CN113151205B

CN113151205B - Alcohol dehydrogenase mutant and application thereof in synthesis of cyclic terpene ketone

Info

Publication number: CN113151205B
Application number: CN202110421331.2A
Authority: CN
Inventors: 李春秀; 占静茹; 寿超; 陈琦; 许建和; 潘江; 钱小龙
Original assignee: Suzhou Baifu Enzyme Technology Co ltd
Current assignee: Suzhou Baifu Enzyme Technology Co ltd
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2024-07-16
Anticipated expiration: 2041-04-20
Also published as: CN113151205A

Abstract

The invention relates to an alcohol dehydrogenase mutant and application thereof in synthesis of cyclic terpene ketone. In particular to an alcohol dehydrogenase mutant with obviously improved catalytic performance, a coding nucleic acid thereof, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid sequence, and application of the alcohol dehydrogenase mutant or the recombinant expression transformant in catalyzing dehydrogenation reaction of hydroxyl compounds, in particular in catalyzing dehydrogenation reaction of cyclic terpene alcohols with hydroxyl groups at 3-and 6-positions so as to prepare cyclic terpene ketone. Compared with wild alcohol dehydrogenase, the alcohol dehydrogenase mutant has a certain degree of improvement on the dehydrogenation reaction activity of a series of hydroxyl compounds, and has a good application prospect.

Description

Alcohol dehydrogenase mutant and application thereof in synthesis of cyclic terpene ketone

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an alcohol dehydrogenase mutant with obviously improved catalytic performance, encoding nucleic acid thereof, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid sequence, and application of the alcohol dehydrogenase mutant or the recombinant expression transformant in preparing cyclic terpene ketone by catalyzing dehydrogenation reaction of hydroxyl compounds, in particular dehydrogenation reaction of cyclic terpene alcohols with 3-and 6-hydroxyl groups.

Background

L- (-) -menthol, also known as L-menthol, is liquid at normal temperature, has poor water solubility, has cool flavor and a certain aroma, and is often used in the processing of products such as wines, seasonings, oral health products, cosmetics and the like (Phytochemistry, 2013, 96:15-25). In nature, (-) -menthol is present in the oil gland secreting cells of mint plant leaves and is obtainable by extraction, atmospheric distillation or the like (Proc.Natl. Acad. Sci.U.S.A,1999, 97:2934-2939). Menthol consumption has increased dramatically year by year in recent years to about 4 ten thousand tons (anhui. Chem. Ind., 2018) in 2018, with about 70% of (-) -menthol being distilled from plant peppermint, which is greatly affected by weather, land, and manpower. About 30% of (-) -menthol is chemically synthesized. So far, the market of the (-) -menthol in China is large, but the large-area planting production area of the menthol in China is less, so that the natural (-) -menthol used in the industries of food and the like is mostly imported from India and Brazil. No menthol chemical method synthesis enterprises with high yield and good technology exist in China.

The current chemical synthesis enterprises of (-) -menthol are unique to both Symbise, japan, and Germany. The Japanese Takasago corporation synthesized (-) -menthol on a large scale by asymmetric synthesis (chem. Biodivers,2014, 11:1688-1699); whereas Symrise in Germany produces (-) -menthol by enantiomer resolution (US 20100249467 A1,2010). However, both of them have drawbacks in that the former catalyst is expensive and serious in pollution, and the latter is difficult to split and has a large loss. Both the Japanese Takasago and Germany Symprise enterprises were removed, leaving a smaller scale chemical synthesis (CN 107056587B, 2017).

The biosynthesis of (-) -menthol has attracted increasing attention from the subjects, since the commercial demands of (-) -menthol obtained by plant extraction and chemical synthesis are still not met. However, currently, the biosynthesis of (-) -menthol is still in the primary stage, most of which are the preparation of (-) -menthol by kinetic resolution of racemic menthol and like derivatives (Tetrahedron asymmetry, 2003, 14:3313-3319). The existing effect is remarkable, namely, the menthyl acetate is split by fixing the esterase of the bacillus subtilis in a double-aqueous-phase system, the substrate loading capacity of the menthyl acetate can be improved to 3M, and meanwhile, the conversion rate of >40% and the e.e. value of 97% are ensured, so that the mass production of the L-menthol is possible (adv. Synth. Catalyst., 2010, 351:405-414). However, these methods are not synthesized from inexpensive compounds such as glycerol, (-) -limonene, and present the same challenges as plant extraction of (-) -menthol (Research on CHEMICAL INTERMEDIATES,2018, 44:6847-6860). Thus the de novo synthetic pathway of (-) -menthol in microorganisms mimics plants is more likely to achieve efficient biosynthesis of (-) -menthol (Journal of Biological Chemistry,1992, 267:7582-7587).

The current approach to de novo construction of (-) -menthol in microorganisms has not been achieved, but has made excellent progress. Nigel S. Scrutton's group of problems coupled the latter two steps of the menthol pathway, simultaneously constructing the alkene reductase (NtDBR) and (-) -menthone reductase (MMR) into E.coli, successfully synthesized (-) -menthol from (+) -menthone by a one-pot enzymatic method, opened the latter two steps of the (-) -menthol biosynthetic pathway (ACS Synth.biol.,2015, 4:1112-1123), which makes the in vitro construction of menthol pathway promising. Subsequently in 2018, this group of subjects replaced isopulegol isomerase (IPGI) by bacterial-derived isomerase (KSI) and achieved the synthesis of (-) -menthol (25.5 mg/L) from (+) -cis-isopulegol. In addition to 2014, the ANDREAS SCHMID group successfully synthesized 2.7g/L of (-) -limonene from E.coli (Biotechnol. J.,2014, 9:1000-1012), the current bottleneck of the microbial (-) -menthol pathway is limonene-3-hydroxylase (L3H) and isomenthol dehydrogenase (IPDH).

