A high-yield theaspirone alcohol dehydrogenase mutant and its application in one-pot method

Abstract

The application discloses an alcohol dehydrogenase mutant with high tea ketone production and application of the mutant in one-pot method, and the mutant is A182L or A182L / V214T. The alcohol dehydrogenase mutant has high catalytic activity on 4-hydroxyisophorone, the yield of tea ketone catalyzed by the mutant A182L / V214T can reach 97%, which is 3.73 times of the original enzyme; the alcohol dehydrogenase mutant has high catalytic activity on synthesis of tea ketone from isophorone in one-pot, the mutant A182L / V214T and cytochrome P450 BM3 The single oxygenase mutant is used in combination to directly obtain tea ketone from isophorone, and the yield of tea ketone can reach 92%; the alcohol dehydrogenase mutant has simple production process, mild reaction condition, and environment-friendly production process in the synthesis of tea ketone, and is favorable for industrialized production of tea ketone, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to an alcohol dehydrogenase and its application, and more particularly to a high-yield tea aroma ketone alcohol dehydrogenase mutant and its application in a one-pot process. Background Technology

[0002] Alcohol dehydrogenases (ADHs) are an important class of enzymes that rely on pyridine nucleotide cofactors (NADH / NADPH) for redox reactions, catalyzing the reversible conversion between alcohols and carbonyl compounds. In the fields of fine chemicals, pharmaceutical synthesis, and flavor compound manufacturing, ADHs are widely used for the green preparation of chiral alcohols and carbonyl compounds due to their excellent recognition and catalytic ability of chiral carbon centers.

[0003] Ketoisophorone is a sesquiterpene compound with floral and fruity aroma characteristics and has important applications in the tea, tobacco, and flavor and fragrance industries. One synthetic route involves using 4-hydroxyisophorone as a substrate and catalyzing a dehydrogenation reaction with alcohol dehydrogenases. However, naturally occurring alcohol dehydrogenases generally exhibit low catalytic efficiency and limited yields for this type of substrate, making it difficult to meet the demands of industrial production. Furthermore, different enzyme sources show significant differences in substrate compatibility, stability, and catalytic activity, resulting in significant shortcomings in reaction rate, yield, and process stability of existing methods.

[0004] Derived from Burkholderia ( Ralstonia alcohol dehydrogenase ( Rs ADH (Alpha-hydroxyhydroxylamine) is a class of oxidoreductases with potential applications, capable of catalyzing the conversion of various cyclic or aliphatic alcohols. However, its natural form, when using 4-hydroxyisophorone as a substrate, still suffers from insufficient catalytic activity and low yield of tea aroma ketones. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an alcohol dehydrogenase mutant that can produce high levels of tea aroma ketones, and to provide the nucleic acid encoding the mutant, the recombinant vector containing the mutant, the recombinant cells or products, and the preparation method and application thereof.

[0006] Technical solution: The high-yield tea aroma ketone alcohol dehydrogenase mutant is obtained by amino acid mutation of the sequence shown in SEQ ID NO.1, and the mutation is A182L or A182L / V214T.

[0007] The wild-type alcohol dehydrogenase used in this invention is derived from Burkholderia (…). Ralstonia ), can use 4-hydroxyisophorone as a substrate and nicotinamide adenine dinucleotide oxidase ( LpNOX) as a coenzyme, catalyzing the synthesis of theaflavone. The amino acid sequence is shown as SEQ ID NO. 1. The present application realizes the change of protein structure and function by site-directed mutagenesis of the amino acids in the active center of the wild-type alcohol dehydrogenase. Through screening, it is found that single mutation or combined mutation of A182L and V214T can obtain alcohol dehydrogenase mutants with high activity, which can significantly increase the yield of theaflavone prepared from 4-hydroxyisophorone. The alcohol dehydrogenase mutant is preferably A182L and A182L / V214T, and the amino acid sequences thereof correspond to SEQ ID NO. 2-3, respectively.

[0008] The nucleic acid encodes the alcohol dehydrogenase mutant.

[0009] The recombinant vector contains the nucleic acid. The recombinant vector can maintain its replication or autonomous replication ability in various host cells such as prokaryotic and / or eukaryotic cells, thereby amplifying or expressing the nucleic acid. The recombinant vector can be various vectors in the art, such as various plasmids, bacteriophages or viral vectors, etc.; preferably PET system expression vectors, such as pET22b(+) plasmid.

