Alcohol dehydrogenase and application thereof in preparation of (1S, 2S)-2, 6-dimethyl-1-hydroxyindan

Abstract

The invention discloses an alcohol dehydrogenase and application thereof in preparation of (1S, 2S)-2, 6-dimethyl-1-hydroxyindan, an amino acid sequence of the alcohol dehydrogenase has at least 95% sequence identity with an amino acid sequence shown as SEQ ID NO: 1, and compared with the amino acid sequence shown as SEQ ID NO: 1, the alcohol dehydrogenase contains amino acid residue difference of a P190 site and / or a D198 site. The conversion rate of the alcohol dehydrogenase substrate is relatively high, the stereoselectivity of the (1S, 2S)-2, 6-dimethyl-1-hydroxyindan is also relatively high, an expensive chiral catalyst does not need to be added, the cost is relatively low, and the method is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis, specifically to an alcohol dehydrogenase and its application in the preparation of (1S,2S)-2,6-dimethyl-1-hydroxyindane. Background Technology

[0002] Indaziflam is a new type of herbicide developed by Bayer Crop Science. It is mainly used to control annual weeds (such as crabgrass, goosegrass, and Kentucky bluegrass), 65 other grass weeds, and broadleaf weeds.

[0003] The structural formula of triazine indoxamine is shown in formula A below:

[0004]

[0005] (1S,2S)-2,6-dimethyl-1-hydroxyindane is one of the important intermediates of triazine indoxachlor. The literature Tetrahedron 2007, 63(29), 6755-6763 reported an asymmetric hydrogenation reaction of 2,6-dimethyl-1-indane as a starting material in the presence of a rhodium or ruthenium-based chiral catalyst to obtain (1S,2S)-2,6-dimethyl-1-hydroxyindane. However, this method is complex and the chiral catalyst is relatively expensive, making it unsuitable for industrial production that requires the addition of large amounts of chiral catalysts.

[0006] Therefore, it is crucial to find a low-cost, industrially viable, and efficient method for preparing (1S,2S)-2,6-dimethyl-1-hydroxyindane. Summary of the Invention

[0007] The technical problem to be solved by the present invention is that the chiral catalysts used in the current methods for preparing the triazine indoxamine intermediate (1S,2S)-2,6-dimethyl-1-hydroxyindoxamine are expensive, resulting in high costs and unsuitability for industrial production. Therefore, the present invention provides an alcohol dehydrogenase and its application in the preparation of (1S,2S)-2,6-dimethyl-1-hydroxyindoxamine.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0009] A first aspect of the present invention provides an alcohol dehydrogenase having an amino acid sequence that is at least 95% sequence identical to the amino acid sequence shown in SEQ ID NO:1, and containing amino acid residue differences at positions P190 and / or D198 compared to the amino acid sequence shown in SEQ ID NO:1, wherein positions 190 and 198 refer to the amino acid residue numbers in SEQ ID NO:1.

[0010] In some embodiments, the P at position P190 is replaced with a sterically hindered amino acid; preferred amino acid residue differences include P190G, P190A, or P190C; and the D at position D198 is replaced with a sterically hindered or neutral amino acid; preferred amino acid residue differences include D198N, D198G, or D198A.

[0011] In this invention, the amino acids with low steric hindrance are selected from: glycine (G), alanine (A), serine (S), valine (V), threonine (T), and cysteine ​​(C).

[0012] In some specific implementations, the sterically hindered amino acid is selected from glycine (G), alanine (A), or cysteine ​​(C).

[0013] In this invention, the neutral amino acid is selected from: alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), cysteine ​​(C), or lysine (K).

[0014] In this invention, the sterically hindered or neutral amino acids are selected from: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), cysteine ​​(C), or lysine (K).

[0015] In some embodiments, the amino acid residue differences also include one or more of the following groups:

[0016] i) T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F;

[0017] ii) P188A or P188G.

[0018] In some embodiments, the amino acid residue differences are selected from the group consisting of:

[0019] (A)P190G, and T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F;

[0020] (B)P190A, and T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F;

[0021] (C)P190C, and T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F;

[0022] (D)P190G, and D198N, D198G or D198A;

[0023] (E)P190A, and D198N, D198G or D198A;

[0024] (F)P190C, and D198N, D198G or D198A;

[0025] Among them, one of T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H and T152F exists; one of D198N, D198G and D198A exists.

