Alcohol dehydrogenase mutant with high nootkatone yield and preparation method thereof

Abstract

The invention discloses an alcohol dehydrogenase mutant with high yield of nootkatone and a preparation method of the alcohol dehydrogenase mutant, alcohol dehydrogenase from alpinia oxyphylla is subjected to site-specific mutagenesis, and the mutagenesis is at least one of S197V, M144C and L196A. The alcohol dehydrogenase mutant provided by the invention has high catalytic activity, and the yield of nootkatone prepared by using the mutant M144C / L196A / S197V to catalyze nocardanol can reach 99%, which is 4.32 times that of an original enzyme; the alcohol dehydrogenase mutant is simple in production process, mild in reaction condition, environment-friendly in production process, beneficial to industrial production of nootkatone and wide in application prospect.

Description

Technical Field

[0001] The invention relates to an alcohol dehydrogenase, in particular to an alcohol dehydrogenase catalyzing nootkatone, and belongs to the technical fields of enzyme engineering and genetic engineering. Background Art

[0002] Nootkatone ((+)-Nootkatone), also known as citronellol, is found in plants such as grapefruit, Alaskan yellow cedar, vetiver and alpinia oxyphylla. Nootkatone has a unique citrus aroma and is an important natural flavor that is widely used in food, beverages, cosmetics and perfume. Nootkatone also has multiple biological activities such as insect repellent, antibacterial and anti-inflammatory, so it also has important application value in the fields of agriculture and medicine. However, the content of nootkatone in plants is low, and the efficiency and cost of direct separation and extraction are low, which cannot meet the needs of industrial applications.

[0003] There are many reports on the research of nootkatone synthesis technology, mainly chemical and biological synthesis methods. Chinese patent application CN 1830936A discloses a synthesis method and application of nootkatone, which adopts chemical synthesis method, first extracts valerian oil by supercritical technology to obtain valerene, then adds sodium hypochlorite / tert-butyl hydroperoxide mixed solution, and produces nootkatone crystals after mixed reaction and separation and purification. Chinese patent application CN 108430464A uses β-pinene as raw material and utilizes ozone oxidation and decomposition to achieve high yield production of nootkatone. However, chemically synthesized nootkatone is a racemate, and its purity and quality are unstable, which limits its application in high value-added products. In addition, the chemical synthesis method also has the disadvantages of complicated operation steps, harsh reaction conditions, and the need to use a large amount of organic solvents and catalysts, resulting in high production costs and serious environmental pollution.

[0004] The biosynthetic method can catalyze the conversion of precursors such as limonene and nootkatol into nootkatone through enzymes. Chinese patent application CN 115976118 A discloses a method and vector for the biosynthesis of nootkatone. By using genetic engineering technology, a full set of enzymes involved in the synthesis of nootkatone in Alpinia oxyphylla were introduced into expression cells to construct a recombinant bacterium capable of synthesizing nootkatone from farnesyl pyrophosphate. Professor Liu Tiangang and his team significantly increased the yield of carbon source-synthesized nootkatol to 10-15 g / L through the selection and optimization of carbon sources, the transformation of microbial strains, and the optimization of fermentation conditions, and synthesized nootkatone in yeast. However, under conventional reaction conditions, the activity of alcohol dehydrogenase that catalyzes the production of nootkatone from nootkatol is low, which has become an important factor limiting the production of nootkatone by biosynthesis. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an alcohol dehydrogenase mutant that can produce high-yield nootkatone, and to provide a nucleic acid molecule encoding the mutant, a vector or recombinant cell or product containing the mutant, and a preparation method and application of the mutant.

[0006] Technical solution: The first aspect of the present invention provides an alcohol dehydrogenase mutant with high production of nootkatone, wherein the amino acid sequence of the alcohol dehydrogenase mutant is obtained by mutation of the sequence shown in SEQ ID NO.1, and the mutation is at least one of S197V, M144C, and L196A.