The isomenthol dehydrogenase (IPDH) belongs to the SDR superfamily. The enzymes can catalyze the oxidation of isomenthol to the corresponding ketone. This enzyme was originally found in plants of the genus Mentha (Archives of Biochemistry & Biophysics,1985, 238:49-60), which is capable of catalyzing both the oxidation of isomenthol (isopiperitenol) and the oxidation of carveol (carveol), and is therefore also known as isomenthol/carveol dehydrogenase (Plant Physiology,2004, 136:4215-4227). In 2005, the enzyme in peppermint was successfully heterologously expressed and characterized in E.coli, but its activity was very low, k _cat/K_m was only 0.027s ^-1·mM^-1. In addition, the enzyme is found in other plants (biol. Pharm. Bull.,2014, 37:847-852), microorganisms (Journal of Biological Chemistry,1999, 274:2692-26304), fungi (fungi Biology,2017, 121:137-144). However, most of these isomenthol dehydrogenases do not characterize the substrate isomenthol, and the crystal structure of the enzyme has not been resolved at present.

IPDH, in addition to having an important role in the microbial synthesis of (-) -menthol, is known to provide a new approach for future synthesis of isomenthone. The isomenthone can be used as an 'alarm element' for warning mites away (Agricultural and Biological Chemistry,1987, 51:3441-3442), which is an important substance in the biological kingdom, and although other applications are not clear at present, more applications of the isomenthone can be found along with the improvement of the synthesis method of the isomenthone. To date, there has been little progress in the preparation of isomenthone, and only chemical synthesis methods exist today, such as oxidation of (-) -limonene to (-) -isomenthone by ruthenium, pyrrole complexes (The Journal of Organic Chemistry,1999, 64:7365-7374). The only preparation to gram-scale was the method used by the task group of Nigel s. Scrutton (j. Nat. Prod.,2018, 81:1546-1552), but in only 35% yield. In summary, the current enzymatic synthesis of isomenthone has not emerged.

As described above, IPDH plays an important role in the microbial synthesis of (-) -menthol and the preparation of isomenthone, but the known isomenthol dehydrogenase MpIPDH has the problems of low catalytic activity, existence in the form of membrane protein, low space-time yield and the like. Therefore, there is a need for an enzyme catalyst with better catalytic performance to meet the demands of efficient menthol synthesis pathways in microorganisms, as well as the demands of enzymatic synthesis of isomenthone.

Disclosure of Invention

The invention aims to solve the technical problem of providing an alcohol dehydrogenase mutant with obviously improved catalytic performance aiming at the defects of the prior art of the isomenthol dehydrogenase.

According to one of the technical schemes of the invention, the alcohol dehydrogenase mutant with obviously improved catalytic performance is obtained.

Protein BLAST was first performed on NCBI bacterial library using MpIPDH (Plant Physiology,2005,137:863-872,NCBI Number:Q5C919.1) as a probe to obtain a sequence with a certain homology to MpIPDH. Secondly, after the homologous sequences are aligned, sequences with key motif are screened out, wherein the key motif is such as SDR catalytic triplets (Ser-Tyr-Lys), NAD (P) ⁺ -binding motif (- (T) GXXXGXG-), SDR conservative motif (NNAG) and NAD ⁺ -dependent binding residues Asp; deleting sequences with over-high homology between every two sequences to ensure that the homology between every two sequences is less than 85 percent, thereby obtaining the enzyme library of the alcohol dehydrogenase. The activity of candidate enzyme is detected, reductase PaIPDH from Pseudomonas aeruginosa is obtained through screening, and the amino acid sequence of the reductase PaIPDH is shown in a sequence table SEQ ID No. 2.

In the invention, the enzyme gene is used as a female parent, and the directed evolution transformation is carried out by adopting strategies such as alanine scanning, crystal structure guidance, site-directed saturation mutation, iterative combination mutation and the like, and the alcohol dehydrogenase mutant with obviously improved activity and thermal stability is obtained by combining with ultraviolet spectrophotometry activity detection re-screening.

The alcohol dehydrogenase mutant is a derivative protein in which 1 or more amino acid residues in 95 th glutamic acid, 97 th glutamic acid, 154 th methionine, 189 th valine, 191 th aspartic acid, 194 th methionine, 195 th phenylalanine, 199 th tyrosine and 208 th phenylalanine of an amino acid sequence shown in SEQ ID No.2 are replaced by other amino acid residues; meanwhile, the derivative protein has higher catalytic performance and stability than those of the protein with the amino acid sequence shown in SEQ ID No. 2.

Compared with wild alcohol dehydrogenase, the alcohol dehydrogenase mutant provided by the invention has higher catalytic activity and stability effect.

Derived proteins in which other amino acid residues are replaced with other amino acid residues that do not affect the catalytic performance of the alcohol dehydrogenase mutant are also within the scope of the claims of the present invention.

The present invention provides a variety of preferred alcohol dehydrogenase mutants which are proteins consisting of any one of the following amino acid sequences:

(1) Substitution of valine (E95V) for glutamic acid at position 95 of the amino acid sequence shown in SEQ ID No. 2;

(2) Substitution of glutamic acid at position 95 of the amino acid sequence shown in SEQ ID No.2 with phenylalanine (E95F);

(3) Substitution of glutamic acid at position 97 of the amino acid sequence shown in SEQ ID No.1 with methionine (E97M);

(4) Replacement of methionine at position 154 of the amino acid sequence shown in SEQ ID No.2 with histidine (M154H);

(5) Valine at position 189 of the amino acid sequence shown in SEQ ID No.2 is replaced by tryptophan (V189W);

(6) Substitution of valine at position 189 of the amino acid sequence shown in SEQ ID No.2 with isoleucine (V189I);

(7) Substitution of valine (D191V) for aspartic acid at position 191 of the amino acid sequence shown in SEQ ID No. 2;

(8) Substitution of methionine at position 194 of the amino acid sequence shown in SEQ ID No.2 with lysine (M194K);

(9) Substitution of methionine at position 194 of the amino acid sequence shown in SEQ ID No.2 with asparagine (M194N);

(10) Substitution of phenylalanine at position 195 of the amino acid sequence shown in SEQ ID No.2 with tryptophan (F195W);

(11) Substitution of phenylalanine at position 195 of the amino acid sequence shown in SEQ ID No.2 with methionine (F195M);

(12) Substitution of tyrosine 199 of the amino acid sequence shown in SEQ ID No.2 with valine (Y199V);

(13) Substitution of tyrosine 199 of the amino acid sequence shown in SEQ ID No.2 with histidine (Y199H);

(14) Substitution of valine for glutamic acid at position 95 (E95V) and histidine for phenylalanine at position 208 (F208H) of the amino acid sequence shown in SEQ ID No. 2;

(15) Substitution of glutamic acid at position 97 of the amino acid sequence shown in SEQ ID No.2 with methionine (E97M), substitution of methionine at position 154 with histidine (M154H);

(16) Substitution of methionine at position 194 of the amino acid sequence shown in SEQ ID No.2 with lysine (M194K) and tyrosine at position 199 with tryptophan (Y199W);

(17) Substitution of methionine at position 194 with lysine (M194K), tyrosine at position 199 with histidine (Y199H), phenylalanine at position 208 with histidine (F208H) of the amino acid sequence shown in SEQ ID No. 2;

(18) Glutamic acid at position 95 of the amino acid sequence shown in SEQ ID No.2 is replaced with phenylalanine (E95F), valine at position 189 with tryptophan (V189W), phenylalanine at position 195 with tryptophan (F195W), tyrosine at position 199 with valine (Y199V).