[0026] In some specific embodiments, the amino acid residue differences are selected from the group consisting of:

[0027] (1)P190G;

[0028] (2) P190G, T152E and D198N;

[0029] (3) P190G, T152E, D198N and P188A;

[0030] (4) P190G, T152A and D198G;

[0031] (5) P190G, T152V and D198A;

[0032] (6) P190G, T152I and D198A;

[0033] (7) P190G, T152G and D198N;

[0034] (8) P190G and T152R;

[0035] (9) P190G, T152E and D198N;

[0036] (10) P190G, T152S and D198A;

[0037] (11) T152D and P190G;

[0038] (12) T152I and P190G;

[0039] (13) T152H and P190G;

[0040] (14) T152F and P190G;

[0041] (15) T152D and P190A;

[0042] (16) T152I and P190A;

[0043] (17) T152H and P190A;

[0044] (18) T152F and P190A;

[0045] (19) T152D and P190C;

[0046] (20)T152I and P190C;

[0047] (21) T152H and P190C;

[0048] (22) T152F and P190C;

[0049] (23)D198A;

[0050] (24)D198N;

[0051] (25)P190C;

[0052] (26)P190A;

[0053] (27) P190G, T152E, D198N and P188G.

[0054] A second aspect of the present invention provides an isolated nucleic acid that encodes an alcohol dehydrogenase as described in any one of the first aspects of the present invention.

[0055] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect of the present invention, wherein the backbone of the recombinant expression vector is preferably pET28a or pET21a.

[0056] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect of the present invention, or a recombinant expression vector as described in the third aspect of the present invention.

[0057] In some embodiments, the host cells used in the construction of the transformant are Escherichia coli or Bacillus subtilis.

[0058] In some specific implementations, the Escherichia coli is Escherichia coli BL21(DE3).

[0059] The fifth aspect of the present invention provides a method for preparing an alcohol dehydrogenase, comprising culturing a transformant as described in the fourth aspect of the present invention to obtain a fermentation product;

[0060] In some specific embodiments, the culture medium used for the culture is selected from LB liquid medium or TB liquid medium.

[0061] In some specific implementations, the culture conditions are: shaking culture at a temperature of 37±1℃;

[0062] In some specific embodiments, the method further includes the step of purifying the fermentation product to obtain the reductase.

[0063] A sixth aspect of the present invention provides an enzyme combination comprising: one or more alcohol dehydrogenases as described in the first aspect of the present invention, and a glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO:3.

[0064] In this invention, the enzyme combination is a combination of separately placed enzymes or a mixture of enzymes placed together.

[0065] The seventh aspect of the present invention provides the use of alcohol dehydrogenases as described in any of the first aspects of the present invention, nucleic acids as described in the second aspect of the present invention, recombinant expression vectors as described in the third aspect of the present invention, transformants as described in the fourth aspect of the present invention, or enzyme combinations as described in the sixth aspect of the present invention in the preparation of (1S,2S)-2,6-dimethyl-1-hydroxyindene.

[0066] In some specific embodiments, the preparation of (1S,2S)-2,6-dimethyl-1-hydroxyindene uses 2,6-dimethyl-1-indene ketone as a substrate.

[0067] An eighth aspect of the present invention provides a method for preparing (1S,2S)-2,6-dimethyl-1-hydroxyindene, the method comprising:

[0068] (1S,2S)-2,6-dimethyl-1-hydroxyindane was prepared by contacting and reacting an alcohol dehydrogenase as described in any of the first aspects of the present invention with a substrate;

[0069] In some specific embodiments, the substrate is 2,6-dimethyl-1-indanone.

[0070] In some specific implementations, the reaction also includes the use of a coenzyme.

[0071] In some specific implementations, the reaction further includes a reaction that regenerates the coenzyme.

[0072] In some embodiments, the coenzyme is NADH, NADPH, or NAD. + and NADP + Any one or both of them;

[0073] In some embodiments, the reaction that regenerates the coenzyme uses NAD. + and NADP + Any one or two of the following, a hydrogen donor and a coenzyme regenerating enzyme; wherein the hydrogen donor and the coenzyme regenerating enzyme are selected from one or more of the following:

[0074] (1) Glucose and glucose dehydrogenase;

[0075] (2) Formic acid or formate and formic acid dehydrogenase;

[0076] (3) Isopropanol and alcohol dehydrogenase;

[0077] In some specific embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:3.

[0078] In some embodiments, the alcohol dehydrogenase or coenzyme regeneration enzyme is used in the form of liquid enzyme, solid enzyme powder, immobilized enzyme, wet cells or bacterial powder.

[0079] In some embodiments, the concentration of 2,6-dimethyl-1-indanone added in the reaction is 1-500 mg / mL.

[0080] In some specific embodiments, the concentration of 2,6-dimethyl-1-indanone added in the reaction is 10-200 mg / mL.