[0007] The wild-type alcohol dehydrogenase used in the present invention is derived from Alpinia oxyphylla, and can use nootkatol as a substrate and nicotinamide adenine dinucleotide oxidase (LpNOX) as a coenzyme to catalyze the synthesis of nootkatone. The present invention achieves changes in protein structure and function by performing site-directed mutagenesis on the amino acids in the active center of the wild-type alcohol dehydrogenase. After screening, it is found that a single mutation or a combined mutation of S197V, M144C or L196A can obtain an alcohol dehydrogenase mutant with high activity, and the mutant catalyzes the production of nootkatone from nootkatol to be significantly increased compared with the wild-type alcohol dehydrogenase.

[0008] The present invention uses the standard single-letter code for amino acids and the standard substitution notation, for example: S197V means that the serine (S) at position 197 at the N-terminus is mutated to valine (V); S197V / M144C means that the serine (S) at position 197 at the N-terminus is mutated to valine (V), and the methionine (M) at position 144 at the N-terminus is mutated to cysteine ​​(C).

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the alcohol dehydrogenase mutant of the first aspect. The nucleic acid molecule is capable of expressing the alcohol dehydrogenase mutant that produces high nootkatone yield.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO.2.

[0011] In a third aspect, the present invention provides a vector comprising the nucleotide sequence described in the second aspect. The vector is a recombinant vector that can maintain its ability to replicate or autonomously replicate in various host cells such as prokaryotic and / or eukaryotic cells, thereby amplifying or expressing the nucleotide sequence. The vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors.

[0012] Furthermore, the vector includes a cloning vector or an expression vector. The expression vector may be a PET series expression vector, preferably a pET22b(+) plasmid.

[0013] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell comprises the vector described in the third aspect. Preferably, Escherichia coli is used as the recombinant cell, such as Escherichia coli C43 or Escherichia coli BL21.

[0014] Furthermore, the method for constructing the recombinant cell comprises the following steps: (1) constructing an expression vector: connecting the nucleic acid molecule described in the second aspect with a plasmid to obtain the expression vector described in the third aspect; (2) constructing a recombinant cell: transferring the expression vector into competent cells, culturing and screening to obtain the recombinant cell.

[0015] In a fifth aspect, the present invention provides a method for preparing the alcohol dehydrogenase mutant according to the first aspect, comprising the following steps:

[0016] (1) Using a plasmid carrying an alcohol dehydrogenase gene as a template, a PCR reaction was performed using a point mutation primer, and the mutant gene fragment and a linearized plasmid were obtained through purification;

[0017] (2) connecting the mutant gene fragment to the linearized plasmid to construct an expression vector, and then transferring the expression vector into the host bacteria for inducing expression;

[0018] (3) Collecting host bacteria expressing the alcohol dehydrogenase mutant, resuspending the bacteria and breaking the cells, and centrifuging and collecting the supernatant to obtain a crude enzyme solution containing the alcohol dehydrogenase mutant.

[0019] Furthermore, based on the preparation method, in step (3), resuspending the bacteria and subsequent operations can be omitted, and the collected wet bacteria can be directly used as whole-cell catalysts.

[0020] The crude enzyme solution or bacterial cell containing the alcohol dehydrogenase mutant obtained by the preparation method has high catalytic activity and can produce high yield of nootkatone.

[0021] Preferably, in step (1), the point mutation primers are as shown in Table 1:

[0022] Table 1 Primers for point mutation

[0023] Primer name Sequence (5'-3') S197V_F gacccgcctgGTTatgccgcatc S197V_R gatgcggcatAACcaggcgggtc M144C_F ctatcgtgagcTGTgctagcgtgag M144C_R ctcacgctagcACAgctcacgatag L196A_F ccgacccgcGCTgttatgccgcatc L196A_R gatgcggcataacAGCgcgggtcgg

[0024] The uppercase letters in the primers indicate the mutation sites, “F” represents the upstream primer, and “R” represents the downstream primer.