The alcohol dehydrogenase mutant provided by the invention is suitable as an isomenthol dehydrogenase mutant.

In a second technical scheme, the invention provides nucleic acid for encoding the alcohol dehydrogenase mutant and a recombinant expression vector containing the nucleic acid sequence of the alcohol dehydrogenase mutant gene.

The nucleic acid codes for and expresses the alcohol dehydrogenase mutant obtained by evolution modification according to the first technical scheme, and the sources of the alcohol dehydrogenase mutant comprise: cloning the gene sequence of the series alcohol dehydrogenase mutant according to the technical scheme I by a genetic engineering technology; alternatively, the nucleic acid molecule encoding the alcohol dehydrogenase mutant according to claim one can be obtained by artificial total sequence synthesis.

The recombinant expression vector can be constructed by connecting the coding nucleic acid sequence of the alcohol dehydrogenase mutant gene of the invention to various commercial empty vectors by a conventional method in the field. The commercially available empty vector may be various plasmid vectors conventional in the art, as long as the recombinant expression vector can normally replicate in a corresponding expression host and express a corresponding alcohol dehydrogenase.

Preferred plasmid vectors are different for different expression hosts. It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. For E.coli hosts, the plasmid vector is preferably a pET-28a (+) plasmid. The escherichia coli recombinant expression vector can be prepared by the following method: the alcohol dehydrogenase mutant gene fragment obtained by PCR amplification is digested with restriction enzymes EcoR I and Hind III, simultaneously the empty plasmid pET-28a (+) is digested with restriction enzymes EcoR I and Hind III, the DNA fragment of the above digested alcohol dehydrogenase mutant and the empty plasmid are recovered, and the DNA fragment and the empty plasmid are ligated by using T4 DNA ligase to construct a recombinant expression vector containing the nucleic acid encoding the alcohol dehydrogenase mutant for E.coli expression.

In a third aspect of the present invention, there is provided a recombinant expression transformant comprising the alcohol dehydrogenase mutant gene of the present invention or a recombinant expression vector thereof. Recombinant expression transformants can be prepared by transforming an already constructed recombinant expression vector into a host cell.

The host cell is a variety of conventional host cells in the art, as long as the recombinant expression vector is capable of stably self-replicating and efficiently expressing the target protein after induction by an inducer. The invention is preferably used as a host cell, and more preferably E.coli BL21 (DE 3) is used for efficiently expressing the alcohol dehydrogenase mutant.

The fourth technical scheme of the invention provides a recombinant alcohol dehydrogenase mutant catalyst, which is any one of the following forms:

(1) Culturing the recombinant expression transformant of the present invention, and isolating a transformant cell containing the alcohol dehydrogenase mutant;

(2) Culturing the recombinant expression transformant of the present invention, and separating a crude enzyme solution containing the alcohol dehydrogenase mutant;

(3) And performing liquid cooling and freeze drying on the crude enzyme of the alcohol dehydrogenase mutant to obtain crude enzyme powder.

The culture method and conditions for the recombinant expression transformant may be those conventional in the art. In one embodiment of the invention, the following steps are provided: the recombinant expression transformant of the present invention is cultured to obtain a recombinant alcohol dehydrogenase. For recombinant E.coli, the preferred medium is LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl and pH 6.5-7.0. The preferred cultivation method is: recombinant E.coli constructed as described above was inoculated into LB medium containing kanamycin, and cultured overnight at 37℃with shaking at 180 rpm. Inoculating 1-2% (v/v) of the recombinant expression vector into a 500mL Erlenmeyer flask containing 100mL of LB culture medium (containing kanamycin), placing the Erlenmeyer flask in a shaking table at 37 ℃ and at 180rpm for shaking culture, adding isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.1-0.5mmol/L as an inducer when the OD ₆₀₀ of the culture solution reaches 0.6-0.8, centrifuging the culture solution after 16-24h induction at 16 ℃, collecting precipitates, and washing twice with physiological saline to obtain recombinant expression transformant cells. And freeze-drying the harvested recombinant cells to obtain freeze-dried cells containing the alcohol dehydrogenase mutant. Suspending the obtained recombinant cells in buffer solution with the volume of 5-10 times (v/w), performing ultrasonic crushing, centrifuging and collecting supernatant, thus obtaining crude enzyme solution of the recombinant alcohol dehydrogenase mutant. And (3) placing the collected crude enzyme solution at the temperature of minus 80 ℃ for freezing, and then drying at low temperature by using a vacuum freeze dryer to obtain the freeze-dried enzyme powder. The obtained freeze-dried enzyme powder is stored in a refrigerator at the temperature of 4 ℃ and can be conveniently used.

The method for measuring the activity of the alcohol dehydrogenase mutant comprises the following steps: 1mL of a reaction system (50 mmol/L glycine-sodium hydroxide buffer, pH 10.0) containing 1mmol/L (-) -trans-isomenthol and 10mmol/L NAD ⁺ was preheated to 40℃and then an appropriate amount of alcohol dehydrogenase mutant was added thereto, the reaction was incubated at 40℃and the absorbance change of NADH at 340nm was detected on a spectrophotometer and the absorbance change over 1 minute was recorded.