[0081] In some embodiments, the molar ratio of the hydrogen donor to the 2,6-dimethyl-1-indanone is 0.5:1 to 5:1.

[0082] In some specific embodiments, the molar ratio of the hydrogen donor to the 2,6-dimethyl-1-indanone is 1:1 to 3:1;

[0083] In some implementations, the "NAD" + and / or NADP + "The mass ratio of the 2,6-dimethyl-1-indanone to the 2,6-dimethyl-1-indanone is 1:1000 to 500:1000."

[0084] In some specific implementations, the NAD + and / or NADP +The mass ratio of the 2,6-dimethyl-1-indanone to the 2,6-dimethyl-1-indanone is 5:1000 or 371:1000;

[0085] In some implementations, the pH of the reaction is 6.5-8.5.

[0086] In some specific implementations, the pH of the reaction is 7.0-8.0.

[0087] In some implementations, the reaction temperature is 25-45°C.

[0088] In some specific implementations, the reaction temperature is 30-40°C.

[0089] In some embodiments, the buffer solution for the reaction is an aqueous phosphate solution with a concentration of 10 mM to 500 mM.

[0090] In some specific embodiments, the buffer solution for the reaction is an aqueous phosphate solution with a concentration of 10 mM to 200 mM.

[0091] In some embodiments, when the alcohol dehydrogenase or the enzyme combination is used as a liquid enzyme, the ratio of the mass of the wet bacterial cells to the mass of the 2,6-dimethyl-1-indanone added in the reaction system is (0.1-2) g:1 g, calculated based on the mass of the wet bacterial cells that produce the reductase.

[0092] In some specific embodiments, when the alcohol dehydrogenase, the transformant, or the enzyme combination is used as a liquid enzyme, the mass ratio of the wet bacterial cells to the 2,6-dimethyl-1-indanone added in the reaction system is 1 g:1 g or 2 g:1 g, calculated based on the mass of the wet bacterial cells that produce the reductase.

[0093] In some embodiments, the coenzyme regenerating enzyme is expressed in a mass ratio of (0.05-1)g:1g based on the mass of the wet bacterial cells used to the hydrogen donor.

[0094] In some specific implementations, the mass ratio of the coenzyme regenerating enzyme to the hydrogen donor, calculated based on the mass of the wet bacterial cells used, is 0.2 g:1 g or 0.6 g:1 g;

[0095] In some implementations, the method also uses a co-solvent.

[0096] In some specific embodiments, the co-solvent is DMSO or isopropanol.

[0097] A ninth aspect of the present invention provides a reaction end product system for the catalytic synthesis of (1S,2S)-2,6-dimethyl-1-hydroxyindane using 2,6-dimethyl-1-indane as a substrate, the reaction end product system comprising:

[0098] (1S,2S)-2,6-dimethyl-1-hydroxyindene;

[0099] 2,6-Dimethyl-1-indanone; and selected from one or more of the following:

[0100] Alcohol dehydrogenases with the amino acid sequence shown in SEQ ID NO:1, alcohol dehydrogenases as described in any of the first aspects of the present invention, and glucose dehydrogenases with the amino acid sequence shown in SEQ ID NO:3.

[0101] The reaction route of this invention is shown in equation B below:

[0102]

[0103] The substrate (i.e., compound 1 shown in formula B) is a racemic mixture of 2,6-dimethyl-1-indanone (or 2,6-dimethyl-2,3-dihydro-1-indanone), which is an equimolar mixture of (2S)-2,6-dimethyl-1-indanone (referred to as the S-configuration substrate) and (2R)-2,6-dimethyl-1-indanone (referred to as the R-configuration substrate).

[0104] The product has four configurations: (1R,2S)-2,6-dimethyl-1-hydroxyindene (abbreviated as R,S configuration product), (1R,2R)-2,6-dimethyl-1-hydroxyindene (abbreviated as R,R configuration product), (1S,2S)-2,6-dimethyl-1-hydroxyindene (abbreviated as S,S configuration product, i.e., compound 2 shown in formula B), and (1S,2R)-2,6-dimethyl-1-hydroxyindene (abbreviated as S,R configuration product).

[0105] ADH stands for alcohol dehydrogenase. Glc stands for glucose. GDH stands for glucose dehydrogenase.

[0106] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. All reagents and raw materials used in the present invention are commercially available.

