen Biotech).

[0072] Example 2: Construction of an IRED random mutation library by random mutation PCR

[0073] Using sequence SEQ ID NO:2 as a template, a random mutant library was constructed using the Agilent GeneMorph II Random Mutagenesis Kit. The sequence of the forward primer IRED-NdeⅠ-F is shown in SEQ ID NO.3, and the sequence of the reverse primer MAON-XhoⅠ-R is shown in SEQ ID NO.4.

[0074] SEQ ID NO.3:

[0075] 5-GAATTCCATATGACCGAACATGGTAAAACCCGG-3'

[0076] SEQ ID NO.4:

[0077] 5'-CCGCTCGAGTTATTTTTTCAGCAGTTCAATCACG-3'.

[0078] The 50 μL PCR system includes: 5 μL of 10×Mutazyme II reaction buffer, 1 μL of 40 mM dNTP mix (200 μM each final), 1 μL each of primers IRED-NdeⅠ-F and IRED-XhoⅠ-R (10 μM), 1 μL of Mutazyme II DNA polymerase (2.5 U / μL), 50 ng IRED-pET28a(+), and water added to a final volume of 50 μL.

[0079] The PCR program was 95℃ for 2 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min 30 s; 30 cycles, 72℃ for 10 min.

[0080] After amplification, gel electrophoresis was performed to detect the 1.5kb random mutant fragment. The fragment was then digested with restriction endonucleases NdeI and XhoI (purchased from New England Biolabs, and the procedure was performed according to the instructions) and recombined into the vector pET 28a(+). The fragment was then processed into E. coli BL21(DE3) chemically competent cells (TransGen) to obtain a random mutant library.

[0081] Example 3: High-throughput screening and enzyme activity assay of mutant libraries

[0082] Transformants from the mutant library were selected using toothpicks and inoculated into 96-well deep-well culture plates containing 300 μL of LB medium with 50 mg / L kanamycin sulfate. The plates were incubated overnight at 37°C with shaking at 220 rpm. 50 μL of the primary seed culture was transferred to a secondary plate containing 600 μL of LB medium and incubated at 37°C for 3 hours until the OD600 reached approximately 1.0. Then, 0.2 mM of thiocyanate was added, and the temperature was lowered to 25°C. The plates were incubated overnight, and the cells were collected by centrifugation (3500 rpm, 10 min). 200 μL of lysis buffer (100 mmol / L phosphate buffer, pH 7.0, containing 750 mg / L lysozyme and 12 mg / L DNase) was added to each well. The cells were levitated and incubated at 37°C with 200 rpm for 2 hours to lyse the cells. After centrifugation at 4°C with 3500 rpm for 30 min, the supernatant was transferred to an ELISA plate to detect cell viability. The activity assay system for the ELISA plate (200 μL) consisted of: 1 mM substrate 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline, 1% (v / v) cosolvent DMSO, 0.15 M NADPH, and 100 mM Tris-HCl buffer (pH 8.5). Activity was determined by the decrease in absorbance of the coenzyme NADPH. From a random mutant library, approximately 10 mutant clones with high reactive enzyme activity were sequenced. Sequencing revealed significant changes in amino acid substitutions at these sites: I122T, D212A, and G228R. Point mutations were then performed on these amino acid sites to obtain the mutant strains shown in Table 1 below.

[0083] Table 1

[0084] mutant strain mutation site Relative activity IRED -- 100％ IRED-M1 I122T 150％ IRED-M2 D212A 200％ IRED-M3 G228R 150％ IRED-M4 I122T+D212A 250％ IRED-M5 D212A+G228R 350％ IRED-M6 I122T+G228R 300％ IRED-M7 I122T+D212A+G228R 1200％

[0085] Example 4 Enzyme activity assay of recombinant IRED and its mutants

[0086] E. coli BL21(DE3) containing the recombinant plasmid IRED-pET28a and its mutant were inoculated into LB medium containing 50 mg / L kanamycin sulfate and cultured overnight at 37°C and 200 rpm. 1% of the culture was then inoculated into fresh LB medium containing 50 mg / L kanamycin sulfate and cultured at 37°C and 200 rpm until OD600 = 0.8. IPTG was then added to a final concentration of 0.1 mmol, and the mixture was cultured at 25°C for another 16 hours to induce the expression of IRED and its mutant.