[0025] Preferably, in step (1), the PCR reaction system is as shown in Table 2:

[0026] Table 2 PCR reaction system

[0027] Composition volume 10×BufferforKOD-Plus- 2.5μL 2mM dNTP 2.5μL <![CDATA[25mMMgSO4]]> 1.5μL DMSO 1μL 10pmol / μLForwardPrimer 0.75μL 10pmol / μLReversePrimer 0.75μL DNA template <100ng KOD-Plus- 1μL <![CDATA[ddH2O]]> up to 25 μL

[0028] Preferably, in step (1), the conditions of the PCR reaction are as shown in Table 3:

[0029] Table 3 PCR reaction conditions

[0030]

[0031] In a sixth aspect, the present invention provides a product, the product comprising the alcohol dehydrogenase mutant described in the first aspect, or the nucleic acid molecule described in the second aspect, or the vector described in the third aspect, or the recombinant cell described in the fourth aspect. The product includes a catalyst.

[0032] In a seventh aspect, the present invention provides an application of the product described in the sixth aspect in the catalytic preparation of nootkatone.

[0033] Further, the catalytic substrate is nokadol. The catalysis can be carried out in a potassium phosphate buffer solution. The reaction conditions of the catalysis include reacting for 8 to 16 hours at a temperature of 20 to 30° C. and a pH of 7.0 to 9.0. Preferably, the temperature is 25° C., the pH is 7.5, and the reaction time is 12 hours.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The alcohol dehydrogenase mutant provided by the present invention has high catalytic activity for nootkadol, and the yield of the mutant M144C / L196A / S197V in catalyzing nootkadol to prepare nootkadone can reach 99%, which is 4.32 times that of the original enzyme; 2. The production process of the alcohol dehydrogenase mutant catalyzing nootkadol to synthesize nootkadone is simple, the reaction conditions are mild, and the production process is environmentally friendly, which is conducive to the industrial production of nootkadone and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the reaction of nootkadol to nootkatone catalyzed by alcohol dehydrogenase;

[0036] Figure 2 is the catalytic yield diagram of wild-type alcohol dehydrogenase and its mutants;

[0037] Figure 3 The catalytic yield of the alcohol dehydrogenase mutant M144C / L196A / S197V at different temperatures;

[0038] Figure 4 The catalytic yield of the alcohol dehydrogenase mutant M144C / L196A / S197V at different pH;

[0039] Figure 5 The catalytic yield of the alcohol dehydrogenase mutant M144C / L196A / S197V at different reaction times. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0041] The wild-type alcohol dehydrogenase and its mutants in the present invention can catalyze the synthesis of nootkatone ((+)-nootkatone) using trans-nootkatol as a substrate and nicotinamide adenine dinucleotide oxidase (LpNOX) as a coenzyme. The reaction process is as follows: Figure 1 shown.

[0042] The acquisition methods of the materials used in the examples are as follows:

[0043] 1. Strains and plasmids

[0044] Plasmids pET-22b(+), E. coli BL21(DE3), and E. coli DH5α are all deposited by the applicant and are all from commercial sources. The wild-type alcohol dehydrogenase is the alcohol dehydrogenase AoSDR1ADH from Alpinia oxyphylla, and LpNOX is from Lactobacillus pentosus. The coding genes of the two enzymes are synthesized by Jinweizhi (Suzhou) Co., Ltd. The site-directed mutation sequence of the enzyme was obtained by the PCR method and constructed by the applicant.

[0045] 2. Reagents and culture media

[0046] DNA gel recovery kit and plasmid extraction kit were both from Sangon Biotech (Shanghai) Co., Ltd.

[0047] The components of LB liquid culture medium are 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0048] The components of LB solid culture medium are 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 15 g / L agar powder.

[0049] The components of TB liquid culture medium are yeast powder 12g / L, trypsin 12g / L, glycerol 4ml / L, dipotassium hydrogen phosphate 12.5g / L, and potassium dihydrogen phosphate 2.3g / L.

[0050] Obtaining LpNOX powder: The pET-22b(+) plasmid containing the LpNOX gene was transformed into the E.coli BL21(DE3) host bacteria and coated on the surface of LB solid culture medium containing 100μg / mL ampicillin, and cultured at 37°C for 8h. Pick a single E. coli colony and inoculate it in 3mL LB liquid culture medium containing 100μg / mL ampicillin, and culture it at 37°C overnight as a seed solution. The seed solution was inoculated at a 5% inoculum into a 250mL conical flask containing 50mL TB culture medium and cultured at 37°C and 200rpm. When the culture reached 8h, IPTG was added to a final concentration of 0.5mM, the culture temperature was set to 18°C, and the culture was continued for 16h. The fermentation broth was centrifuged, the bacteria were collected, and resuspended with 200mM phosphate buffer at pH 7.5. The obtained crude enzyme solution was ultrasonically crushed and centrifuged, the supernatant was collected, and lyophilized to obtain LpNOX powder.