[0107] The positive and progressive effects of this invention are as follows:

[0108] The alcohol dehydrogenase of the present invention uses the racemic mixture of 2,6-dimethyl-1-indanone as a substrate to prepare (1S,2S)-2,6-dimethyl-1-hydroxyindanone. It has a high substrate conversion rate and high stereoselectivity for (1S,2S)-2,6-dimethyl-1-hydroxyindanone. It does not require the addition of expensive chiral catalysts, has a low cost, and is suitable for industrial production. Detailed Implementation

[0109] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0110] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0111] The reagents used in this invention are as follows:

[0112] TB liquid culture medium: tryptone 10g / L, yeast extract 18g / L, glycerol 4mL / L, KH2PO4 (anhydrous) 2.31g / L, K2HPO4 (trihydrate) 16.43g / L.

[0113] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0114] LB solid medium (Kan resistant): tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L, kanamycin (Kan) 50 μg / mL.

[0115] LB agar plates: Plate culture dishes containing LB solid culture medium.

[0116] Escherichia coli BL21 was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0117] pET28a plasmid was purchased from Novagen.

[0118] IPTG was purchased from Bangtai Biotechnology Co., Ltd.

[0119] The Minerva Super Fusion Cloning Kit was purchased from Suzhou Yuheng Biotechnology Co., Ltd., product codes M2026S and M2026L.

[0120] The measurement method of the present invention is as follows:

[0121] The method for measuring the substrate and product configuration ratios (HPLC, %a / a) is as follows:

[0122] Column: CHIRALPAK OD-H (5μm, 250×4.6mm);

[0123] Mobile phase: n-hexane:isopropanol = 98:2; flow rate: 0.6 mL / min; injection volume: 10 μL; column temperature: 25℃; wavelength: 274 nm; run time: 40 min.

[0124] The blank solution and diluent were prepared as hexane:isopropanol = 90:10. Weigh 10 mg of the substrate, S,S configuration product reference, R,S configuration product reference, and S,R configuration product reference, and place them in volumetric flasks respectively. Dissolve and dilute with diluent, shake well, and filter through an organic filter membrane to obtain the respective reference solutions.

[0125] Weigh a certain amount of product into a volumetric flask, dissolve and dilute with diluent, and then filter through an organic filter membrane to obtain the test solution. Inject 10 μL of the blank solution, each reference solution, and the test solution into a high-performance liquid chromatograph (HPLC), measure and record the peak areas. The amount of products with different configurations is calculated using the area normalization method based on the peak area ratio of each product in the HPLC chromatogram.

[0126] Substrate configuration: The retention time of the R-configuration product reference was 10.336 min, and the retention time of the S-configuration product reference was 11.359 min.

[0127] Product configurations: The retention time of the R,R configuration product reference was 16.135 min, the S,S configuration product reference was 17.434 min, the R,S configuration product reference was 19.243 min, and the S,R configuration product reference was 20.627 min.

[0128] The formula for calculating the proportion of S,S configuration products is as follows:

[0129] S,S configuration product proportion Among them, A S,S Peak area of ​​S,S configuration product, A R,S Peak area of ​​R,S configuration product, A S,R Peak area of ​​S,R configuration product, A R,R : Peak area of ​​R,R configuration products.

[0130] The conversion rates of substrate R configuration and substrate S configuration were calculated using the configuration ratios of the substrate and product after the reaction.

[0131] The formula for calculating the total conversion rate of the substrate is: Total conversion rate of substrate = Amount of substrate converted into product / Total amount of substrate × 100% = Conversion rate of R configuration in substrate + Conversion rate of S configuration in substrate.

[0132] Yield = Total conversion × Percentage of target configuration in product (S, S configuration product percentage) × 100%.

[0133] Example 1: Preparation of crude enzyme solution

[0134] 1.1 Synthesis of Recombinant Plasmids

[0135] In this embodiment, crude enzyme solutions of alcohol dehydrogenase Enz.1 and glucose dehydrogenase Enz.2 were prepared. The enzyme numbers and sequences are shown in Table 1. According to Table 1, the gene fragments of the above enzymes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the expression vector pET28a (Kan resistant), respectively, to obtain the recombinant plasmids pET28a-Enz.1 and pET28a-Enz.2. The restriction sites were NdeI / BamHI.

[0136] Table 1 Enzyme Sequence

[0137] Enzyme number enzyme name amino acid sequence nucleotide sequence Enz.1 alcohol dehydrogenase SEQ ID NO:1 SEQ ID NO:2 Enz.2 glucose dehydrogenase SEQ ID NO:3 SEQ ID NO:4

[0138] 1.2 Transformation of enzyme genes

[0139] The synthesized recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, and the transformed bacterial cultures were plated on LB agar plates containing 50 μg / mL kanamycin (Kan) and incubated overnight at 37°C.

