A morphinone reductase mutant

Abstract

The present invention discloses a morphinone reductase mutant, as well as polynucleotides, expression vectors, host cells, and production methods related to the morphinone reductase mutant. The morphinone reductase mutant provided by the present invention, or cells expressing the morphinone reductase mutant, can convert 7,8-didehydronaloxone into naloxone through a specific bioconversion reaction, effectively removing impurities while increasing naloxone production.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzymes, and in particular relates to a morphinone reductase mutant. Background Art

[0002] Naloxone is a highly effective opioid receptor antagonist that rapidly restores a patient's breathing and consciousness by blocking the inhibitory effects of opioids on the central nervous system. It is widely used in the treatment of opioid poisoning, alcoholism, and analgesic overdose. Its unique pharmacological effects and broad application prospects have attracted considerable attention to its synthesis process.

[0003] The traditional naloxone synthesis process uses thebaine as the starting material and proceeds through multiple chemical reactions, including oxidation, hydrogenation, acylation, deoxymethylation, hydrolysis, and N-allylation, to ultimately produce the naloxone product. However, this process inevitably produces a variety of impurities, among which 7,8-didehydronaloxone (EP impurity D), a key mutagenic impurity, poses a serious threat to the drug's quality and safety. The production of 7,8-didehydronaloxone primarily results from side reactions of unsaturated bonds or easily oxidizable groups in the synthetic raw materials, as well as improper control of synthesis conditions. Its presence can reduce the purity of naloxone, thereby affecting the drug's quality and efficacy. Studies have shown that 7,8-didehydronaloxone may have potential toxicity or mutagenicity. Even at low concentrations, these impurities can cause DNA damage, potentially leading to carcinogenesis.

[0004] Therefore, removing the impurity 7,8-didehydronaloxone is crucial to ensuring the safety and efficacy of naloxone. While traditional chemical methods can remove this impurity to a certain extent, they often suffer from complex procedures, high costs, and environmental concerns. Therefore, there is still a need for simpler, more cost-effective, and environmentally friendly methods for removing 7,8-didehydronaloxone. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention provides a morphinone reductase mutant, which removes 7,8-didehydronaloxone impurities in the naloxone synthesis process through a biocatalytic conversion method.

[0006] In one aspect, the present invention provides a morphinone reductase mutant having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0007] Preferably, the morphinone reductase mutant has the amino acid sequence shown in SEQ ID NO: 1.

[0008] In another aspect, the present invention provides a polynucleotide encoding the morphinone reductase mutant according to any embodiment herein.

[0009] Preferably, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0010] More preferably, the polynucleotide has the nucleotide sequence shown in SEQ ID NO:5.

[0011] In another aspect, the present invention provides an expression vector comprising the polynucleotide according to any embodiment herein.

[0012] Preferably, the expression vector comprises a lactose operon element; more preferably, the expression vector is pET21a.

[0013] In another aspect, the present invention provides a host cell comprising the morphinone reductase mutant, polynucleotide, or expression vector according to any embodiment herein.

[0014] Preferably, the host cell is E. coli BL21.

[0015] In another aspect, the present invention provides a method for producing the morphinone reductase mutant according to any embodiment herein, the method comprising:

[0016] introducing a K97D mutation, an R266D mutation, or both a K97D and a R266D mutation into wild-type morphinone reductase; or

[0017] using a polynucleotide as described in any embodiment herein for expression; or

[0018] Expressed using an expression vector as described in any embodiment herein; or

[0019] Expression is performed using a host cell as described in any embodiment herein.