[0010] The recombinant cell contains the recombinant vector. Preferably, Escherichia coli is used as the recombinant cell, such as Escherichia coli C43 or Escherichia coli BL21.

[0011] The preparation method is as follows: using whole plasmid PCR to mutate the wild-type alcohol dehydrogenase gene, obtaining the target mutant gene and constructing the recombinant vector; then transforming the recombinant vector into competent cells to obtain the recombinant cell; and inducing the expression of the alcohol dehydrogenase mutant. After the expression is completed, the cells are preferably collected, and the collected wet bacteria are used as whole-cell catalysts; or the cells are broken, and the crude enzyme solution or pure enzyme is collected. Preferably, the collected cells or pure enzyme are prepared into immobilized cells or immobilized enzyme by immobilization technology.

[0012] The product contains the alcohol dehydrogenase mutant, or the nucleic acid, or the recombinant vector, or the recombinant cell.

[0013] The application is: catalyzing the preparation of theaflavone with 4-hydroxyisophorone as the substrate, or preparing theaflavone in one-pot method. The reaction conditions of the catalysis include reacting at a temperature of 20-40 ℃ and a pH of 5.0-10.0 for 12-20 h. The preferred temperature is 30 ℃, the pH is 9.0, and the reaction time is 16 h. The one-pot method is that the alcohol dehydrogenase mutant is used in combination with cytochrome P450 BM3 monooxygenase. BM3 The monooxygenase has the function of catalyzing isophorone to generate 4-hydroxyisophorone; and the cytochrome P450 BM3 monooxygenase is preferably cytochrome P450BM3 Monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F, the amino acid sequence of which is shown as SEQ ID NO. 9.

[0014] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0015] 1. The alcohol dehydrogenase mutant provided by the present application has high catalytic activity for 4-hydroxyisophorone, and the yield of tea aroma ketone catalyzed by the mutant A182L / V214T can reach 97% (40mM), which is 3.73 times that of the original enzyme;

[0016] 2. The alcohol dehydrogenase mutant provided by the present application has high catalytic activity for one-pot synthesis of tea aroma ketone from isophorone, and the mutant A182L / V214T and cytochrome P450 BM3 The yield of tea aroma ketone obtained by direct oxidation of isophorone by the combination of the monooxygenase mutant can reach 92% (25mM), which is superior to the existing production level of tea aroma ketone biosynthesized from isophorone;

[0017] 3. The alcohol dehydrogenase mutant provided by the present application has simple production process, mild reaction conditions, and environmentally friendly production process for catalytic synthesis of tea aroma ketone, which is conducive to the industrialized production of tea aroma ketone and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a schematic diagram of the reaction of alcohol dehydrogenase catalyzing 4-hydroxyisophorone to synthesize tea aroma ketone;

[0019] Figure 2 is a catalytic yield diagram of wild-type alcohol dehydrogenase and its mutants A182L, A182L / V214T;

[0020] Figure 3 is a catalytic yield diagram of alcohol dehydrogenase mutant A182L / V214T at different pH values;

[0021] Figure 4 is a catalytic yield diagram of alcohol dehydrogenase mutant A182L / V214T at different temperatures;

[0022] Figure 5 is a schematic diagram of one-pot synthesis of tea aroma ketone from isophorone by the combination of alcohol dehydrogenase and cytochrome P450 BM3 Monooxygenase. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be further described below with reference to the accompanying drawings.

[0024] The amino acid sequence of the wild-type alcohol dehydrogenase used in the examples is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 4. The wild-type alcohol dehydrogenase can catalyze the synthesis of theaflavone using 4-hydroxyisophorone as a substrate, coenzyme NOX, and the reaction process is shown in Lp NOX as a coenzyme, and the reaction process is shown in Figure 1

[0025] Obtaining route of the materials used in the examples:

[0026] Strains and plasmids

[0027] The plasmid pET-22b (+), E. coli BL21 (DE3), E. coli DH5α are all preserved by the applicant and are all obtained from commercial channels. The wild-type alcohol dehydrogenase is an alcohol dehydrogenase from Burkholderia sp. Ralstonia ADH, Rs NOX is from Lactobacillus pentosus, and the coding genes of the two enzymes are synthesized by Jinweizhi (Suzhou) Company. The site-directed mutation sequence of the enzyme is obtained by PCR method and is constructed by the applicant. Lp Lactobacillus pentosus Reagents and culture medium

[0028] The DNA gel recovery kit and the plasmid extraction kit are both from Shenguo Bioengineering (Shanghai) Co., Ltd.