[0140] 1.3 Expression of enzyme genes

[0141] Single colonies from LB agar plates were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C for 4 h. Then, they were inoculated at a 1% (v / v) inoculation rate into TB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 250 rpm until the bacterial concentration reached OD500. 600 =0.6~0.8, add IPTG inducer to a final concentration of 0.1mM, induce overnight at 25℃ and 250rpm, centrifuge at 4000rpm for 20min, and collect wet cells.

[0142] 1.4 Preparation of crude enzyme solution

[0143] The wet bacterial cells were resuspended in 50mM pH7.0 PBS buffer at a ratio of 1g:10mL (i.e., homogenization ratio), homogenized, flocculant was added, and the precipitate was discarded by centrifugation. The supernatant enzyme solution obtained was the crude enzyme solution. Enz.1 crude enzyme solution and Enz.2 crude enzyme solution were obtained respectively and then stored at -20℃ for later use.

[0144] Example 2: Construction of an alcohol dehydrogenase mutant library

[0145] 2.1 Using alcohol dehydrogenase Enz.1 (amino acid sequence SEQ ID NO:1) as the starting enzyme, simulation analysis was performed using protein three-dimensional structure analysis software and molecular docking software. Considering the docking results between alcohol dehydrogenase Enz.1 and its substrate, the characteristics of the enzyme's substrate binding pocket, the structural characteristics of the enzyme's stereoselective substrate recognition, and the enzyme's catalytic mechanism, it was ultimately deduced that sites P190, T152, D198, and P188 may be sites affecting the enzyme activity of alcohol dehydrogenase Enz.1.

[0146] 2.2 Based on the nucleotide sequence of alcohol dehydrogenase Enz.1 (SEQ ID NO:2), corresponding mutant primers were designed, and the sequences are shown in Table 2. Using the pET28a-Enz.1 recombinant plasmid containing the nucleotide sequence of alcohol dehydrogenase Enz.1 as a template, PCR amplification was performed using the primers in Table 2. The PCR amplification system is shown in Table 3, and the PCR amplification program is shown in Table 4. Sequences SEQ ID NO:5–SEQ ID NO:12 are single-point mutation primers, SEQ ID NO:13–SEQ ID NO:14 are primers for iterative mutation based on the P190 mutation, and SEQ ID NO:15–SEQ ID NO:16 are primers for iterative mutation based on the P190G-T152E-D198N mutation.

[0147] Table 2 Primers for alcohol dehydrogenase mutation

[0148]

[0149]

[0150] Where N represents any one of the nucleotides A, G, C, and T, and K represents either G or T.

[0151] Table 3. PCR amplification system (total volume 50 μL)

[0152] Table 4 PCR amplification program

[0153]

[0154] 2.3 Seamless cloning / in-fusion cloning technology was used, and a fusion cloning kit was employed to ligate and recombine the PCR amplification products. The ligated and recombined plasmids were transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Colony PCR and sequencing verification confirmed the presence of multiple mutant strains with mutations at the target site.

[0155] 2.4 Prepare crude enzyme solutions of various alcohol dehydrogenase mutants corresponding to the mutant strains by following the steps in Section 1.3 of Example.

[0156] Example 3: Screening of alcohol dehydrogenase mutants

[0157] This embodiment constructed an enzyme catalytic system for preparing (1S,2S)-2,6-dimethyl-1-hydroxyindane using (2S)-2,6-dimethyl-1-indane as a substrate, in order to screen various alcohol dehydrogenase mutants obtained in the previous embodiment. The enzyme catalytic system is shown in Table 5.

[0158] The enzyme-catalyzed reaction conditions were as follows: 1 mL of the enzyme-catalyzed reaction system was placed at 30℃ and 200 rpm for 16 h. After the reaction was completed, an appropriate amount of product was taken for detection to obtain the proportion of products with different configurations. Enzyme mutants with a higher proportion of S,S configuration products were screened, and the mutation sites relative to Enz.1 and the proportion of S,S configuration products obtained by catalysis are shown in Table 6.

[0159] Table 5 Enzyme catalysis system (total volume 1 mL)

[0160] Material Name Added amount 2,6-Dimethyl-1-indanone racemic mixture (substrate) 10mg (62.42mM) <![CDATA[NADP + (Oxidized Coenzyme) 3.717 mg (5 mmol) Isopropanol (co-solvent) 50μL Glucose (hydrogen donor) 34.59 mg (192 mM) Enz.1 crude enzyme solution or crude enzyme solution of alcohol dehydrogenase mutant 200 μL (equivalent to 20 mg of wet bacterial cells) Enz.2 crude enzyme solution (coenzyme regeneration enzyme) 60 μL (equivalent to 6 mg of wet bacterial cells) PBS solution (pH 7.0; 0.2M) Adjust the volume to 1000 μL

[0161] In Table 5, the crude enzyme solutions of Enz.1 and Enz.2 were from Example 1, and the crude enzyme solution of the alcohol dehydrogenase mutant was from Example 2. Enz.2 was used to catalyze NADP. + The reduced coenzyme NADPH required to catalyze substrate reactions when producing crude enzyme solution of Enz.1 or crude enzyme solution of alcohol dehydrogenase mutant.