The enzyme activity was calculated using the following formula:

Enzyme activity (U) =ew×v×10 ³/(6220×l) where EW is the change in absorbance at 340nm within 1 minute; v is the volume of the reaction solution, and the unit is mL;6220 is the molar extinction coefficient of NADH, in L/(mol cm); l is the optical path distance in cm.1 enzyme activity unit (U) is defined as the amount of enzyme required to catalytically reduce 1. Mu. Mol NAD ⁺ per minute under the conditions described above.

The fifth technical scheme of the invention provides application of the alcohol dehydrogenase mutant or the recombinant alcohol dehydrogenase mutant catalyst in catalyzing dehydrogenation reaction of hydroxyl compounds.

Namely, the present invention provides a method for preparing the corresponding ketone by catalyzing the dehydrogenation reaction of the hydroxyl compound by using the alcohol dehydrogenase mutant or the recombinant alcohol dehydrogenase mutant catalyst.

Wherein the hydroxy compound may be selected from any one of the following compounds:

in the application, the concentration of the hydroxyl compound can be 1-5 mmol/L, and the dosage of the alcohol dehydrogenase mutant in the alcohol dehydrogenase mutant or recombinant alcohol dehydrogenase mutant catalyst can be 1-50U/mmol of the hydroxyl compound.

In one embodiment of the present invention, there is provided the use of the alcohol dehydrogenase mutant or recombinant alcohol dehydrogenase mutant catalyst to catalyze the dehydrogenation of cyclic terpene alcohols having hydroxyl groups in the 3-and 6-positions to produce cyclic terpene ketones.

In one embodiment of the invention, the cyclic terpene ketone comprises (-) -isomenthone, (-) -carvone.

In one embodiment of the invention, there is provided the use of the alcohol dehydrogenase mutant or recombinant alcohol dehydrogenase mutant catalyst to catalyze a (-) -trans-isomenthol dehydrogenation reaction to produce (-) -isomenthone.

In one embodiment of the invention, NADH or NAD ⁺ is used in the reaction mixture in an amount of 0.1 to 0.5mmol/L. In the reaction process, O ₂ can be used as an auxiliary substrate, the coenzyme circulation of NAD ⁺ in the reaction system is realized through the reaction catalyzed by NADH dehydrogenase, and the phosphate buffer solution of the reaction system is a phosphate buffer solution which is conventional in the art, such as potassium phosphate buffer solution, and the concentration of the phosphate buffer solution is preferably 50mmol/L. The dehydrogenation reaction is carried out under the condition of shaking or stirring. The dehydrogenation reaction is carried out at a temperature of 20 to 40 ℃, preferably 25 ℃. The dehydrogenation reaction takes place over a period of time based on complete conversion of the substrate or self-termination of the reaction, preferably a reaction time of less than 24 hours.

In catalyzing the dehydrogenation of (-) -trans-isomenthol to prepare (-) -isomenthone, it is not desirable to prepare (-) -isomenthone directly from (-) -trans-isomenthol due to the commercial unavailability of the substrate (-) -trans-isomenthol. Thus (-) -isomenthone is indirectly synthesized by introducing a hydroxylation step. The method comprises the following steps: by coexpression of P450cam _Y96F/Y247L (chem. Commun.,2001, 635-636) with its indispensable electron-transporting moieties Pdx, pdR on ampicillin-resistant pET21a, inexpensive (-) -limonene can be mostly hydroxylated to (-) -trans-isomenthol, which is then oxidized to (-) -isomenthol produced by the last step by the protein expressed by kanamycin-resistant pET28a-PaIPDH mutant. Thus, the first step P450cam portion of the cascade consumes NADH to produce NAD ⁺, while the second step PaIPDH mutant consumes NAD ⁺ to produce NADH, a self-circulating coenzyme. Thus, no external auxiliary enzyme is required to recycle the coenzyme. And through whole cell reaction, the activity of P450 can be kept to the maximum degree without adding coenzyme NAD ⁺/NADH.

Wherein, the reaction process of the alcohol dehydrogenase mutant and P450cam combined conversion to synthesize (-) -isomenthone (compound 3 a) is shown in figure 1.

Wherein the DNA sequence of the coexpression protein P450cam _Y96F/Y247L -Pdx-PdR is shown in a sequence table SEQ ID No. 3.

After the oxidation reaction is finished, the cyclic terpene ketone which is an oxidation product in the reaction liquid is separated and extracted by adopting a conventional method.

The method for separating and extracting the oxidation product cyclic terpene ketone in the reaction liquid is preferably to add ethyl acetate into the reaction liquid for extraction, add anhydrous sodium sulfate into an organic phase obtained by extraction for overnight drying, concentrate an extraction liquid by adopting a reduced pressure distillation method at 20-25 ℃ to remove a solvent due to the volatility of the product, and obtain a crude product of the product ketone. Preferably, separating crude ketone product by silica gel column chromatography, adopting 200-300 mesh silica gel, and eluting with petroleum ether: ethyl acetate = 50:1 (or 100:1), detecting the purity by GC after the elution is finished, collecting and combining elution tubes meeting the purity requirement, removing the solvent by a reduced pressure distillation method, and purifying to obtain the target (-) -isomenthone pure product.

Compared with the prior art, the innovation and improvement effect of the invention is as follows:

the invention provides an alcohol dehydrogenase mutant with better catalytic performance, which efficiently catalyzes hydroxyl dehydrogenation reaction of cyclohexane 3-position/6-position-alcohol to prepare optically pure ketone compounds such as: (-) -isomenthone, (-) -carvone. The (-) -isomenthone and (-) -carvone are cyclic terpene ketone compounds, and the cyclic terpene ketone compounds have unique fragrance and antibacterial and anticancer effects, so that the (-) -isomenthone and (-) -carvone have wide application values in spice markets and medicine markets. The alcohol dehydrogenase can catalyze the conversion of hydrophobic (-) -trans-isomenthol substrate with the concentration of up to 8mM, achieves the conversion rate of more than 80%, and the yield reaches 128.6mg L ^-1, thus being the highest yield of the (-) -isomenthone synthesized by the current enzyme method. Compared with the maternal alcohol dehydrogenase PaIPDH, the alcohol dehydrogenase mutant obtained by the invention has the advantages of higher catalytic activity, high stability and the like, so that the alcohol dehydrogenase mutant is more suitable for being applied to construction of a high-efficiency (-) -menthol synthesis path in microorganisms, and has a far-reaching prospect.