[0087] The bacterial sludge was collected by centrifugation at 10,000 rpm for 10 min at 4 °C, and resuspended in sodium phosphate buffer (50 mM, pH 7.5). The cells were then sonicated in an ice bath (4 s working, 4 s rest, 10 min sonication). The supernatant enzyme solution was collected by centrifugation at 10,000 rpm for 10 min at 4 °C. IRED and its mutants were purified by Ni-NTA affinity chromatography (Shanghai Sangon Biotech) to obtain pure enzyme solution.

[0088] Enzyme activity assay (1 mL): 10 μL of DMSO solution of substrate 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline (final substrate concentration 1 mmol / L), NADPH (10 μL, final concentration 0.15 mmol / L), 10 μL of enzyme solution, and 970 μL of 100 mM Tris-HCl buffer (pH 8.5) were used. The absorbance at 340 nm was measured at 25 °C. Enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute under the above conditions. The initial IRED enzyme activity was 5 U / mL.

[0089] The measurement results are shown in Table 2.

[0090] Table 2

[0091] mutant strain Relative activity IRED 100％ IRED-M1 150％ IRED-M2 200％ IRED-M3 150％ IRED-M4 250％ IRED-M5 350％ IRED-M6 300％ IRED-M7 1200％

[0092] As shown in Table 2, compared with the original imine reductase, the enzyme activity of the mutants was significantly increased, with relative enzyme activities all exceeding 150%, and even reaching 1200%. These results indicate that the present invention successfully improved the enzyme's reactivity by introducing mutations into imine reductase, and has significant practical application value.

[0093] Example 5: Fermentation of mutant imine reductase

[0094] A) Shake-flask fermentation

[0095] Shake flask seed culture medium components: yeast extract: 5g / L; peptone: 10g / L; NaCl: 10g / L; kanamycin (abbreviated as kanamycin) 50ug / mL.

[0096] Preparation of shake flask seed culture medium: Take 5g yeast extract, 10g peptone, and 10g NaCl, dissolve them in 800mL distilled water, adjust the pH to 7, and make up to 1000mL with distilled water. Keep at 121℃ for 15min. After the solution is cooled to below 60℃, add kanamycin to a final concentration of 50ug / mL.

[0097] Fermentation steps:

[0098] Streak the original bacterial culture on an LB agar plate and incubate overnight at 37°C with the plate inverted position. Pick a single colony from the agar plate and inoculate it into 3 mL of seed culture medium (10 mL test tube). Incubate at 37°C with shaking at 200 rpm for 18 hours until the OD600 reaches approximately 1.8. Then, inoculate 1% of the culture into 300 mL of seed culture medium (1 L Erlenmeyer flask) and incubate at 37°C with shaking at 200 rpm for 6 hours until the OD600 reaches approximately 1.5.

[0099] B) Fermentation culture

[0100] Culture media are divided into fermentation media and fed-batch media.

[0101] The fermentation medium was M9 medium with the following components: Na2HPO4 6g / L, KH2PO4 3g / L, MgSO4·7H2O 0.246g / L, (NH4)2SO4 2.24g / L, NaCl 0.5g / L, and glucose 20g / L.

[0102] Fermentation medium preparation: Na2HPO4, KH2PO4, MgSO4·7H2O, (NH4)2SO4, NaCl, and glucose were stirred and dissolved, kept at 121℃ for 30 min, cooled and set aside for use. Kanamycin was sterilized by filtration through a sterile membrane, and then the sterile kanamycin was added to the fermentation medium.

[0103] The fed-batch culture medium composition is as follows: glucose 600 g / L.

[0104] Preparation of fed-batch culture medium: Dissolve glucose in water, keep at 115℃ for 30 min, and cool before use.

[0105] Fermentation control in fermenter: Throughout the process, DO is controlled to be above 20%, the ventilation ratio is 1:3 (VVM), the fermentation temperature is controlled at 37℃, pH is 7.0, dissolved oxygen changes after 8 hours of culture, feeding begins, OD600 is around 30 for induction, the final IPTG concentration is 1mM, the induction temperature is controlled at 25℃, and the fermenter is discharged after 21 hours of culture.

[0106] Example 6: Preparation of whole-cell and enzyme solutions of imine reductase

[0107] After fermentation, imine reductase cells were collected by centrifugation at 5000g for 30 min. The centrifuged bacterial cells were resuspended in 3 volumes of 50mmol PBNa (pH 7.5) buffer, and then the bacterial cells were lysed by ultrasonication and centrifuged (4℃, 10000g, 10 min). The supernatant was collected as imine reductase solution.