[0162] Table 6. Proportion and yield of different conformations of products obtained by different alcohol dehydrogenase mutants

[0163]

[0164]

[0165] Table 6 shows that the proportion of S,S configuration products obtained by Enz.1-1 to Enz.1-25 catalysis was higher than that obtained by Enz.1 catalysis of the same substrate (79.94%). Enz.1-3 catalysis yielded the highest proportion of S,S configuration products, reaching 98.3%. Compared with Enz.1, the mutant enzymes Enz.1-1 to Enz.1-25 effectively improved the yield of the target configuration, with Enz.1-3 achieving the highest yield at 43.74%. The proportion of S,S configuration products obtained by Enz.1-30 catalysis was higher than that obtained by Enz.1 catalysis of the same substrate.

[0166] Example 4 utilizes an alcohol dehydrogenase mutant to catalyze the production of (1S,2S)-2,6-dimethyl-1-hydroxyindane from a substrate.

[0167] The wet cell Enz.1-3 obtained according to the method in Examples 1.1-1.3 was resuspended in 50 mM pH 7.0 PBS buffer at a ratio of 1 g: 4 mL (i.e., homogenization ratio), homogenized, flocculant added, centrifuged and the precipitate was discarded to obtain the supernatant enzyme solution as the crude enzyme solution of Enz.1-3. The wet cell Enz.2 obtained according to the method in Examples 1.1-1.3 was homogenized at a ratio of 1:4 to obtain the crude enzyme solution of Enz.2. In a 0.25 L three-necked flask, while stirring, 80 mL of Enz.1-3 crude enzyme solution (1:4 homogenization, 20 g wet cell), 16 mL of Enz.2 crude enzyme solution (1:4 homogenization, 4 g wet cell), and 1.38 mL of NADP were added sequentially. + A 72 g / L aqueous solution (99.4 mg, 133.7 mmol) and 22.5 g glucose (125 mmol) were dissolved by stirring. Then, 20 g of substrate (2,6-dimethyl-1-indanone racemic mixture, 125 mmol) was added, and the pH was controlled at approximately 7.5 with 5% sodium hydroxide. The reaction mixture was approximately 100 ml. The reaction mixture was placed at 30 °C and 200 rpm for 16 h. After the reaction was complete, a suitable amount of product was taken for analysis to obtain the proportion of different configurations. The same catalytic reaction was performed on alcohol dehydrogenases Enz.1, Enz.1-2, and Enz.1-12. The proportions and yields of different configurations of products catalyzed by different alcohol dehydrogenase mutants are shown in Table 7.

[0168] Table 7. Proportion and yield of different conformations of products obtained by different alcohol dehydrogenase mutants

[0169]

[0170] As shown in Table 7, the mutant enzymes can effectively improve the yield of the target conformation, with Enz.1-3 showing the highest yield at 43.15%. The proportion of the S,S conformation product obtained by Enz.1-3 catalysis was the highest, reaching 98.58%.

[0171] SEQ ID NO:1

[0172] MTDRLKGKVAIVTGGTLGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGLVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDDLEGWEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ

[0173] SEQ ID NO:2

[0174] ATGACCGATCGCCTGAAGGGTAAAGTGGCCATTGTTACCGGCGGCACCCTGGGTATTGGCCTGGCCATTGCCGATAAGTTTGTGGAGGAAGGCGCCAAAGTGGTGATTACCGGTCGCCATGCCGATGTGGGTGAAAAAGCCGCCAAAAGCATCGGCGGTACCGATGTGATCCGCTTCGTGCAGCATGATGCCAGTGATGAAGCCGGCTGGACCAAACTGTTCGATACCACCGAAGAGGCATTCGGCCCGGTGACCACCGTTGTTAACAACGCCGGCATTGCAGTGAGCAAAAGCGTTGAAGACACCACCACCGAGGAGTGGCGCAAACTGCTGAGCGTGAACCTGGACGGCGTTTTCTTTGGTACCCGCCTGGGTATTCAGCGCATGAAGAACAAGGGTCTGGGTGCCAGCATCATCAACATGAGCAGCATCGAGGGTCTGGTGGGCGATCCGACCCTGGGTGCCTATAACGCCAGCAAGGGCGCCGTTCGTATCATGAGCAAGAGCGCAGCCCTGGATTGCGCCCTGAAGGACTATGATGTGCGCGTGAATACCGTGCACCCGGGTCCGATTAAAACACCGCTGCTGGATGACCTGGAAGGCTGGGAAGAGATGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATCGGCGAACCGAATGACATCGCCTGGATCTGCGTTTACCTGGCCAGCGATGAAAGCAAATTTGCCACCGGCGCAGAGTTTGTGGTGGATGGTGGCTATACCGCCCAG