Drawings

FIG. 1 is a schematic diagram showing the reaction process of the alcohol dehydrogenase mutant of the present invention combined with P450cam to synthesize (-) -isomenthone (Compound 3 a).

Detailed Description

The individual reaction or detection conditions described in the context of the present invention may be combined or modified in accordance with common general knowledge in the art and may be verified experimentally. The technical solutions and technical effects of the present invention will be clearly and completely described in the following in conjunction with specific embodiments, but the scope of the present invention is not limited to these embodiments, and all changes or equivalent substitutions without departing from the concept of the present invention are included in the scope of the present invention.

The sources of materials in the following examples are:

Genome Pseudomonas aeruginosa, which contains the nucleic acid sequence shown in SEQ ID No.1 of the sequence table, is extracted in the laboratory where the inventors are located.

The empty plasmid vector pET28a was purchased from Novagen.

E. coli BL21 (DE 3) competent cells, PRIMESTAR (HS), taq DNA polymerase, agarose gel DNA recovery kit were all purchased from Beijing Tiangen Biochemical technology Co.

The restriction enzymes EcoR I and Hind III are commercially available from NEW ENGLAND Biolabs (NEB).

MM in the examples are in the shorthand of mmol/L.

Unless otherwise indicated, the specific experiments in the following examples were performed according to methods and conditions conventional in the art, or following the commercial specifications of the kit.

Example 1 construction of alcohol dehydrogenase PaIPDH

The alcohol dehydrogenase PaIPDH with the amino acid sequence shown in the sequence table SEQ ID No.2 is constructed by adopting a PCR technology.

The primers used were:

upstream primer sequence: CCGGAATTCATGAGCAAACTTCTTTCCGGCCAGG.

Downstream primer sequence: CCCAAGCTTTCAGATCGCCGTCGC.

Wherein the GAATTC sequence in the upstream primer is EcoR I cleavage site, and the AAGCTT sequence in the downstream primer is HindIII cleavage site.

PCR was performed using the genome Pseudomonas aeruginosa as a template and 2×Taq DNA polymerase. PCR System (20. Mu.L): 10. Mu.L of 2 XTaq DNA polymerase, 1. Mu.L of each of the upstream and downstream primers (10. Mu.M), 1. Mu.L of DMSO, and Pseudomonas aeruginosa. Mu.L of genome were added and sterilized distilled water was added to make up to 20. Mu.L. PCR reaction procedure: (1) pre-denaturation at 94℃for 10min; (2) denaturation at 94℃for 1min; (3) annealing at 55 ℃ for 30s; (4) extending at 72 ℃ for 1min; steps (2) - (4) are carried out for 30 cycles altogether; finally, the product is preserved at 72 ℃ for 10min and 4 ℃. The PCR product is purified and recovered by agarose gel electrophoresis analysis and verification, and the recovered target gene DNA fragment and empty plasmid pET28a are respectively subjected to double enzyme digestion for 6 hours at 37 ℃ by using restriction enzymes EcoR I and Hind III. And (3) performing agarose gel electrophoresis analysis and verification on the double-enzyme-digested product, then performing gel-digested purification and recovery, and connecting the obtained linearized pET28a plasmid with the purified target gene DNA fragment at 16 ℃ by using T4 DNA ligase for overnight. The ligation product was transformed into E.coli BL21 (DE 3) competent cells, and uniformly spread on LB agar plates containing 50. Mu.g/ml kanamycin, and placed in a 37℃incubator for stationary culture for about 12 hours.

Transformants on the transformation plates were picked up by a lance tip into 4mL LB tubes and incubated overnight at 37℃in a shaker at 220 rpm. After verification by sequencing, 50% of glycerol is added for sterilization, thus obtaining the pET28a-PaIPDH recombinant bacteria.

Example 2 construction of the alcohol dehydrogenase PaIPDH mutant by semi-rational design

Based on example 1, paIPDH and its coenzyme NAD ⁺ were subjected to protein crystallization to obtain PaIPDH-NAD ⁺ crystal complex, the PaIPDH structure was subjected to molecular docking with a substrate molecule, and then alanine scanning was performed on the amino acid near the substrate pocket. The amino acid residues around the binding site of the substrate (-) -trans-isopiperitenol in the spatial stereo structure of the alcohol dehydrogenase PaIPDH having the amino acid sequence shown in SEQ ID No.2 of the sequence Listing, aided by software such as Pymol, swiss-model, autodockvina, etc., include: glutamic acid at position 95, glutamic acid at position 97, glutamine at position 98, methionine at position 154, valine at position 189, aspartic acid at position 191, threonine at position 192, methionine at position 194, phenylalanine at position 195, tyrosine at position 199, phenylalanine at position 208. After the sensitive site is obtained from alanine scanning, the activity of the enzyme is further improved through site-directed saturation mutation and combined mutation. Site-directed mutagenesis was performed on the amino acid residues at these sites using site-directed mutagenesis techniques.

The primers used are shown in Table 1:

TABLE 1 primer list

PCR amplification was performed using PRIMESTAR (HS) premix using pET28a-PaIPDH as template. The PCR system is as follows: 2X PRIMESTAR HS premix. Mu.L, 1. Mu.L of each of the upstream and downstream primers, 40ng of pET28a-PaIPDH plasmid, 1. Mu.L of DMSO, and 20. Mu.L of sterilized distilled water were added. PCR reaction procedure: (1) pre-denaturation at 95℃for 3min; (2) denaturation at 98℃for 10s; (3) annealing at 55 ℃ for 15s; (4) extending at 72 ℃ for 6min 20s; steps (2) - (4) are carried out for 18 cycles altogether; finally, the extension is carried out for 10min at 72 ℃. After the reaction, 1. Mu.L of restriction enzyme Dpn I and 2. Mu.L of 2X Cutsmart to 20. Mu.L of PCR product were added, and incubated at 37℃for 2 hours to allow the template to be digested and degraded sufficiently, and the digested product was transformed into E.coli BL21 (DE 3) competent cells, and spread uniformly on LB agar plates containing 50. Mu.g/ml kanamycin, and allowed to stand in an incubator at 37℃for about 12 hours. The resulting monoclonal colonies were picked up into 4mL of LB medium and cultured overnight at 37℃in a 220rpm shaker. After sequencing verification, the recombinant strain of the pET28a-PaIPDH mutant is obtained.