[0020] Preferably, the production method comprises:

[0021] S1: Synthesize target gene: Based on the amino acid sequence of MOR with NCBI accession number AAC43569.1, synthesize target gene containing K97D mutation, R266D mutation, or K97D and R266D mutations;

[0022] S2: Obtain expression vectors: clone the target genes into the vector plasmid pET21a to obtain expression vectors pET21a-K97D, pET21a-R266D or pET21a-K97D / R266D;

[0023] S3: Obtain expression strains: Transform the expression vector pET21a-K97D, pET21a-R266D, or pET21a-K97D / R266D into competent cells by heat shock transformation, and screen positive strains by colony PCR to obtain expression strains E-K97D, E-R266D, or E-K97D / R266D;

[0024] S4: Obtaining fermentation broth: The expression strain was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain primary seeds; the primary seeds were inoculated into LB liquid medium at a 1% (v / v) inoculum, cultured at 37°C and 220 rpm for 4-6 hours, and then IPTG was added to a final concentration of 0.1 mmol / L. The culture was continued at 25°C and 220 rpm for 20 hours to obtain fermentation broth;

[0025] S5: Obtain crude enzyme solution: Centrifuge the fermentation broth at 3000 × g for 10 min at 4°C, discard the supernatant to obtain wet cells; resuspend the wet cells in potassium phosphate buffer, and centrifuge at 7200 rpm and 4°C for 40 min to obtain crude enzyme solution;

[0026] S6: Obtaining pure enzyme solution: After filtering the crude enzyme solution with a 0.45 μm water filter membrane, slowly add the sample to a Ni-NTA affinity chromatography column and perform gradient elution; collect the eluate and purify it with a QFF anion exchange chromatography column to obtain pure enzyme solution.

[0027] More preferably, in step S5, the wet bacterial resuspension is subjected to high-pressure homogenization treatment at a working pressure of 850 bar and a motor operating frequency of 40 Hz and then to centrifugation treatment.

[0028] In another aspect, the present invention provides use of the morphinone reductase mutant according to any embodiment herein for catalyzing the conversion of 7,8-didehydronaloxone into naloxone.

[0029] In another aspect, the present invention provides a method for converting 7,8-didehydronaloxone into naloxone, the method comprising using the morphinone reductase mutant according to any embodiment herein.

[0030] The morphinone reductase mutants provided herein, or cells expressing them, can convert 7,8-didehydronaloxone into naloxone through a specific biotransformation reaction, effectively removing impurities while increasing naloxone production. Compared to traditional chemical methods, whole-cell catalytic biological methods offer advantages such as ease of operation, low cost, and environmental friendliness, providing a more efficient and safer means of controlling impurities in the synthesis of naloxone. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the nucleic acid electrophoresis identification result of the colony PCR product after Escherichia coli was transformed with the morphinone reductase mutant expression plasmid.

[0032] Figure 2 This is the SDS-PAGE electrophoresis result of the pure enzyme solution of the morphinone reductase mutant.

[0033] Figure 3 The figure shows the liquid chromatography results of the reaction products of crude naloxone catalyzed by the whole cell suspension of the strain expressing the mutant morphinone reductase.

[0034] Figure 4 This is the mass spectrum result of pure EP impurity D.

[0035] Figure 5 This is the mass spectrometry result of pure naloxone.

[0036] Figure 6 The chromatographic mass spectrometry results of the reaction products of crude naloxone catalyzed by the whole cell suspension of the strain expressing the mutant morphinone reductase are shown in FIG. Figure 6 A is a chromatographic result of the reaction product of crude naloxone catalyzed by the whole cell suspension of the morphinone reductase mutant expressing strain at a detection wavelength of 210.4 nm; Figure 6 B is the total ion current chromatogram of the reaction products of crude naloxone catalyzed by the whole cell suspension of the strain expressing the morphinone reductase mutant in the positive mode; Figure 6 C is the total ion current chromatogram of the reaction products of crude naloxone catalyzed by the whole cell suspension of the strain expressing the morphinone reductase mutant in the negative charge mode; Figure 6 D is the mass spectrum of the main product extracted from the total ion current of the reaction products of the whole cell suspension of the morphinone reductase mutant expressing strain catalyzing the conversion of crude naloxone in positive mode.

[0037] Figure 7 This is a liquid chromatography result of the reaction product of EP impurity D catalyzed by the whole cell suspension of the morphinone reductase mutant expression strain.