[0051] Example 1 Alcohol dehydrogenase mutant S197V

[0052] In this example, alcohol dehydrogenase AoSDR1ADH is used as the original enzyme, and its amino acid sequence is shown in SEQ ID No. 1. The serine (S) at position 197 of its N-terminus is mutated to valine (V), which is the sequence of the mutant S197V. The nucleotide sequence of the original enzyme is shown in SEQ ID No. 2. The specific preparation method is as follows:

[0053] 1. Construction of recombinant plasmid

[0054] Using the pET-22b(+) plasmid carrying the alcohol dehydrogenase AoSDR1ADH gene as a template, a PCR reaction was performed using point mutation primers, and the mutant gene fragment and linearized plasmid were obtained after purification. The point primers are shown in Table 4, the PCR reaction system is shown in Table 5, and the PCR reaction conditions are shown in Table 6.

[0055] Table 4 Primers for S197V mutant gene fragment and linearized plasmid primers

[0056] Primer name Sequence (5'-3') S197V_F gacccgcctgGTTatgccgcatc S197V_R gatgcggcatAACcaggcgggtc

[0057] Note: The uppercase letters in the primers indicate the mutation sites, “F” represents the upstream primer and “R” represents the downstream primer.

[0058] Table 5 PCR reaction system

[0059] Composition volume 10×BufferforKOD-Plus- 2.5μL 2mM dNTP 2.5μL <![CDATA[25mMMgSO4]]> 1.5μL DMSO 1μL 10pmol / μLForwardPrimer 0.75μL 10pmol / μLReversePrimer 0.75μL DNA template <100ng KOD-Plus- 1μL <![CDATA[ddH2O]]> up to 25 μL

[0060] Table 6 PCR reaction conditions

[0061]

[0062]

[0063] After the PCR product was verified by nucleic acid electrophoresis, it was purified using a DNA gel recovery kit. The reaction system shown in Table 7 was prepared, wherein the mutant gene fragment was a gene fragment in which the serine at position 197 was mutated to valine, and the linearized plasmid was a linearized plasmid containing a gene fragment in which the serine at position 197 was mutated to valine. One-step cloning was performed at 37°C for 1 hour to obtain a recombinant plasmid containing the mutant enzyme gene.

[0064] Table 7 One-step cloning system

[0065] Composition content Linearized plasmid 0.03pmol Mutated gene fragment 0.06pmol 5×CEIIBuffer 4μL ExnaseII 2μL <![CDATA[ddH2O]]> to20μL

[0066] 2. Construction of recombinant strains and expression of enzyme mutants

[0067] Transform the recombinant plasmid into E.coli DH5α competent cells, spread on the surface of LB solid culture medium containing 100μg / mL ampicillin, and culture at 37℃ for 12h. Scrape all single colonies grown on the surface of the plate culture medium into a test tube containing 5mL LB liquid culture medium containing 100μg / mL ampicillin, and culture at 37℃ and 200rpm for 12h. Extract the plasmid using a plasmid extraction kit, and store the recombinant plasmid in a -20℃ refrigerator.

[0068] The recombinant plasmid was transformed into the host bacteria of E.coli BL21 (DE3), and spread on the surface of LB solid culture medium containing 100μg / mL ampicillin, and cultured at 37℃ for 8h. A single E. coli colony was picked and inoculated into 3mL LB liquid culture medium containing 100μg / mL ampicillin, and cultured at 37℃ overnight as seed liquid. The seed liquid was inoculated at 5% and transferred to a 250mL conical flask containing 50mL TB culture medium, and cultured at 37℃ and 200rpm. When the culture reached 8h, IPTG was added to a final concentration of 0.5mM, the culture temperature was set to 18℃, and the culture was continued for 16h. The fermentation broth was centrifuged, the bacteria were collected, and resuspended with 200mM phosphate buffer at pH 7.5 to obtain a whole-cell catalyst (i.e., wet bacteria) containing the alcohol dehydrogenase mutant S197V.