The PaIPDH mutant protein is purified from the broken supernatant of pET28a-PaIPDH mutant recombinant bacteria, and the activity of the expressed protein is measured in a cuvette of 1mL by taking NAD ⁺ as a coenzyme. 1mL of a reaction system (50 mmol/L glycine-sodium hydroxide buffer, pH 10.0) containing 1mmol/L (-) -trans-isomenthol and 10mmol/L NAD ⁺ was preheated to 40 ℃, then an appropriate amount of alcohol dehydrogenase mutant was added, the reaction was incubated at 40 ℃, the absorbance change of NADH at 340nm was detected on a spectrophotometer, the absorbance change value within 1 minute was recorded, and the enzyme activity was calculated.

The activity of the obtained serial alcohol dehydrogenase mutants was found to be improved by site-directed mutagenesis to replace glutamic acid at position 95 of alcohol dehydrogenase PaIPDH with phenylalanine (E95F), valine at position 189 with tryptophan (V189W), phenylalanine at position 195 with tryptophan (F195W), tyrosine at position 199 with valine (Y199V), and the resulting serial alcohol dehydrogenase mutants were found to have improved activity on (-) -trans-isomenthol. Based on this, saturation mutation was performed on some sites and the mutation points were combined to obtain mutants having significantly improved activity on (-) -trans-isomenthol, the sequences of which and the activities of which on (-) -trans-isomenthol are shown in table 2.

TABLE 2 alcohol dehydrogenase mutant sequences and corresponding list of activity improvement

The alcohol dehydrogenase PaIPDH mutant amino acid has one of the following sequences:

EXAMPLE 3 expression and purification of recombinant PaIPDH mutant E95F/V189W/F195W/Y199V

The expression strain E.coli/pET28a-PaIPDH _{E95F/V189W/F195W/Y199V} obtained in example 2 was inoculated into LB liquid medium (tryptone: 10g/L, yeast extract: 5g/L, naCl:10 g/L) containing 50. Mu.g/mL kanamycin, shake-cultured at 37℃for 24 hours at 220rpm, inoculated into 100mL of TB liquid medium (tryptone: 12g/L, yeast extract: 24g/L, glycerol: 4mL/L, KH ₂PO₄：2.31g/L,K₂HPO₄ 12.54.54 g/L) containing 50. Mu.g/mL kanamycin at 1% of inoculum size, shake-cultured at 37℃at 220rpm, and when the optical density OD ₆₀₀ of the culture broth reached 0.6-0.8, induction was performed by adding 0.2mM IPTG, and continuous culture at 16℃for 20-24 hours. After the completion of the culture, the culture broth was centrifuged at 8000 Xg at 4℃to remove the supernatant, and the cells were washed twice with 0.9% physiological saline. Then the cells were resuspended in purified buffer A(0.59g/LNaH₂PO₄·2H₂O,5.8g/L Na₂HPO₄·12H₂O,500mM NaCl,5mM imidazole, 375. Mu.L/L beta-mercaptoethanol, pH 7.4) at a ratio of 1:20 (g/mL). Cells were sonicated after the end of the resuspension (260W, work 4s, intermittent 6 s). The crushed solution was centrifuged at 8000 Xg at 4℃to remove the precipitate, thereby obtaining a supernatant containing the protein. The supernatant containing the target protein was poured onto a nickel column equilibrated in advance with purification buffer a. The purification buffer B(0.59g/L NaH₂PO₄·2H₂O,5.8g/L Na₂HPO₄·12H₂O,500mM NaCl,500mM imidazole, 375 mu L/L beta-mercaptoethanol, pH 7.4-7.6) and the purification buffer A are premixed to be eluted in a gradient with the imidazole concentration of 0-250 mM. SDS-PAGE was performed using a vertical electrophoresis apparatus at a concentration of 12.5% and the eluted proteins collected were detected, which showed that the mutants obtained by the above method had a purity of more than 90% and a molecular weight of about 30 kDa. Collecting proper eluent, ultrafiltering and concentrating at 4 ℃ by using a 10kDa ultrafiltration membrane, removing imidazole by using a purification buffer C(0.59g/L NaH₂PO₄·2H₂O,5.8g/L Na₂HPO₄·12H₂O,200mM NaCl,2mM DTT), ultrafiltering and concentrating to 1mL of protein solution at 4 ℃ by using the 10kDa ultrafiltration membrane, adding 20% glycerol, and standing at-80 ℃ for later use.

EXAMPLE 4 dehydrogenation Activity of recombinant hydroxyl dehydrogenase PaIPDH and mutant E95F/V189W/F195W/Y199V catalytic substrate

The enzyme activity assay was performed in a 1mL cuvette, to 740. Mu.L glycine-NaOH buffer (50 mM, pH 10.0) was added 10. Mu.L 100mM series of substrates (structure shown in Table 3), 200. Mu.L 50mM NAD ⁺, 50. Mu.L diluted pure enzyme. Detecting the absorbance change of NADH at 340nm on a spectrophotometer at 40 ℃, recording the absorbance change value within 1 minute, and calculating the enzyme activity.

TABLE 3 PaIPDH Activity of catalytic series substrates

EXAMPLE 5 catalytic Synthesis of (-) -isomenthone by recombinant hydroxy dehydrogenase PaIPDH _{E95F/V189W/F195W/Y199V}