[0038] Figure 8This is the liquid chromatography result of the morphinone reductase mutant MOR-K97D / R266D pure enzyme solution catalyzing the conversion of EP impurity D. DETAILED DESCRIPTION

[0039] Example 1: Design and synthesis of morphinone reductase mutant expression plasmid

[0040] The amino acid sequence of morphinone reductase (MOR) with NCBI accession number NO.AAC43569.1 was used as the basic sequence. Lysine at position 97 and arginine at position 266 were used as mutation sites respectively. The mutation schemes were that lysine at position 97 was mutated to aspartic acid (K97D), arginine at position 266 was mutated to aspartic acid (R266D), and a mutation scheme (K97D / R266D) comprising two mutations was used simultaneously. The sequence information of the wild type and three mutation schemes was submitted to GenScript for synthesis of the MOR mutant target gene. Finally, the MOR mutant target gene was cloned into the vector plasmid pET21a (commercially available) to obtain expression vectors pET21a-K97D, pET21a-R266D and pET21a-K97D / R266D comprising the MOR mutant target genes K97D, R266D and K97D / R266D respectively. Since the vector plasmid pET21a contains a lactose operon element, it can be used to regulate the expression of the MOR mutant.

[0041] In the mutation scheme, the amino acid sequence of the MOR mutant MOR-K97D / R266D is:

[0042] MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTRSRTPDSVPGRLQQIYYGQRASAGLIISEATNISPTARGYVYTPGIWTDAQEAGWKGVVEAVHA D GGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELD D RGLAYLHFNEPDWIGGDITYPEGFREQMRQRFKGGLIYCGNYDAGRAQARLDDNTADAVAFGRPFIANPDLPERFRLGAALNEPDPSTFYGGAEVGYTDYPFLDNGHDRLG (SEQ ID NO: 1)

[0043] The amino acid sequence of the MOR mutant MOR-K97D is as follows:

[0044] MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTRSRTPDSVPGRLQQIYYGQRASAGLIISEATNISPTARGYVYTPGIWTDAQEAGWKGVVEAVHA D GGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAKRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELDRRGLAYLHFNEPDWIGGDITYPEGFREQMRQRFKGGLIYCGNYDAGRAQARLDDNTADAVAFGRPFIANPDLPERFRLGAALNEPDPSTFYGGAEVGYTDYPFLDNGHDRLG (SEQ ID NO: 2)

[0045] The amino acid sequence of the MOR mutant MOR-R266D is as follows:

[0046] MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTRSRTPDSVPGRLQQIYYGQRASAGLIISEATNISPTARGYVYTPGIWTDAQEAGWKGVVEAVHAKGGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAKRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELD D RGLAYLHFNEPDWIGGDITYPEGFREQMRQRFKGGLIYCGNYDAGRAQARLDDNTADAVAFGRPFIANPDLPERFRLGAALNEPDPSTFYGGAEVGYTDYPFLDNGHDRLG (SEQ ID NO: 3)

[0047] The amino acid sequence of the wild-type MOR is as follows:

[0048] MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTRSRTPDSVPGRLQQIYYGQRASAGLIISEATNISPTARGYVYTPGIWTDAQEAGWKGVVEAVHA K GGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELD R RGLAYLHFNEPDWIGGDITYPEGFREQMRQRFKGGLIYCGNYDAGRAQARLDDNTADAVAFGRPFIANPDLPERFRLGAALNEPDPSTFYGGAEVGYTDYPFLDNGHDRLG(SEQ ID NO: 4)

[0049] In the mutation scheme, the coding base sequence of the MOR mutant MOR-K97D / R266D after codon optimization is:

[0050]

[0051] The coding base sequence of the MOR mutant MOR-K97D after codon optimization is:

[0052]

[0053]

[0054] The coding base sequence of the wild-type MOR after codon optimization is:

[0055]

[0056] Example 2: Construction of a morphinone reductase mutant expression strain

[0057] The expression vectors pET21a-K97D, pET21a-R266D, and pET21a-K97D / R266D were transformed into E. coli BL21 (DE3) competent cells by heat shock transformation. The transformed cells were incubated at 37°C with shaking for 1 hour. The incubated bacterial suspension was spread on plates containing ampicillin antibiotics and incubated inverted at 37°C overnight. Colonies on the transformed plates were picked for colony PCR, and the PCR products were identified by nucleic acid electrophoresis. The identification results of the colonies corresponding to pET21a-K97D / R266D are as follows: Figure 1 As shown (M is a DNA molecular weight marker; CK is a negative control; lanes 1 and 2 are the positive results of two parallel samples), the colony PCR identification results of the corresponding colonies of pET21a-K97D and pET21a-R266D also obtained positive results (not shown in the figure), indicating that the expression plasmid has been successfully transformed into the bacteria.