[0069] Example 2 An alcohol dehydrogenase mutant S197V / M144C

[0070] In this example, the plasmid with the enzyme mutant S197V constructed in Example 1 was used as a template, and the methionine (M) at position 144 of the N-terminal amino acid sequence was mutated to cysteine ​​(C). The rest of the preparation method was the same as that in Example 1. The point mutation primers and plasmid construction primers are shown in Table 8.

[0071] Table 8 S197V / M144C mutant gene fragment primers and linearized plasmid primers

[0072]

[0073]

[0074] Example 3 Alcohol dehydrogenase mutant M144C / L196A / S197V

[0075] In this example, the plasmid with the enzyme mutant S197V / M144C constructed in Example 2 was used as a template, and the leucine (L) at position 196 of the N-terminal amino acid sequence was mutated to alanine (A). The rest of the preparation method was the same as that in Example 2. The point mutation primers and plasmid construction primers are shown in Table 9.

[0076] Table 9 Primers for M144C / L196A / S197V mutant gene fragments and linearized plasmid primers

[0077] Primer name Sequence (5'-3') L196A_F ccgacccgcGCTgttatgccgcatc L196A_R gatgcggcataacAGCgcgggtcgg

[0078] Example 4 Comparison of catalytic yields of wild-type alcohol dehydrogenase and its mutants

[0079] The wild-type alcohol dehydrogenase AoSDR1ADH and the whole-cell catalyst containing the alcohol dehydrogenase mutant prepared in Example 1-3 were respectively added to the catalyst OD 600 =60, substrate nootkatol concentration of 100mM, LpNOX final concentration of 5mg / ml, NAD+ final concentration of 80μM and potassium phosphate buffer solution of pH=7.5, react at 400rpm at room temperature (about 25°C) overnight (about 12h), and obtain a reaction solution containing nootkatol after the reaction is completed. Take 500μL of the reaction solution containing nootkatol and add 500μL of ethyl acetate to extract, centrifuge at 1,2000rpm for 1min, and take 300μL of the supernatant for gas chromatography detection.

[0080] Gas phase analysis conditions: Agilent HP-5 chromatographic column. Gas phase program: injection pressure 23psi, flow rate 2.5ml / min, column temperature increased from 60℃ to 240℃ at a rate of 50℃ / min and maintained for 4min, single sample sampling for a total of 7.6min. Nootkatone retention time is 5.24min.

[0081] The test results are as follows Figure 2 The yields of the alcohol dehydrogenase mutants obtained in Examples 1-3 were all higher than those of the wild-type enzyme. The enzyme activity of the alcohol dehydrogenase mutant M144C / L196A / S197V obtained in Example 3 was significantly improved, and the catalytic yield of nootkadone was 4.32 times that of the original enzyme, which was conducive to industrial production.

[0082] Example 5 Catalytic yield of alcohol dehydrogenase mutant M144C / L196A / S197V at different temperatures

[0083] The alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was used as a catalyst, and the same reaction system as in Example 4 was used, and the reaction was carried out at 400 rpm at 15°C, 20°C, 25°C, 30°C, 35°C and 40°C overnight, and a reaction solution containing nootkatone was obtained after the reaction was completed. The detection method was the same as in Example 4.

[0084] The test results are as follows Figure 3 Different temperatures have a significant effect on the catalytic effect of the alcohol dehydrogenase mutant enzyme M144C / L196A / S197V. The catalytic efficiency is better in the range of 15-30℃, and the enzyme activity decreases when the temperature is higher than 30℃.

[0085] Example 6 Catalytic yield of alcohol dehydrogenase mutant M144C / L196A / S197V at different pH

[0086] The alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was used as a catalyst. Based on the reaction system of Example 4, the catalyst OD 600 =40, the substrate nootkatol concentration is 80mM, and the potassium phosphate buffer solution with pH=6.0, 7.0, 8.0, 9.0 and 10.0 is reacted at 400rpm at room temperature overnight to obtain a reaction solution containing nootkatol after the reaction is completed. The detection method is the same as that in Example 4.