[0175] SEQ ID NO:3:

[0176] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGMTQYPSFQAGRG

[0177] SEQ ID NO:4

[0178] ATGTACCCTGATCTTAAAGGCAAAGTTGTTGCTATCACAGGCGCTGCTTCTGGCCTTGGCAAAGCTATGGCTATCCGTTTCGGCAAAGAACAAGCTAAAGTTGTTATCAACTACTCTAACAAACAAGATCCTAACGAAGTTAAAGAAGAAGTTATCAAAGCTGGCGGCGAAGCTGTTGTTGTTCAAGGCGAT GTTACAAAAGAAGAAGATGTTAAAAACATCGTTCAAACAGCTATCAAAGAATTCGGCACACTTGATATCATGATCAACAACGCTGGCCTTGAAAACCCTGTTCCTTCTCATGAAATGCCTCTTAAAGATTGGGATAAAGTTATCGGCACAAACCTTACAGGCGCTTTCCTTGGCTCTCGTGAAGCTATCAAATACT TCGTTGAAAACGATATCAAAGGCAACGTTATCAACATGTCTTCTGTTCATGAAGTTATCCCTTGGCCTCTTTTCGTTCATTACGCTGCTTCTAAAGGCGGCATCAAACTTATGACAGAAACACTTGCTCTTGAATACGCTCCTAAAGGCATCCGTGTTAACAACATCGGCCCTGGCGCTATCAACACACCTATCAA CGCTGAAAAATTCGCTGATCCTAAACAAAAAGCTGATGTTGAATCTATGATCCCTATGGGCTACATCGGCGAACCTGAAGAAATCGCTGCTGTTGCTGCTTGGCTTGCTTTAAAGAAGCTTCTTACGTACAGGCATCACACTTTTCGCTGATGGCGGCATGACACAATACCCTTCTTTCCAAGCTGGCCGTGGC

[0179] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An alcohol dehydrogenase, characterized in that, The amino acid sequence of the alcohol dehydrogenase has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and contains amino acid residue differences at positions P190 and / or D198 compared with the amino acid sequence shown in SEQ ID NO:1, wherein positions 190 and 198 refer to the amino acid residue numbers in SEQ ID NO:

1. Preferably, the P at position P190 is replaced with a sterically hindered amino acid; preferred amino acid residue differences include P190G, P190A, or P190C; and the D at position D198 is replaced with a sterically hindered or neutral amino acid; preferred amino acid residue differences include D198N, D198G, or D198A.

2. The alcohol dehydrogenase according to claim 1, characterized in that, The amino acid residue differences also include one or more of the following groups: i) T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F; ii) P188A or P188G; Preferably, the amino acid residue difference is selected from the group consisting of: (A)P190G, and T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F; (B)P190A, and T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F; (C)P190C, and T152E, T152A, T152V, T152I, T152G, T152R, T152S, T152D, T152H or T152F; (D)P190G, and D198N, D198G or D198A; (E)P190A, and D198N, D198G or D198A; (F)P190C, and D198N, D198G or D198A.

3. The alcohol dehydrogenase according to claim 1 or 2, characterized in that, The differences in amino acid residues are selected from the following group: (1)P190G; (2) P190G, T152E and D198N; (3) P190G, T152E, D198N and P188A; (4) P190G, T152A and D198G; (5) P190G, T152V and D198A; (6) P190G, T152I and D198A; (7) P190G, T152G and D198N; (8) P190G and T152R; (9) P190G, T152E and D198N; (10) P190G, T152S and D198A; (11) T152D and P190G; (12) T152I and P190G; (13) T152H and P190G; (14) T152F and P190G; (15) T152D and P190A; (16) T152I and P190A; (17) T152H and P190A; (18) T152F and P190A; (19) T152D and P190C; (20)T152I and P190C; (21) T152H and P190C; (22) T152F and P190C; (23)D198A; (24)D198N; (25)P190C; (26)P190A; (27) P190G, T152E, D198N and P188G.

4. An isolated nucleic acid encoding an alcohol dehydrogenase as described in any one of claims 1-3.

5. A recombinant expression vector comprising the nucleic acid as described in claim 4, wherein the backbone of the recombinant expression vector is preferably pET28a or pET21a.