[0058] Transformants corresponding to positive results were streaked onto plates containing ampicillin, and the streaked plates were incubated inverted at 37°C overnight. Single colonies on the streaked plates were picked and inoculated into LB liquid medium. The culture was shaken and incubated at 37°C for 4-6 h. The cultured bacterial liquid and glycerol were mixed at a ratio of 1:1 (v / v) and aliquoted into cryovials (1 mL / tube). This yielded the MOR mutants MOR-K97D, MOR-R266D, and the MOR-K97D / R266D expression strain E-K97D. Glycerol tubes of E-R266D and E-K97D / R266D were stored at -80°C.

[0059] Example 3: Inducible expression and protein purification of morphinone reductase mutants

[0060] Glycerol tubes of expression strains E-K97D, E-R266D, and E-K97D / R266D were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm with shaking to obtain primary seeds. The primary seeds were inoculated into new LB liquid medium at a 1% (v / v) inoculum and cultured at 37°C and 220 rpm with shaking for 4-6 hours. After that, LIPTG was added to a final concentration of 0.1 mmol / L and cultured at 25°C and 220 rpm for another 20 hours to induce expression of each MOR mutant. Fermentation broth was obtained after the culture was completed.

[0061] The fermentation broth was centrifuged at 3000 × g for 10 min at 4°C, and the supernatant was discarded to obtain wet cells. The wet cells were resuspended in 50 mM potassium phosphate buffer and subjected to high-pressure homogenization at 850 bar and 40 Hz. The suspension was then centrifuged at 7200 rpm and 4°C for 40 min, and the supernatant was collected to obtain a crude MOR mutant enzyme solution. The crude MOR mutant enzyme solution was filtered through a 0.45 μm aqueous filter and slowly loaded onto a Ni-NTA affinity chromatography column. After loading, the solution was eluted with buffer B (20 mM Tris-HCl, 1 M imidazole). During the elution process, the imidazole concentration in buffer B was continuously reduced, and eluate C corresponding to 300 mmol / L imidazole was collected. The imidazole in eluate C was then removed using a QFF anion exchange chromatography column to obtain a pure MOR mutant enzyme solution. SDS-PAGE protein electrophoresis was used to detect the proteins in the crude enzyme solution of the MOR mutant, the flow-through of the MOR mutant affinity chromatography and the pure enzyme solution of the MOR mutant. The detection results of MOR-K97D / R266D were as follows: Figure 2 As shown (M is a protein molecular weight marker, lane 1 is a crude enzyme solution of MOR-K97D / R266D; lanes 2 and 4 are the flow-throughs of MOR-K97D / R266D affinity chromatography; lanes 3 and 5 are pure enzyme solutions of MOR-K97D / R266D), significant MOR-K97D / R266D protein expression can be detected in both the crude enzyme solution and the pure enzyme solution corresponding to E-K97D / R266D; significant MOR-K97D and MOR-R266D protein expression can also be detected in the crude enzyme solution and the pure enzyme solution corresponding to E-K97D and E-R266D, respectively (not shown in the figure), thereby verifying the ability of the above-mentioned expression vector and host cells to express the target protein MOR mutant.

[0062] Example 4: Morphinone reductase mutant whole cell catalyzes crude naloxone

[0063] To a 1.5 mL centrifuge tube, 745 μL of 50 mM potassium phosphate buffer (pH 6.4), 5 μL of 200 g / L glucose solution, 150 μL of 100 g / L crude naloxone, and 100 μL of a 500 g / L whole-cell bacterial suspension (E-WT, E-K97D, E-R266D, or E-K97D / R266D, cell mass is measured by wet weight, the whole-cell bacterial suspension is prepared by resuspending the wet cells in Example 3 in 50 mM potassium phosphate buffer (pH 6.4) to a concentration of 500 g / L) were added to prepare a 1 mL reaction system; a control group CK was prepared by adding 100 μL of 50 mM potassium phosphate buffer (pH 6.4) instead of the whole-cell suspension of the expression strain.