[0108] Application Example 1

[0109] In an application example, (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline was prepared by the following method, the schematic diagram of which is shown below. Figure 1 As shown, the specific steps include:

[0110] 10 mL of the IRED-M7 enzyme solution (50 U / mL) prepared in Example 6 was added to 100 mL of Tris-HCl buffer (100 mM, pH 8.5). 1 g of glucose, 5 mg of NADP+, 1 mL of GDH enzyme solution, and 1 g of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline were added. The pH was controlled at 8.5 using 10% sodium carbonate solution. The reaction was carried out at 25°C under nitrogen protection for 24 hours. HPLC analysis showed that the reaction was complete (using a variable UV detector, detection wavelength of 210 nm, and a [missing information - likely a specific column type]). A Phenomen C18 column (4.6 mm × 50 mm, 5 μm) was used. Mobile phase A: 0.1% phosphate buffer, mobile phase B: acetonitrile, flow rate 0.8 mL / min. Purity was calculated from the peak area ratio of HPLC chromatograms. The solution was extracted with methyl tert-butyl ester (100 mL × 3), dried over anhydrous sodium sulfate, and then evaporated to dryness. The extract was purified by column chromatography to obtain 0.8 g of (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline, with a yield of 80% and an ee value of 99.5%.

[0111] The chirality detection method was performed by high performance liquid chromatography for quantitative analysis. A variable ultraviolet detector was used with a detection wavelength of 230 nm. The chromatographic column was a Daicel CHIRALPAK IC column (4.6 mm × 250 mm, 5 μm). The mobile phase was n-hexane-ethanol-diethylamine (50:50:0.05) and the flow rate was 1 mL / min.

[0112] ee = ([S] - [R] / [S] + [R]) × 100%, where [S] represents (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline, and [R] represents (R)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline.

[0113] Application Example 2

[0114] 20 mL of the IRED-M7 enzyme solution (50 U / mL) prepared in Example 6 was added to 100 mL of Tris-HCl buffer (100 mM, pH 8.5). 3 g of glucose, 10 mg of NADP+, 3 mL of GDH enzyme solution, and 3 g of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline were added. The pH was controlled at 8.5 using 10% sodium carbonate solution. The reaction was carried out at 25 °C under nitrogen protection for 24 hours. HPLC analysis showed that the reaction was complete. The pH was adjusted to 11 with 5.0 M NaOH solution. The mixture was extracted with methyl tert-butyl ester (150 mL × 3), dried over anhydrous sodium sulfate, and then evaporated to dryness. 2.46 g of (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline was obtained by column chromatography, with a yield of 82% and an ee value of 99.4%.

[0115] Application Example 3

[0116] 50 mL of the IRED-M7 enzyme solution (50 U / mL) prepared in Example 6 was added to 500 mL of Tris-HCl buffer (100 mM, pH 8.5). 10 g of glucose, 20 mg of NADP+, 5 mL of GDH enzyme solution, and 10 g of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline were added. The pH was controlled at 8.5 using 10% sodium carbonate solution. The reaction was carried out at 25 °C under nitrogen protection for 24 hours. HPLC analysis showed that the reaction was complete. The pH was adjusted to 11 with 5.0 M NaOH solution. Extraction was performed with methyl tert-butyl ester (250 mL × 3). After drying with anhydrous sodium sulfate, the extract was evaporated to dryness and purified by column chromatography to obtain 8.60 g of (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline, with a yield of 86% and an ee value of 99.6%.

[0117] Application Example 4

[0118] The only difference from Application Example 1 is that IRED-M7 is replaced with IRED-M1, while the other materials and preparation conditions are the same as in Application Example 1.

[0119] Application Example 5

[0120] The only difference from Application Example 1 is that IRED-M7 is replaced with IRED-M2, while the other materials and preparation conditions are the same as in Application Example 1.

[0121] Application Example 6

[0122] The only difference from Application Example 1 is that the IRED-M7 enzyme solution is replaced with IRED-M3, while the other materials and preparation conditions are the same as in Application Example 1.

[0123] Application Example 7

[0124] The only difference from Application Example 1 is that IRED-M7 is replaced with IRED-M4, while the other materials and preparation conditions are the same as in Application Example 1.

[0125] Application Example 8

[0126] The only difference from Application Example 1 is that IRED-M7 is replaced with IRED-M5, while the other materials and preparation conditions are the same as in Application Example 1.