[0087] The test results are as follows Figure 4 As shown. The enzyme has good catalytic efficiency in the range of pH = 8.0 to 9.0. Under acidic buffer conditions (pH = 6.0 to 7.0), the catalytic activity of the alcohol dehydrogenase mutant M144C / L196A / S197V is relatively limited. When the buffer pH is greater than 9.0, the enzyme activity gradually weakens.

[0088] Example 7 Catalytic yield of alcohol dehydrogenase mutant M144C / L196A / S197V at different reaction times

[0089] The alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was used as a catalyst, and the same reaction system as in Example 4 was used, and the reaction was carried out at 400 rpm at room temperature for 1 h, 2 h, 4 h, 8 h and 16 h, respectively, to obtain a reaction solution containing nootkatone after the reaction was completed. The detection method was the same as in Example 4.

[0090] The test results are as follows Figure 5The optimal reaction time of the enzyme is 8 to 12 h. As the reaction time increases, the catalytic yield of nootkatone gradually increases and gradually levels off after 12 h.

[0091] Example 8 Catalytic yield of alcohol dehydrogenase mutant M144C / L196A / S197V at different cell concentrations and substrate concentrations

[0092] The alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was used as a catalyst. Based on the reaction system of Example 4, the catalyst OD 600 =20, 40, 60, the substrate nootkatol concentration is 60mM, 80mM, 100mM, and the reaction is carried out at 400rpm overnight at room temperature to obtain a reaction solution containing nootkatol after the reaction is completed. The detection method is the same as that of Example 4.

[0093] The test results are shown in Table 10.

[0094] Table 10 The yield of nootkatone synthesized by alcohol dehydrogenase mutant M144C / L196A / S197V

[0095]

[0096] Example 9 Determination of kinetic parameters of alcohol dehydrogenase mutant M144C / L196A / S197V

[0097] The specific activity of the alcohol dehydrogenase mutant M144C / L196A / S197V in Example 3 was determined for different substrate concentrations (2 mM, 5 mM, 10 mM, and 20 mM of nocardol), and a double reciprocal curve was drawn based on the reciprocal of the specific activity and substrate concentration. The kinetic parameter Km was calculated to be 1.37 × 10 -15 mM, Kcat is 11.136S -1 .

Claims

1. A mutant of alcohol dehydrogenase with high production of nootkatone, characterized in that: The amino acid sequence of the alcohol dehydrogenase mutant is obtained by mutation of the sequence shown in SEQ ID NO.1, and the mutation is at least one of S197V, M144C, and L196A.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the alcohol dehydrogenase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO.

2.

4. A carrier, characterized in that Comprising the nucleotide sequence of claim 3.

5. The carrier according to claim 4, characterized in that The vector includes a cloning vector or an expression vector.

6. A recombinant cell, characterized in that The recombinant cell comprises the vector of claim 4.

7. The method for preparing the alcohol dehydrogenase mutant according to claim 1, characterized in that: The steps include: (1) Using the plasmid carrying the alcohol dehydrogenase gene as a template, a PCR reaction was performed using a point mutation primer, and the mutant gene fragment and linearized plasmid were obtained after purification; (2) Connect the mutant gene fragment to the linearized plasmid to construct an expression vector, and then transfer the expression vector into the host bacteria for inducing expression; (3) Collect the host bacteria expressing the alcohol dehydrogenase mutant, resuspend the bacteria, break the cells, and centrifuge to obtain the supernatant to obtain the crude enzyme solution containing the alcohol dehydrogenase mutant.

8. A product, characterized in that The product comprises the alcohol dehydrogenase mutant according to claim 1 or the nucleic acid molecule according to any one of claims 2 to 3 or the vector according to claim 4 or the recombinant cell according to claim 6.

9. Use of the product according to claim 8 in the catalytic preparation of nootkatone.

10. The use according to claim 9, characterized in that: The catalytic substrate is nootkaol.

Images