[0064] Table 1: Reaction system of crude naloxone catalyzed by whole cells of morphinone reductase mutants

[0065]

[0066] The reaction system was placed in a shaking environment at 40°C and 220 rpm / min for 2 hours. After the reaction, the reaction mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was collected. The supernatant was analyzed by HPLC-MS to detect the reaction products. The test results were as follows: Figure 3 and Figure 6 As shown (CK is negative control, WT is wild type).

[0067] The HPLC-MS analysis method parameters are as follows:

[0068] Octadecylsilane bonded silica gel was used as the filler (4.6 mm × 250 mm, 5 μm); Solution A: 1.58 g of sodium bicarbonate was dissolved in 950 mL of water, the pH was adjusted to 9.0 with aqueous ammonia, and the volume was made up to 1000 mL with water. The mobile phase was acetonitrile-solution A (40:60), the detection wavelength was 210 nm, the flow rate was 1.0 mL / min, the column temperature was 40°C, and the injection volume was 10 μL.

[0069] Depend on Figure 3 As can be seen, a system with only 50 g / L of E-K97D / R266D added can completely convert the EP impurity D in 15 g / L of crude naloxone into naloxone, with a conversion rate of 100%. The conversion rate of EP impurity D is calculated by the ratio of its reduction, i.e., conversion rate (%) = (reduction in EP impurity D / initial amount of EP impurity D) × 100%. Under the same conditions, the conversion rates of mutants K97D, R266D, and the wild type were 78.32%, 89.31%, and 20.53%, respectively.

[0070] Figure 4 The mass spectrometry results of pure EP impurity D are shown, and its mass-to-nuclear ratio is 325.8.

[0071] Figure 5 The mass spectrometry results of pure naloxone are shown, with a mass-to-nuclear ratio of 327.8.

[0072] Figure 6 The chromatographic mass spectrometry results of the reaction products of E-K97D / R266D are shown, wherein Figure 6 A shows the chromatographic mass spectrometry results at a wavelength of 210.4 nm. To reduce the interference of impurities, the total ion chromatograms of the reaction products in positive and negative modes were detected, and the results were as follows: Figure 6 B and Figure 6 C; at the same time, the ion mass spectrum of the main product extracted from the total ion flow in the positive mode was also detected, and the results were as follows Figure 6D. The molecular weight of the main component in the reaction product of E-K97D / R266D was verified by HPLC-MS. Figures 3 to 6 It can be seen that the molecular weight of the main component is completely consistent with that of naloxone, indicating that the EP impurity D in the reaction product has been completely reduced to naloxone.

[0073] Example 5: Morphinone reductase mutant whole cell catalysis of EP impurity D

[0074] To a 1.5 mL centrifuge tube, 885 μL of 50 mM potassium phosphate buffer (pH 6.4), 5 μL of 200 g / L glucose solution, 10 μL of 5 g / L EP impurity D, and 100 μL of 500 g / L whole-cell bacterial suspension (E-WT, E-K97D, E-R266D, or E-K97D / R266D, cell mass is measured by wet weight, and the whole-cell bacterial suspension is resuspended from the wet cells in Example 3 using 50 mM potassium phosphate buffer (pH 6.4) to a concentration of 500 g / L) was added to prepare a 1 mL reaction system; a system in which no whole-cell suspension of the expression strain was added but 100 μL of 50 mM potassium phosphate buffer (pH 6.4) was added was used as the control group CK.

[0075] Table 2: Reaction system of EP impurity D catalyzed by whole cell of morphinone reductase mutant

[0076]

[0077] The reaction system was placed in a shaking environment at 40°C and 220 rpm / min for 2 hours. After the reaction, the reaction mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was collected. The supernatant was analyzed by HPLC-MS to detect the reaction products. The test results were as follows: Figure 7 As shown (CK is negative control, WT is wild type).

