Morphone reductase mutant

Abstract

The invention discloses a morphone reductase mutant, and also discloses a polynucleotide related to the morphone reductase mutant, an expression vector, a host cell and a production method. The morphone reductase mutant provided by the invention or a cell for expressing the morphone reductase mutant can be used for converting 7, 8-didehydronaloxone into naloxone through a specific biological conversion reaction, so that not only is the effective removal of impurities realized, but also the yield of naloxone is increased.

Description

Technical Field

[0001] The 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 the 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, alcohol poisoning, and analgesic overdose. Its unique pharmacological effects and broad application prospects have attracted much attention to the synthesis process of naloxone.

[0003] The traditional naloxone synthesis process uses thebaine as the starting material and undergoes multiple chemical reactions, including oxidation, hydrogenation, acylation, deoxymethylation, hydrolysis and N-allylation, to finally obtain the naloxone product. However, this process will inevitably produce a variety of impurities, among which 7,8-didehydronaloxone (EP impurity D) is a key mutagenic impurity that poses a serious threat to the quality and safety of the drug. The production of 7,8-didehydronaloxone mainly stems from the side reactions of unsaturated bonds or easily oxidized groups in the synthetic raw materials during the reaction process, as well as improper control of the synthesis conditions. Its presence will reduce the purity of naloxone, thereby affecting the quality and efficacy of the drug. Studies have shown that 7,8-didehydronaloxone may have potential toxicity or mutagenicity. Such impurities may cause DNA damage at low levels, which may lead to carcinogenesis.

[0004] Therefore, removing the impurity 7,8-didehydronaloxone is crucial to ensure the safety and effectiveness of naloxone. Although traditional chemical methods can remove this impurity to a certain extent, they often have problems such as complex operation, high cost, and environmental unfriendliness. It can be seen that there is still a need to develop a method for removing 7,8-didehydronaloxone that is simpler to operate, cheaper, and more environmentally friendly. Summary of the invention

[0005] In order to solve the defects 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 an amino acid sequence as shown in SEQ ID NO:1.

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

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

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

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

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

[0018] Use the expression vector as described in any embodiment of the present invention for expression; or

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

[0020] Preferably, the production method comprises:

[0021] S1: Synthesize the target gene: Based on the amino acid sequence of MOR with NCBI accession number NO.AAC43569.1, synthesize the target gene containing K97D mutation, R266D mutation, or K97D and R266D mutation;

[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 method, and screen positive strains by colony PCR to obtain expression strains E-K97D, E-R266D or E-K97D / R266D;

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

[0025] S5: Obtain crude enzyme solution: centrifuge the fermentation broth at 4°C and 3000×g for 10 min, 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: Filter 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 heavy suspension is subjected to high-pressure homogenization treatment at a working pressure of 850 bar and a motor operating frequency of 40 Hz and then centrifuged.

[0028] In another aspect, the present invention provides use of the morphinone reductase mutant as described in any embodiment herein in 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 mutant or the cell expressing the morphinone reductase mutant provided by the present invention can convert 7,8-didehydronaloxone into naloxone through a specific bioconversion reaction, thereby achieving effective removal of impurities and increasing the yield of naloxone. Compared with traditional chemical methods, the whole-cell catalytic biological method has the advantages of simple operation, low cost, and environmental friendliness, and provides a more efficient and safe impurity control method for the synthesis process of naloxone. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 result of the reaction products of crude naloxone catalyzed by the whole cell suspension of the strain expressing the mutant of 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 result of the reaction product of the whole cell suspension of the morphinone reductase mutant expressing strain catalyzing the conversion of crude naloxone. Figure 6 A is a chromatographic result of the reaction product of crude naloxone catalytically converted 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 products 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 the positive mode.

[0037] Figure 7 The figure 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, and the lysine at position 97 and the arginine at position 266 were used as mutation sites, respectively, and the mutation schemes were respectively that the lysine at position 97 was mutated to aspartic acid (K97D), the arginine at position 266 was mutated to aspartic acid (R266D), and the mutation schemes (K97D / R266D) containing two mutations at the same time. The sequence information of the wild type and the three mutation schemes was submitted to GenScript for the synthesis of the MOR mutant target gene. Finally, the MOR mutant target gene was cloned in the vector plasmid pET21a (commercially available), and expression vectors pET21a-K97D, pET21a-R266D and pET21a-K97D / R266D containing the MOR mutant target genes K97D, R266D and K97D / R266D were obtained. 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 GGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAKRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELD 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 GGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAKRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELD 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 strain expressing a morphinone reductase mutant

[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 method, and the transformed bacteria were incubated at 37°C for 1 hour with shaking; the incubated bacterial liquid was taken and spread on plates containing ampicillin antibiotics, and the plates were incubated inverted at 37°C overnight. The colonies on the transformation 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] The transformants corresponding to the positive results were inoculated on plates containing ampicillin antibiotics by streaking, and the streaked plates were inverted and cultured overnight at 37°C. Single colonies on the streaked plates were picked and inoculated into LB liquid medium, and cultured at 37°C with shaking for 4 to 6 hours. The cultured bacterial solution and glycerol were mixed evenly at a ratio of 1:1 (v / v), and dispensed into cryopreservation tubes (1 mL / tube) to obtain the expression strains E-K97D of the MOR mutants MOR-K97D, MOR-R266D, and MOR-K97D / R266D, and the glycerol tube strains 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] The glycerol tube strains of the expression strains E-K97D, E-R266D and E-K97D / R266D were inoculated into LB liquid culture medium respectively, and cultured overnight at 37°C and 220rpm to obtain primary seeds. The primary seeds were inoculated into new LB liquid culture medium at an inoculum of 1% (v / v), and cultured at 37°C and 220rpm for 4 to 6 hours, and then 0.1mmol / LIPTG was added to induce the expression of each MOR mutant at 25°C and 220rpm for 20 hours to induce the expression of each MOR mutant; after the culture was completed, the fermentation liquid was obtained.