[0127] Application Example 9

[0128] The only difference from Application Example 1 is that IRED-M7 is replaced with IRED-M6, while the other materials and preparation conditions are the same as in Application Example 1.

[0129] Comparative Example 1

[0130] The only difference from Application Example 1 is that IRED-M7 is replaced with IRED, while the other materials and preparation conditions are the same as in Application Example 1.

[0131] The statistical results of the yield and ee value of the product (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline in Application Examples 1-9 and Comparative Example 1 are shown in Table 3.

[0132] Table 3

[0133] Group Yield (%) ee value (%) Application Example 1 80 99.5 Application Example 2 82 99.4 Application Example 3 86 99.6 Application Example 4 53 95.2 Application Example 5 61 94.1 Application Example 6 55 94.6 Application Example 7 64 90.4 Application Example 8 71 95.6 Application Example 9 68 92.8 Comparative Example 1 42 90.2

[0134] As can be seen from Table 3, the present invention introduces a mutation on the basis of the original imine reductase, which significantly improves the enzyme activity and enhances the stereoselectivity and product yield in the preparation of (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline.

[0135] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An imine reductase mutant, characterized in that, The imine reductase mutant is an imine reductase that has undergone amino acid mutations based on the wild-type imine reductase; The amino acid sequence of the wild-type imine reductase is the amino acid sequence shown in SEQ ID NO:1; The amino acid mutation type is any one of D212A, I122T+D212A, D212A+G228R, or I122T+D212A+G228R.

2. The imine reductase mutant according to claim 1, characterized in that, The coding sequence of the wild-type imine reductase is the nucleotide sequence shown in SEQ ID NO.

2.

3. The imine reductase mutant according to claim 1, characterized in that, The wild-type imine reductase is derived from Amycolatopsis regifaucium .

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an imine reductase mutant as described in any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector contains at least one copy of the nucleic acid molecule as described in claim 4.

6. An imine reductase mutant transformant, characterized in that, The imine reductase mutant transformant is a genetically engineered strain expressing the imine reductase mutant as described in any one of claims 1-3.

7. The imine reductase mutant transformant according to claim 6, characterized in that, The imine reductase mutant transformant contains the nucleic acid molecule described in claim 4.

8. The imine reductase mutant transformant according to claim 6, characterized in that, The imine reductase mutant transformant contains the expression vector described in claim 5.

9. The imine reductase mutant transformant according to claim 6, characterized in that, The genetically engineered strain is selected from any one of Escherichia coli, Pichia pastoris, or Bacillus subtilis.

10. A method for preparing an imine reductase mutant according to any one of claims 1-3, characterized in that, The preparation method includes: An expression vector was constructed and transformed into recipient cells to create an imine reductase mutant transformant. The imine reductase mutant transformant was cultured, and the culture was collected to obtain the imine reductase mutant.

11. A method for synthesizing (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline using an imine reductase mutant as described in any one of claims 1-3, characterized in that, Includes the following steps: 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline was mixed with an imine reductase mutant enzyme solution and reacted to obtain the (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline. The above reaction system also contains glucose dehydrogenase, coenzyme, and glucose.

12. The method according to claim 11, characterized in that, The concentration of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline in the reaction system is 10 mM-100 mM.

13. The method according to claim 11, characterized in that, The amount of imine reductase mutant used in the reaction system is 5-10 U / mL.

14. The method according to claim 11, characterized in that, The amount of glucose dehydrogenase used in the reaction system is 10-25 U / mL.

15. The method according to claim 11, characterized in that, The coenzyme is NADP+.

16. The method according to claim 11, characterized in that, The concentration of the coenzyme in the reaction system is 0.05 mM-0.5 mM.

17. The method according to claim 11, characterized in that, The concentration of glucose in the reaction system is 50 mM-500 mM.

18. The method according to claim 11, characterized in that, The solvent for the reaction is Tris-HCl buffer solution with a concentration of 10-100 mM and a pH of 8.0-8.

5.

19. The method according to claim 11, characterized in that, The reaction was carried out at pH 8.0-8.

5.

20. The method according to claim 11, characterized in that, The reaction temperature is 20-25℃, and the reaction time is 12-24 hours.

21. The use of the imine reductase mutant according to any one of claims 1-3, or the imine reductase mutant transformant according to any one of claims 6-9, or the method according to any one of claims 11-20 in the preparation of dextromethorphan.

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