[0029] The composition of the LB liquid culture medium is tryptone 10 g / L, yeast powder 5 g / L, and sodium chloride 10 g / L.

[0030] The composition of the LB solid culture medium is tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, and agar powder 15 g / L.

[0031] The composition of the FB liquid culture medium is dipotassium hydrogen phosphate 14.66 g / L, sodium dihydrogen phosphate 2.48 g / L, ammonium sulfate 2.5 g / L, anhydrous sodium sulfate 2 g / L, trisodium citrate dihydrate 1.2 g / L, ammonium chloride 0.5 g / L, and glycerol 5 ml / L.

[0032] The composition of the TB liquid culture medium is yeast powder 12 g / L, tryptone 12 g / L, glycerol 4 ml / L, dipotassium hydrogen phosphate 12.5 g / L, and potassium dihydrogen phosphate 2.3 g / L.

[0033] Obtaining of NOX powder: the pET-22b plasmid containing the NOX gene is transformed into

[0034] BL21 (DE3) and DH5α. Lp Lp E. coli ​​​BL21(DE3) host bacteria, and coated on the surface of LB solid medium containing 100 μg / mL ampicillin, 37 ℃ for 8 h. Pick single E. coli colonies inoculated in 3 mL of LB liquid medium containing 100 μg / mL ampicillin, 37 ℃ overnight culture as seed liquid. The seed liquid was inoculated into a 250 mL conical flask containing 50 mL TB medium at a 5% inoculation amount, and cultured at 37 ℃, 200 rpm. When the culture reached 8 h, IPTG was added to a final concentration of 0.5 mM, and the culture temperature was set to 18 ℃, and the culture was continued for 16 h. The fermentation broth was centrifuged, and the bacterial cells were collected and resuspended with pH 7.5 200 mM phosphate buffer. The resulting crude enzyme solution was sonicated and centrifuged, and the supernatant was collected and freeze-dried to obtain Lp NOX powder.

[0035] ls The preparation method of the GDH powder is as follows Lp NOX. ls The GDH is a glucose dehydrogenase (GDH) from Gluconobacter oxydans (ATCC 621H). Gluconobacter oxydans ).

[0036] Example 1

[0037] This example provides an alcohol dehydrogenase mutant A182L, which is obtained by mutating alanine (A) at the N-terminal 182nd position of the sequence shown in SEQ ID NO. 1 to leucine (L). The specific preparation method is as follows:

[0038] 1. Constructing a nucleic acid encoding the alcohol dehydrogenase mutant A182L and an expression vector encoding the above nucleic acid

[0039] The pET-22b plasmid with wild-type alcohol dehydrogenase Rs ADH as a template, the full plasmid PCR method is used for gene mutation to obtain the target mutant gene (i.e. the nucleic acid encoding the alcohol dehydrogenase mutant A182L), and construct on the pET-22b plasmid (i.e. the expression vector encoding the above nucleic acid).

[0040] Among them, the PCR primer of the nucleic acid encoding the alcohol dehydrogenase mutant A182L is SEQ ID NO. 5 and SEQ ID NO. 6.

[0041] The PCR reaction system of the full plasmid PCR is shown in Table 1.

[0042] Table 1

[0043] Components Volume 10x Buffer for KOD-Plus- 2.5 μL 2 mM dNTP 2.5 μL 25 mM MgSO4 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75 μL 10 pmol / μL Reverse Primer 0.75 μL DNA template < 100 ng KOD-Plus- 1 μL ddH2O up to 25 μL

[0044] The PCR reaction program (cycle 32 times) of the full plasmid PCR is shown in Table 2.

[0045] Table 2

[0046] Reaction temperature Time 95 ℃ 3 min 95 ℃ 20 s 57 ℃ 10 s 70 ℃ 4 min 12 ℃ 10 min .

[0047] After the PCR amplification of the target fragment, the amplification product was detected by 1% agarose gel electrophoresis, and the results showed that the amplification product was a single band with a size of about 6000 bp. The amplification product was purified and recovered by using a DNA gel recovery and purification kit.

[0048] Finally, the pET22b plasmid of the alcohol dehydrogenase mutant A182L was obtained, which was the expression vector of the alcohol dehydrogenase mutant A182L.

[0049] 2. Constructing a host cell transfected with the expression vector.