[0061] The fermentation broth was centrifuged at 4°C and 3000×g for 10 min, and the supernatant was discarded to obtain wet cells. The wet cells were resuspended in 50mM potassium phosphate buffer, and the wet cell resuspension was subjected to high-pressure homogenization at a working pressure of 850bar and a motor working frequency of 40Hz, and then centrifuged at 7200rpm and 4°C for 40min, and the supernatant was collected to obtain the crude enzyme solution of the MOR mutant. The crude enzyme solution of the MOR mutant was filtered with a 0.45μm water filter membrane and slowly loaded onto a Ni-NTA affinity chromatography column. After loading, the solution was eluted with buffer B (20mM Tris-HCl, 1M imidazole). During the elution process, the concentration of imidazole in buffer B was continuously reduced, and the eluate C corresponding to 300mmol / L imidazole was collected; the imidazole in the eluate C was removed with a QFF anion exchange chromatography column to obtain the pure enzyme solution of the MOR mutant. 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 are 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 cell to achieve the expression of the target protein MOR mutant.

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

[0063] 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 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 bacteria in Example 3 with 50 mM potassium phosphate buffer (pH 6.4) to a concentration of 500 g / L) were added to a 1.5 mL centrifuge tube to prepare a 1 mL reaction system; the 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 the control group CK.

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

[0065]

[0066] The reaction system was placed 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 are 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 um); 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 It can be seen that the system with only 50 g / L E-K97D / R266D added can completely convert the EP impurity D in 15 g / L crude naloxone into naloxone, and the conversion rate can reach 100%; the conversion rate of EP impurity D is calculated by the ratio of its reduction amount, that is, conversion rate (%) = (reduction amount of EP impurity D / initial amount of EP impurity D) × 100%. The conversion rates of mutant K97D, R266D and wild type under the same conditions are 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; in order to reduce the interference of impurities, the total ion chromatograms of the reaction products in the positive and negative modes were detected respectively, 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 are shown in Figure 6D. The molecular weight of the main component in the reaction product of E-K97D / R266D was verified by HPLC-MS. Figure 3 to Figure 6 It can be seen that the molecular weight of the main component is completely consistent with the molecular weight 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 EP impurity D

[0074] 885 μL 50 mM potassium phosphate buffer (pH 6.4), 5 μL 200 g / L glucose solution, 10 μL 5 g / L EP impurity D and 100 μL 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 made by resuspending the wet bacteria in Example 3 using 50 mM potassium phosphate buffer (pH 6.4) to a concentration of 500 g / L) in a 1.5 mL centrifuge tube to prepare a 1 mL reaction system; the system without adding the whole cell suspension of the expression strain and supplemented with 100 μL 50 mM potassium phosphate buffer (pH 6.4) was used as the control group CK.

[0075] Table 2: Morphinone reductase mutant whole cell catalyzed EP impurity D reaction system

[0076]

[0077] The reaction system was placed 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 are 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 It can be seen that the system with only 50 g / L E-K97D / R266D whole cell suspension added can completely hydrogenate and reduce 50 mg / L EP impurity D to naloxone, and the conversion rate can reach 100%; the conversion rate of EP impurity D is calculated by the ratio of its reduction amount, that is, conversion rate (%) = (reduction amount of EP impurity D / initial amount of EP impurity D) × 100%. The conversion rates of mutant K97D, R266D and wild type under the same conditions are 16.34%, 69.37% and 9.86%, respectively.

[0080] Example 6: Catalysis of EP impurity D by pure enzyme solution of morphinone reductase mutant K97D / R266D

[0081] In a 1.5 mL centrifuge tube, 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 were added to prepare a 1 mL reaction system; the control group CK was a system without adding pure enzyme solution but supplemented 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 heated at 100°C for 10 minutes to terminate the reaction, and centrifuged at 12000 rpm for 3 minutes to collect the supernatant; the supernatant was analyzed by HPLC-MS to detect the reaction product 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 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 suspension of the MOR mutant expression strain (E-K97D, E-R266D or E-K97D / R266D) and the pure enzyme solution of the MOR mutant (MOR-K97D, MOR-R266D or MOR-K97D / R266D) obtained by the present invention have significant activity in converting 7,8-didehydronaloxone (EP impurity D) into naloxone. In terms of the conversion activity of the whole cell suspension of the expression strain, the conversion activity of the expression strains of the three MOR mutants is significantly higher than that of the wild-type expression strain.

Claims

1. A morphinone reductase mutant, characterized in that: The morphinone reductase mutant has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

2. The morphinone reductase mutant according to claim 1, characterized in that The morphinone reductase mutant has an amino acid sequence as 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, a R266D mutation, or a K97D and R266D mutation into a wild-type morphinone reductase; or Using the polynucleotide of claim 3 for expression; or Using the expression vector of claim 6 for expression; or The host cell according to claim 7 is used for expression.

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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