An alcohol dehydrogenase mutant and application thereof in synthesis of 4-hydroxy-2-butanone

Abstract

This invention discloses an alcohol dehydrogenase mutant and its application in the synthesis of 4-hydroxy-2-butanone, belonging to the field of enzyme engineering technology. Based on alcohol dehydrogenase, this invention performs site-directed mutagenesis on amino acids at positions 246 and 296, mutating glutamic acid at position 246 to proline and threonine at position 296 to proline. The amino acid sequence of the mutant is shown in SEQ ID NO.1. After incubation at 60°C for 48 hours, its residual enzyme activity is approximately 40.27% of the initial enzyme activity, about 5.75 times that of the wild type. This is similar to the NADH oxidase mutant LrNox from *Lactobacillus rhamnosus*. L179S The coupling synthesis of 4-hydroxy-2-butanone, combined with a substrate addition strategy, resulted in a final accumulation of 2.2 g / L of 4-hydroxy-2-butanone, laying the foundation for subsequent research on the efficient biosynthesis of 4-hydroxy-2-butanone and its derivatives.

Description

Technical Field

[0001] This invention relates to an alcohol dehydrogenase mutant and its application in the synthesis of 4-hydroxy-2-butanone, belonging to the field of enzyme technology. Background Technology

[0002] 4-Hydroxy-2-butanone (4H2B) and its derivative 3-buten-2-one are important intermediates in the synthesis of steroids, pesticides, anticancer drugs, and camptothecin. They can form complexes with Ziegler-Natta catalysts for the stereospecific polymerization of butadiene and are intermediates in organic synthesis. They are also intermediates in the synthesis of fragrances such as raspberry ketone and anisylacetone, and can be used to synthesize the important chiral intermediate (R)-3-aminobutanol, an anti-HIV drug dulutevir. Currently, the industrial production of 4H2B is mainly based on chemical methods, but these methods have drawbacks such as cumbersome reaction steps, numerous byproducts, and the generation of toxic compounds. There are few reports on the biological synthesis of 4H2B; therefore, there is an urgent need for an environmentally friendly biocatalyst with good activity to produce 4H2B.

[0003] Alcohol dehydrogenases catalyze reversible reactions between aldehydes (ketones) and alcohols, typically at temperatures between 30 and 65°C. However, within these temperatures, alcohol dehydrogenases exhibit limited thermostability and are prone to inactivation. Therefore, the thermostability of enzymes is crucial for industrial production. The optimal temperature for alcohol dehydrogenases derived from *Methanococcus faecium* is 60°C. After incubation at this temperature for 4 hours, the residual enzyme activity is only about 50% of the initial activity. Incubation at 37°C for 12 hours reaches the enzyme's half-life, which is unfavorable for industrial production. Therefore, improving the thermostability of alcohol dehydrogenases is urgently needed to enhance their catalytic efficiency and promote industrial applications. Summary of the Invention

[0004] Technical Problem: To solve the above-mentioned technical problem, the present invention provides an alcohol dehydrogenase mutant and its application in the production of 4-hydroxy-2-butanone.

[0005] Technical solution: This invention provides an alcohol dehydrogenase mutant of *Methanococcus methanans* as the parent, a recombinant bacterium containing the alcohol dehydrogenase mutant, and its application. The thermostability of the alcohol dehydrogenase mutant of this invention is improved. When it is constructed into *Escherichia coli*, it can undergo long-term enzymatic conversion to synthesize 4H2B, laying the foundation for subsequent research on efficient biosynthesis of 4H2B and its derivatives.

[0006] This invention provides an alcohol dehydrogenase mutant, wherein the mutant is formed by mutating glutamic acid at position 246 of the alcohol dehydrogenase shown in SEQ ID NO.3 to proline, and is named MeADH. E246P .

[0007] This invention provides an alcohol dehydrogenase mutant, wherein the mutant is formed by mutating the threonine at position 296 of the alcohol dehydrogenase enzyme as shown in SEQ ID NO.3 to proline, MeADH. T296P .

[0008] This invention provides an alcohol dehydrogenase mutant, wherein the mutant has its amino acid sequence as shown in SEQ ID NO.3, consisting of mutating glutamic acid at position 246 to proline and simultaneously mutating threonine at position 296 to proline, and is named MeADH. E246P / T296P The alcohol dehydrogenase mutant MeADH E246P / T296P The amino acid sequence is shown in SEQ ID NO.1; encoding the alcohol dehydrogenase mutant MeADH. E246P / T296P The nucleotide sequence is shown in SEQ ID NO.2.