[0050] The purified gene fragment was transformed into the E. coli DH5α strain to construct a recombinant mutant expression strain E. coli DH5α / pET22b- Rs ADH.

[0051] Specifically, the purified gene fragment was digested with EasyCut endonuclease-DpnI to remove the template, and then recombined with recombinase. The recombination product was transformed into E. coli DH5α competent cells, spread on the surface of LB solid medium containing 100 mg / mL ampicillin, and incubated at 37°C for 16 h. Single colonies were picked and inoculated in LB liquid medium containing 100 mg / mL ampicillin, and incubated at 37°C, 220 rpm for 16 h. After incubation, a portion of the bacterial solution was added to sterile glycerol to a final glycerol concentration of 15-20%, numbered, and stored at 80°C for future use. The recombinant mutant plasmid clone strain E. coli DH5α / pET22b- Rs ADH; a portion of the bacterial solution was centrifuged at 8,000 rpm for 3 min and the cells were collected. The plasmid was extracted from E. coli DH5α / pET22b- Rs ADH using a high-purity plasmid extraction kit, and the correctness of the mutant site was verified by sequencing.

[0052] Finally, the recombinant mutant plasmid clone strain of the alcohol dehydrogenase mutant A182L and the corresponding recombinant mutant plasmid, i.e., the host cell transfected with the expression vector of the alcohol dehydrogenase mutant A182L, were obtained.

[0053] 3. Constructing a recombinant mutant protein expression strain to obtain a crude enzyme solution containing the alcohol dehydrogenase mutant A182L.

[0054] The sequencing successful pET22b plasmid was transformed into E. coli BL21(DE3) strain, E. coli BL21(DE3) is an expression host (i.e. host cell), and a recombinant mutant protein expression strain is constructed E. coli BL21(DE3) / pET22b- Rs ADH.

[0055] Specifically, the successfully constructed recombinant mutant plasmid was transformed into E. coli BL21(DE3) competent cells, and then coated on a plate containing 100 mg / mL ampicillin at a final concentration. Single colonies were picked and inoculated into a culture tube containing 10 mL of LB liquid medium containing 100 mg / mL ampicillin, and then cultured at 37°C and 220 rpm for 16 h. The seed liquid was obtained to obtain a recombinant mutant protein expression strain E. coli BL21(DE3) / pET22b- Rs ADH. The seed liquid was inoculated into a 50 mL TB medium containing 100 mg / mL ampicillin at a 5% inoculation amount, and then cultured at 37°C and 200 rpm. When the culture reached 8 h, IPTG was added to a final concentration of 0.5 mM, and the culture temperature was set to 20°C. The culture was continued for 16 h. The fermentation broth was centrifuged, the bacterial cells were collected, and then resuspended with 100 mM Tris-hydrochloric acid buffer at pH 9.0 to obtain a whole-cell catalyst containing alcohol dehydrogenase mutant A182L (i.e. wet bacterial cells).

[0056] Example 2

[0057] This example provides an alcohol dehydrogenase mutant A182L / V214T. The valine (V) at the 214th position of the N-terminal amino acid sequence of the plasmid with the enzyme mutant A182L constructed in Example 1 was mutated to threonine (T). The remaining preparation method is the same as that of Example 1. Among them, the PCR primers for encoding the nucleic acid of the alcohol dehydrogenase mutant V214T are SEQ ID NO. 7 and SEQ ID NO. 8.

[0058] Example 3

[0059] This example compares the yield of tea aroma ketone generated by wild-type alcohol dehydrogenase and the mutants prepared in Examples 1-2.

[0060] The wild-type alcohol dehydrogenase ADH, the whole-cell catalysts containing alcohol dehydrogenase mutants prepared in Examples 1-2 were respectively used to catalyze 4-hydroxyisophorone to generate tea aroma ketone. Rs ADH, the whole-cell catalysts containing alcohol dehydrogenase mutants prepared in Examples 1-2 were respectively used to catalyze 4-hydroxyisophorone to generate tea aroma ketone. 600 =60, the substrate 4-hydroxyisophorone concentration was 40 mM, LpNOX final concentration of 5 mg / ml, NAD + The reaction system was composed of Tris-hydrochloric acid buffer solution with a final concentration of 0.8 mM and pH=9.0, and the reaction was carried out at 30 °C overnight (about 16 h) at 400 rpm. After the reaction, the reaction solution containing theamyrin was obtained. 500 µL of the reaction solution containing theamyrin was extracted with 500 µL of ethyl acetate, centrifuged at 1,2000 rpm for 1 min, and 300 µL of the supernatant was detected by gas chromatography.