[0078] The HPLC-MS analysis method parameters are the same as in Example 4.

[0079] Depend on Figure 7 As can be seen, a system supplemented with only 50 g / L of E-K97D / R266D whole-cell suspension can completely hydrogenate and reduce 50 mg / L of EP impurity D to naloxone, with a conversion rate of 100%. The conversion rate of EP impurity D is calculated by the ratio of its reduced amount: conversion rate (%) = (reduced amount of EP impurity D / initial amount of EP impurity D) × 100%. Under the same conditions, the conversion rates of mutants K97D, R266D, and the wild type were 16.34%, 69.37%, and 9.86%, respectively.

[0080] Example 6: Catalysis of EP Impurity D by Pure Enzyme of Morphinone Reductase Mutant K97D / R266D

[0081] To a 1.5 mL centrifuge tube, add 870 μL of 50 mM potassium phosphate buffer (pH 6.4), 10 μL of 90 mM NADH, 20 μL of 5 g / L EP impurity D, and 100 μL of 1 g / L pure enzyme solution to prepare a 1 mL reaction system. A control group (CK) was prepared without the addition of pure enzyme solution but with 100 μL of 50 mM potassium phosphate buffer (pH 6.4).

[0082] Table 3: Reaction system of EP impurity D catalyzed by pure enzyme solution of morphinone reductase mutant K97D / R266D

[0083]

[0084] The reaction system was placed at 40°C for 2 hours, the reaction mixture was incubated at 100°C for 10 minutes to terminate the reaction, and centrifuged at 12000 rpm for 3 minutes, and the supernatant was collected; the supernatant was analyzed by HPLC-MS to detect the reaction products therein. The detection results are as follows: Figure 6 As shown (CK is the negative control).

[0085] Depend on Figure 6 It can be seen that the system with only 0.1 g / L of the MOR mutant MOR-K97D / R266D added can completely hydrogenate and reduce 100 mg / LEP impurity D to naloxone, and the conversion rate can reach 100%.

[0086] In summary, the whole-cell suspensions of the MOR mutant-expressing strains (E-K97D, E-R266D, or E-K97D / R266D) and the pure enzyme solutions of the MOR mutants (MOR-K97D, MOR-R266D, or MOR-K97D / R266D) obtained in the present invention all exhibited significant activity in converting 7,8-didehydronaloxone (EP impurity D) to naloxone. Regarding the conversion activity of the whole-cell suspensions of the expression strains, the conversion activity of the expression strains of the three MOR mutants was significantly higher than that of the wild-type expression strain.

Claims

1. A morphinone reductase mutant, characterized in that: The amino acid sequence of the morphinone reductase mutant is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

2. The morphinone reductase mutant according to claim 1, wherein The amino acid sequence of the morphinone reductase mutant is shown in SEQ ID NO:

1.

3. A polynucleotide, characterized in that The polynucleotide encodes the morphinone reductase mutant according to claim 1.

4. The polynucleotide according to claim 3, wherein The polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:

7.

5. The polynucleotide according to claim 3, wherein The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

5.

6. An expression vector, characterized in that The expression vector comprises the polynucleotide according to claim 3.

7. A host cell, characterized in that The host cell comprises the morphinone reductase mutant according to claim 1 , the polynucleotide according to claim 3 , or the expression vector according to claim 6 .

8. A method for producing the morphinone reductase mutant according to claim 1, characterized in that: The method comprises: introducing a K97D mutation, an R266D mutation, or both a K97D and a R266D mutation into wild-type morphinone reductase; or Using the polynucleotide of claim 3 for expression; or Use the expression vector according to claim 6 for expression; or The expression is carried out using the host cell according to claim 7.

9. Use of the morphinone reductase mutant according to claim 1 in catalyzing the conversion of 7,8-didehydronaloxone into naloxone.

10. A method for converting 7,8-didehydronaloxone into naloxone, characterized in that: The method comprises using the morphinone reductase mutant according to claim 1 for catalysis.

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