6. A transformant comprising the nucleic acid as described in claim 4, or the recombinant expression vector as described in claim 5; Preferably, the host cell used in the construction of the transformant is Escherichia coli or Bacillus subtilis, and the Escherichia coli is more preferably Escherichia coli BL21(DE3).

7. A method for preparing alcohol dehydrogenase, comprising culturing the transformant as described in claim 6 to obtain a fermentation product; Preferably, the culture medium used for the culture is selected from LB liquid medium or TB liquid medium, and / or the culture conditions are: shaking culture at a temperature of 37±1℃; More preferably, the method further includes the step of purifying the fermentation product to obtain the reductase.

8. An enzyme combination, characterized in that, The enzyme combination comprises: one or more alcohol dehydrogenases as described in any one of claims 1-3, and glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO:

3.

9. The use of the alcohol dehydrogenase as described in any one of claims 1-3, the nucleic acid as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, or the enzyme combination as described in claim 8 in the preparation of (1S,2S)-2,6-dimethyl-1-hydroxyindene; Preferably, the preparation of (1S,2S)-2,6-dimethyl-1-hydroxyindene uses 2,6-dimethyl-1-indene as a substrate.

10. A method for preparing (1S,2S)-2,6-dimethyl-1-hydroxyindane, characterized in that, The method includes: At least one of the alcohol dehydrogenases as described in any one of claims 1-3 is contacted with a substrate and reacted to prepare (1S,2S)-2,6-dimethyl-1-hydroxyindane; Preferably, the substrate is 2,6-dimethyl-1-indanone; And / or, the reaction further includes the use of a coenzyme; and / or, the reaction further includes a reaction that regenerates the coenzyme.

11. The method as described in claim 10, characterized in that, The coenzyme is NADH, NADPH, NAD. + and NADP + Any one or both of them; Preferably, the reaction that regenerates the coenzyme uses NAD. + and NADP + Any one or two of the following, a hydrogen donor and a coenzyme regenerating enzyme; wherein the hydrogen donor and the coenzyme regenerating enzyme are selected from one or more of the following: (1) Glucose and glucose dehydrogenase; (2) Formic acid or formate and formic acid dehydrogenase; (3) Isopropanol and alcohol dehydrogenase; More preferably, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:

3.

12. The method as described in claim 10 or 11, characterized in that, The alcohol dehydrogenase or coenzyme regeneration enzyme is used in the form of liquid enzyme, solid enzyme powder, immobilized enzyme, wet cell or bacterial powder. And / or, the concentration of 2,6-dimethyl-1-indanone added in the reaction is 1-500 mg / mL; preferably 10-200 mg / mL; And / or, the molar ratio of the hydrogen donor to the 2,6-dimethyl-1-indanone is 0.5:1-5:1; preferably 1:1-3:1; And / or, the "NAD" + and / or NADP + "The mass ratio of the 2,6-dimethyl-1-indanone to the 2,6-dimethyl-1-indanone is 1:1000 to 500:1000; for example, 5:1000 or 371:1000." And / or, the pH of the reaction is 6.5-8.5; preferably 7.0-8.0; And / or, the reaction temperature is 25-45°C; preferably 30-40°C; And / or, the buffer solution for the reaction is an aqueous phosphate solution, the concentration of which is 10mM-500mM, preferably 10mM-200mM.

13. The method according to any one of claims 10-12, characterized in that, When the alcohol dehydrogenase or coenzyme regenerating enzyme is used in the form of a liquid enzyme, the ratio of the mass of the wet bacterial cells that produce the liquid enzyme to the mass of the added 2,6-dimethyl-1-indanone is (0.1-2) g:1 g, for example, 1 g:1 g or 2 g:1 g. And / or, the mass ratio of the coenzyme regenerating enzyme to the hydrogen donor, calculated based on the mass of the wet bacterial cells used, is (0.05-1) g:1 g, for example, 0.2 g:1 g, 0.6 g:1 g; And / or, the method also uses a co-solvent; the co-solvent is preferably DMSO or isopropanol.

14. A reaction end product system for the catalytic synthesis of (1S,2S)-2,6-dimethyl-1-hydroxyindanone from 2,6-dimethyl-1-indanone, characterized in that, The reaction end product system includes: (1S,2S)-2,6-dimethyl-1-hydroxyindene; 2,6-Dimethyl-1-indanone; and selected from one or more of the following: Alcohol dehydrogenases with the amino acid sequence shown in SEQ ID NO:1, alcohol dehydrogenases as described in any one of claims 1-3, and glucose dehydrogenases with the amino acid sequence shown in SEQ ID NO:3.

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