Two plasmids of different resistances, pET21a-P450cam _Y96F/Y247L -Pdx-Pdr and pET28a-PaIPDH mutant pET28a-PaIPDH _{E95F/V189W/F195W/Y199V}, were introduced into E.coli BL21 (DE), plated onto double-resistant plates and incubated at 37℃for 12h. The resulting monoclonal colonies were picked up and cultured in 4mL LB medium containing 100. Mu.g/mL of ampicillin and 50. Mu.g/mL of kanamycin, and cultured overnight in a shaker at 37℃and 220 rpm. Bacterial liquid was aspirated from the tube at 1% inoculum size and inoculated into shake flask LB medium containing 100. Mu.g/mL of ampicillin and 50. Mu.g/mL of kanamycin, and after incubation at 37℃until OD ₆₀₀ was 0.8, 0.2mM IPTG,0.1mM FeCl ₃ and 0.2mM of 5-aminolevulinic acid were added for induction, followed by further incubation at 180rpm at 16℃for 24 hours. After the completion of the culture, the culture broth was centrifuged at 8000 Xg at 4℃to remove the supernatant, and the cells were washed twice with 0.9% physiological saline. The harvested bacterial pellet was resuspended in 50mM KPB (pH 7.6) at a cell content of 20g/L, and 1L of the cell suspension was added to a 5L three-necked flask, followed by the addition of 158.9mg of (-) -limonene (final concentration 8 mM), reacted at 25℃at 200rpm for 18 hours, with a (-) -isomenthone product concentration of 128.6mg L ^-1. After the reaction, the product was extracted with an equal volume of ethyl acetate, dried over anhydrous sodium sulfate for 8 hours, and the solvent was removed by rotary evaporation under reduced pressure at 20℃to give a yellow crude product. All crude products obtained in the 9 batches of reactions are combined and purified by silica gel column chromatography to obtain the product (-) -isomenthone, wherein the mobile phase is petroleum ether/ethyl acetate, and the ratio is 100:1. The yellowish liquid 529.5mg is separated, the separation yield is 41%, and the purity is more than 92%.

The above examples 4-5 illustrate specific applications of the mutant E95F/V189W/F195W/Y199V (i.e., substitution of phenylalanine at position 95, tryptophan at position 189, tryptophan at position 195, and valine at position 199) in the catalytic substrate dehydrogenation activity, catalytic synthesis of (-) -isomenthone, and the like. It should be noted that: as shown in Table 2, the mutant E95F/V189W/F195W/Y199V is only one of the various alcohol dehydrogenase mutants of the invention, and as can be seen from Table 2, other types of alcohol dehydrogenase mutants have much higher specific activities than those of proteins composed of the amino acid sequences shown in SEQ ID No.2, so those skilled in the art know that other types of alcohol dehydrogenase mutants can catalyze the dehydrogenation reaction activities of substrates, catalyze the synthesis of (-) -isomenthone, and have technical effects superior to those of proteins composed of the amino acid sequences shown in SEQ ID No. 2. The examples of the present invention are described by way of example only with respect to the mutant E95F/V189W/F195W/Y199V.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Sequence listing

<110> University of eastern China, bruce Fu Anse technology Co., ltd

<120> Alcohol dehydrogenase mutant and its use in cyclic terpene ketone synthesis