[0061] Gas analysis conditions: Agilent CYCCOSIL-B chromatographic column. Gas program: injection pressure 23 psi, flow rate 2.5 ml / min, column temperature from 60 °C for 1 min, then increased to 160 °C at a rate of 20 °C / min, then kept for 6 min, then increased to 240 °C at a rate of 20 °C / min, then kept for 2 min, and a single sample was sampled for a total of 18 min.

[0062] The test results are shown in Table 1. Figure 2 The yield of the alcohol dehydrogenase mutants obtained in Examples 1-2 was higher than that of the wild-type enzyme. The enzyme activity of the alcohol dehydrogenase mutant A182L / V214T obtained in Example 2 was significantly improved, the catalytic yield of theamyrin reached 97%, which was 3.73 times that of the original enzyme, and was conducive to the realization of industrial production.

[0063] Example 4

[0064] In this example, the catalytic effect of the alcohol dehydrogenase mutant A182L / V214T with the best yield in Example 3 at different pH values was explored.

[0065] The alcohol dehydrogenase mutant A182L / V214T was used as a catalyst, and on the basis of the reaction system of Example 3, the concentration of the substrate 4-hydroxyisophorone was adjusted to 20 mM, and the citric acid-sodium citrate buffer solution with pH=5.0 and 6.0 and a concentration of 0.1 M, the potassium phosphate dibasic-potassium phosphate monobasic buffer solution with pH=7.0, 7.5 and 8.0 and a concentration of 0.1 M, and the glycine-sodium hydroxide buffer solution with pH=9.0 and 10.0 and a concentration of 0.1 M were used, respectively, and the reaction was carried out at 30 °C overnight (about 16 h) at 400 rpm. After the reaction, the reaction solution containing theamyrin was obtained. The detection method was the same as that of Example 3.

[0066] The test results are shown in Table 2. Figure 3 The alcohol dehydrogenase mutant A182L / V214T had good catalytic efficiency in the pH range of 7.0-9.0. In the acidic buffer solution (pH=5.0-7.0), the catalytic activity of the enzyme was limited.

[0067] Example 5

[0068] This example investigates the catalytic effect of the alcohol dehydrogenase mutant A182L / V214T, which yielded the best results in Example 3, at different temperatures.

[0069] Using the alcohol dehydrogenase mutant A182L / V214T as a catalyst, the reaction system of Example 3 was further modified by reacting overnight (approximately 16 h) at 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C at 400 rpm. The resulting reaction solution contained tea aroma ketones was obtained after the reaction. The detection method was the same as in Example 3.

[0070] Test results are as follows Figure 4 As shown, different temperatures significantly affect the catalytic activity of the alcohol dehydrogenase mutant A182L / V214T. The catalytic efficiency is better in the range of 25–30 °C, while the enzyme activity decreases significantly below 25 °C. Above 30 °C, it is speculated that the yield gradually decreases as the product overflows upon heating.

[0071] Example 6

[0072] This embodiment is based on Figure 5 The cascade reaction shown compares three methods for preparing tea aroma ketones from isophorone: the one-pot method, the equivalent glucose method, and the whole-cell GDH catalytic method. Cytochrome P450 was used. BM3 monooxygenase (P450) BM3 The mutant of this enzyme is A74G / V78A / F87V / L188Q / T327A / A328F, and its amino acid sequence is shown in SEQ ID NO.9.

[0073] One-pot method: With a substrate isophorone concentration of 25 mM and the whole-cell catalyst OD of the alcohol dehydrogenase mutant A182L / V214... 600 =60, Cytochrome P450 BM3 The monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F had a final catalyst concentration of 4 µM and NADP in its lysis buffer. + In a reaction system consisting of a phosphate buffer solution with a final concentration of 0.8 mM and pH=7.5, the reaction was carried out overnight at 30 °C at 400 rpm (approximately 16 h). After the reaction was completed, a reaction solution containing tea aroma ketones was obtained.