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 762

<212> DNA

<213> Pseudomonas aeruginosa

<400> 1

atgagcaaac ttctttccgg ccaggtcgcg ctggtcactg gcggtgcggc gggcatcggc 60

cgcgctaccg cgcaggcctt cgccgccgcc ggggtcaagg tggtggtcgc cgacctggac 120

agcgccggcg gcgagggcac ggtcgaagcg atccgccagg caggtggcga agccgtcttc 180

attcgctgcg acgtcacccg cgacgccgag gtcaaggcgc tggtagaggg ttgcgcggcg 240

gcctacggcc gtctcgacta cgccttcaac aacgccggta tcgagatcga gcagggcaag 300

ctggccgacg gcaacgaagc cgagttcgac gccatcatgg ctgtcaacgt gaagggcgtc 360

tggttgtgca tgaagcacca gatcccgctg atgctggccc agggcggtgg cgcgatcgtc 420

aacaccgcct cggtcgccgg gctcggcgcg gcgccgaaga tgagcatcta cgctgcttcc 480

aagcacgcgg tgatcggcct gaccaaatcg gcggcgatcg agtacgcgaa gaagggcatc 540

cgcgtcaacg ccgtgtgtcc ggcggtgatc gacaccgaca tgttccgccg cgcctacgag 600

gccgatccgc gcaaggccga gttcgccgcc gcgatgcatc cgctgggtcg ggtcgggcgg 660

gtcgaagaaa tcgccgccgc ggtgctctat ctgtgcagcg acaacgcagg cttcaccacc 720

ggtatcgcct tgccggtgga cggcggggcg acggcgatct ga 762

<210> 2

<211> 253

<212> PRT

<213> Pseudomonas aeruginosa

<400> 2

Met Ser Lys Leu Leu Ser Gly Gln Val Ala Leu Val Thr Gly Gly Ala

1 5 10 15

Ala Gly Ile Gly Arg Ala Thr Ala Gln Ala Phe Ala Ala Ala Gly Val

20 25 30

Lys Val Val Val Ala Asp Leu Asp Ser Ala Gly Gly Glu Gly Thr Val

35 40 45

Glu Ala Ile Arg Gln Ala Gly Gly Glu Ala Val Phe Ile Arg Cys Asp

50 55 60

Val Thr Arg Asp Ala Glu Val Lys Ala Leu Val Glu Gly Cys Ala Ala

65 70 75 80

Ala Tyr Gly Arg Leu Asp Tyr Ala Phe Asn Asn Ala Gly Ile Glu Ile

85 90 95

Glu Gln Gly Lys Leu Ala Asp Gly Asn Glu Ala Glu Phe Asp Ala Ile

100 105 110

Met Ala Val Asn Val Lys Gly Val Trp Leu Cys Met Lys His Gln Ile

115 120 125

Pro Leu Met Leu Ala Gln Gly Gly Gly Ala Ile Val Asn Thr Ala Ser

130 135 140

Val Ala Gly Leu Gly Ala Ala Pro Lys Met Ser Ile Tyr Ala Ala Ser

145 150 155 160

Lys His Ala Val Ile Gly Leu Thr Lys Ser Ala Ala Ile Glu Tyr Ala

165 170 175

Lys Lys Gly Ile Arg Val Asn Ala Val Cys Pro Ala Val Ile Asp Thr

180 185 190

Asp Met Phe Arg Arg Ala Tyr Glu Ala Asp Pro Arg Lys Ala Glu Phe

195 200 205

Ala Ala Ala Met His Pro Leu Gly Arg Val Gly Arg Val Glu Glu Ile

210 215 220

Ala Ala Ala Val Leu Tyr Leu Cys Ser Asp Asn Ala Gly Phe Thr Thr

225 230 235 240

Gly Ile Ala Leu Pro Val Asp Gly Gly Ala Thr Ala Ile

245 250

<210> 3

<211> 2879

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60

gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120

gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180

ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240

cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagcctt tgacttcatt 300

cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360

ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420

gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480

atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540

ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600

ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660

atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720

aggatgtgtg gcctgttact gctgggcggc ctggatacgg tggtcaattt cctcagcttc 780

agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840

cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900

atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960

ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020

ttcagtcgcc aaaaggtttc acacaccacc tttggccacg gcagccatct gtgccttggc 1080

cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140

gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200

caggcactcc ctctggtctg ggatccggcg actaccaaag cggtatgaga attcaaggag 1260

atataccatg tctaaagtag tgtatgtgtc acatgatgga acgcgtcgcg aactggatgt 1320

ggcggatggc gtcagcctga tgcaggctgc agtctccaat ggtatctacg atattgtcgg 1380

tgattgtggc ggcagcgcca gctgtgccac ctgccatgtc tatgtgaacg aagcgttcac 1440

ggacaaggtg cccgccgcca acgagcggga aatcggcatg ctggagtgcg tcacggccga 1500

actgaagccg aacagcaggc tctgctgcca gatcatcatg acgcccgagc tggatggcat 1560

cgtggtcgat gttcccgata ggcaatggta agagctcaag gagatatacc atgaacgcaa 1620

acgacaacgt ggtcatcgtc ggtaccggac tggctggcgt tgaggtcgcc ttcggcctgc 1680

gcgccagcgg ctgggaaggc aatatccggt tggtggggga tgcgacggta attccccatc 1740

acctaccacc gctatccaaa gcttacttgg ccggcaaagc cacagcggaa agcctgtacc 1800

tgagaacccc agatgcctat gcagcgcaga acatccaact actcggaggc acacaggtaa 1860

cggctatcaa ccgcgaccga cagcaagtaa tcctatcgga tggccgggca ctggattacg 1920

accggctggt attggctacc ggagggcgtc caagacccct accggtggcc agtggcgcag 1980

ttggaaaggc gaacaacttt cgatacctgc gcacactcga ggacgccgag tgcattcgcc 2040

ggcagctgat tgcggataac cgtctggtgg tgattggtgg cggctacatt ggccttgaag 2100

tggctgccac cgccatcaag gcgaacatgc acgtcaccct gcttgatacg gcagcccggg 2160

ttctggagcg ggttaccgcc ccgccggtat cggcctttta cgagcaccta caccgcgaag 2220

ccggcgttga catacgaacc ggcacgcagg tgtgcgggtt cgagatgtcg accgaccaac 2280

agaaggttac tgccgtcctc tgcgaggacg gcacaaggct gccagcggat ctggtaatcg 2340

ccgggattgg cctgatacca aactgcgagt tggccagtgc ggccggcctg caggttgata 2400

acggcatcgt gatcaacgaa cacatgcaga cctctgatcc cttgatcatg gccgtcggcg 2460

actgtgcccg atttcacagt cagctctatg accgctgggt gcgtatcgaa tcggtgccca 2520

atgccttgga gcaggcacga aagatcgccg ccatcctctg tggcaaggtg ccacgcgatg 2580

aggcggcgcc ctggttctgg tccgatcagt atgagatcgg attgaagatg gtcggactgt 2640

ccgaagggta cgaccggatc attgtccgcg gctctttggc gcaacccgac ttcagcgttt 2700

tctacctgca gggagaccgg gtattggcgg tcgatacagt gaaccgtcca gtggagttca 2760

accagtcaaa acaaataatc acggatcgtt tgccggttga accaaaccta ctcggtgacg 2820

aaagcgtgcc gttaaaggaa atcatcgccg ccgccaaagc tgaactgagt agtgcctga 2879

Claims

1. An alcohol dehydrogenase mutant characterized in that the amino acid sequence is selected from any one of the following:

(1) The 199 th tyrosine of the amino acid sequence shown in SEQ ID No.2 is replaced by valine;

(2) Substitution of methionine at position 194 with lysine and tyrosine at position 199 with tryptophan of the amino acid sequence shown in SEQ ID No. 2;

(3) Substitution of methionine at position 194 with lysine, tyrosine at position 199 with histidine, phenylalanine at position 208 with histidine of the amino acid sequence shown in SEQ ID No. 2;

(4) The 95 th glutamic acid of the amino acid sequence shown in SEQ ID No.2 is replaced by phenylalanine, the 189 th valine is replaced by tryptophan, the 195 th phenylalanine is replaced by tryptophan, and the 199 th tyrosine is replaced by valine.

2. An isolated nucleic acid, wherein the nucleic acid is a nucleic acid molecule encoding the alcohol dehydrogenase mutant of claim 1.

3. A recombinant expression plasmid comprising the nucleic acid of claim 2.

4. A recombinant expression transformant comprising the recombinant expression plasmid of claim 3.

5. A recombinant alcohol dehydrogenase mutant catalyst, wherein the recombinant alcohol dehydrogenase mutant catalyst is in any one of the following forms:

(1) Culturing the recombinant expression transformant according to claim 4, and isolating a transformant cell containing the alcohol dehydrogenase mutant;

(2) Culturing the recombinant expression transformant according to claim 4, and isolating a crude enzyme solution containing the alcohol dehydrogenase mutant;

(3) Culturing the recombinant expression transformant according to claim 4, separating a crude enzyme solution containing the alcohol dehydrogenase mutant, and freeze-drying the crude enzyme solution of the alcohol dehydrogenase mutant to obtain a crude enzyme powder.

6. Use of the alcohol dehydrogenase mutant according to claim 1 or the recombinant alcohol dehydrogenase mutant catalyst according to claim 5 for catalyzing the dehydrogenation of hydroxyl compounds;

the hydroxyl compound is selected from any one of the following compounds:

7. the use according to claim 6, wherein the alcohol dehydrogenase mutant or recombinant alcohol dehydrogenase mutant catalyst is used for catalyzing the dehydrogenation of cyclic terpene alcohols having hydroxyl groups in the 3-and 6-positions to produce cyclic terpene ketones.

8. The use according to claim 6, wherein the alcohol dehydrogenase mutant or recombinant alcohol dehydrogenase mutant catalyst is used to catalyze the dehydrogenation of (-) -trans-isomenthol to produce (-) -isomenthone.