[0074] Equivalent glucose method: In the cytochrome P450 BM3 The monooxygenase mutant lysis buffer had a final catalyst concentration of 4 µM, a substrate isophorone concentration of 25 mM, and a coenzyme concentration of [missing information]. ls The final concentration of GDH was 5 mg / ml, and the cofactor NADP was... +A reaction system consisting of a potassium phosphate buffer solution with a final concentration of 0.8 mM, a final glucose concentration of 25 mM, and a pH of 7.5 was used. The reaction was carried out overnight (approximately 12 h) at 30°C with 400 rpm. After the reaction, a reaction solution containing 4-hydroxyisophorone was obtained. Then, a final concentration of OD was added to this reaction solution. 600 =60 alcohol dehydrogenase mutant A182L / V214 whole-cell catalyst, final concentration 5 mg / ml Lp NOX, NAD at a final concentration of 0.8 mM + The reaction was carried out overnight at 30 °C at 400 rpm (approximately 16 h), and a reaction solution containing tea ketones was obtained after the reaction was completed.

[0075] Whole-cell GDH catalysis: In the cytochrome P450 BM3 The monooxygenase mutant lysis buffer had a final catalyst concentration of 4 µM, a substrate isophorone concentration of 25 mM, and a coenzyme concentration of [missing information]. ls The final concentration of GDH was 5 mg / ml, and the cofactor NADP was... + A reaction system consisting of a potassium phosphate buffer solution with a final concentration of 0.8 mM, a final glucose concentration of 50 mM, and a pH of 7.5 was used. The reaction was carried out overnight (approximately 12 h) at 30 °C with 400 rpm. After the reaction, a reaction solution containing 4-hydroxyisophorone was obtained. This reaction solution was centrifuged at 12000 rpm for 2 min to remove the whole-cell GDH catalyst, and the supernatant containing 4-hydroxyisophorone was collected. Then, a final concentration of OD was added to this supernatant. 600 =60 alcohol dehydrogenase mutant A182L / V214 whole-cell catalyst, final concentration 5 mg / ml Lp NOX, NAD at a final concentration of 0.8 mM + The reaction was carried out overnight at 30 °C at 400 rpm (approximately 16 h), and a reaction solution containing tea ketones was obtained after the reaction was completed.

[0076] The detection method is the same as that of Example 3. Among them, the one-pot method can directly oxidize isophorone to obtain thearubinone, and the yield can reach 92% (25mM), which is better than the existing production level of biosynthesis of thearubinone from isophorone, and is the best embodiment; and when only the equivalent of glucose is put in to accurately regulate the coenzyme regeneration, the GDH-mediated coenzyme cycle system just completes the reaction from isophorone to 4-hydroxyisophorone, limits GDH to participate in the oxidation to thearubinone in the second step, and reduces the competition of GDH compared with ADH, but the yield of thearubinone is less than 20%, which is speculated to be that the non-excess glucose limits the reaction efficiency of the first step; in the whole-cell GDH catalysis method, even if the centrifugation eliminates the influence of GDH on the second step, the yield is also not ideal, which is also less than 20%, which is speculated to be that part of the substrate is removed during centrifugation, so as to cause loss.

Claims

1. A mutant of an alcohol dehydrogenase that produces tea aroma ketones, characterized in that, The sequence shown in SEQ ID NO.1 was obtained by amino acid mutation, wherein the mutation is A182L or A182L / V214T, and the amino acid sequences correspond to SEQ ID NO.2-3 respectively.

2. A nucleic acid, characterized in that, Encodes the alcohol dehydrogenase mutant of claim 1.

3. A recombinant vector, characterized in that, It includes the nucleic acid described in claim 2.

4. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 3.

5. A method for preparing the alcohol dehydrogenase mutant according to claim 1, characterized in that, The wild-type alcohol dehydrogenase gene was mutated using whole plasmid PCR to obtain the target mutant gene and construct a recombinant vector; the recombinant vector was then transformed into competent cells to obtain recombinant cells; and the expression of the alcohol dehydrogenase mutant was induced.

6. A product producing tea aroma ketones, characterized in that, The product comprises the alcohol dehydrogenase mutant of claim 1, or the nucleic acid of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4.

7. The use of the product of claim 6 in the catalytic preparation of tea aroma ketone using 4-hydroxyisophorone as a substrate.

8. The application of the product of claim 6 in a one-pot process, characterized in that, The one-pot method involves using an alcohol dehydrogenase mutant and cytochrome P450. BM3 The monooxygenase was used in combination to synthesize tea aroma ketone in a one-pot catalysis by isophorone.

9. The application according to claim 8, characterized in that, The cytochrome P450 BM3 Monooxygenase is cytochrome P450 BM3 The monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F has the amino acid sequence shown in SEQ ID NO. 9.

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