[0009] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme of the alcohol dehydrogenase is shown in SEQ ID NO.4.

[0010] The present invention also provides a gene encoding the above-mentioned alcohol dehydrogenase mutant.

[0011] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0012] In one embodiment of the present invention, the recombinant expression vector uses pET28a as the expression vector.

[0013] The present invention also provides recombinant cells expressing the above-mentioned dipeptidase mutant, or carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

[0014] In one embodiment of the present invention, the recombinant cells are expressed using bacteria or fungi as expression hosts.

[0015] In one embodiment of the present invention, the recombinant cells are expressed using Escherichia coli as the expression host.

[0016] In one embodiment of the present invention, the recombinant cells use Escherichia coli BL21(DE3) as the host strain.

[0017] This invention also provides a method for preparing 4H2B, wherein the method comprises using the above-mentioned mutant or the above-mentioned recombinant cell or the recombinant alcohol dehydrogenase expressed by the recombinant cell as a catalyst, 1,3-butanediol as a substrate, and simultaneously introducing the NADH oxidase mutant LrNox from Lactobacillus rhamnosus. L179S Building NADP + The coenzyme cycle system was used to prepare 4H2B through a reaction.

[0018] In one embodiment of the present invention, the LrNox L179S The amino acid sequence is shown in SEQ ID NO.5, encoding the LrNox. L179S The base sequence is shown in SEQ ID NO.6.

[0019] In one embodiment of the present invention, the amount of mutant or recombinant alcohol dehydrogenase added is 5-10 U.

[0020] In one embodiment of the present invention, the amount of the mutant or the recombinant alcohol dehydrogenase added is 10U.

[0021] In one embodiment of the present invention, the reaction conditions are: reaction at 37°C for 24–48 h.

[0022] In one embodiment of the present invention, the reaction conditions are: reaction at 37°C for 48 hours.

[0023] The present invention also provides the application of the above-mentioned mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned preparation method in the preparation of 4H2B and its derivatives.

[0024] Beneficial effects:

[0025] (1) The present invention obtains a mutant by replacing glutamic acid at position 246 and threonine at position 296 in the sequence SEQ ID NO.3 with proline, wherein the mutant MeADH E246P / T296P After incubation at 60℃ for 48 hours, its residual enzyme activity is about 40.27% of the initial enzyme activity, which is about 5.36 times that of the wild type, making it more suitable for industrial production.

[0026] (2) MeADH, the alcohol dehydrogenase mutant of the present invention E246P / T296P The thermal stability of the 4-hydroxy-2-butanone was significantly improved compared to the wild type, and it was applied to the synthesis of 4H2B. After optimization of enzyme conversion conditions and substrate addition strategy, the final synthesis yield of 4-hydroxy-2-butanone reached 2.2 g / L, laying the foundation for subsequent research on the efficient biosynthesis of 4-hydroxy-2-butanone and its derivatives. Attached Figure Description

[0027] Figure 1 SDS-PAGE images of wild-type and mutant cells: Lane 1: Protein Marker; Lane 2: Control of E. coli BL21 / pET-28a empty vector cell lysate supernatant; Lane 3: Control of E. coli BL21 / pET28a-MeADH cell lysate supernatant; Lane 4: Control of E. coli BL21 / pET28a-MeADH cell lysate. E246PCell lysis supernatant; Lane 5: E. coli BL21 / pET28a-MeADH G222D Cell lysis supernatant; Lane 6: E. coli BL21 / pET28a-MeADH T310K Cell lysis supernatant; Lane 7: E. coli BL21 / pET28a-MeADH A307R Cell lysis supernatant; Lane 8: E. coli BL21 / pET28a-MeADH G292T Cell lysis supernatant; Lane 9: E. coli BL21 / pET28a-MeADH N117D Cell lysis supernatant; Lane 10: E. coli BL21 / pET28a-MeADH K201P Cell lysis supernatant; Lane 11: E. coli BL21 / pET28a-MeADH D51P Cell lysis supernatant; Lane 12: E. coli BL21 / pET28a-MeADH D97R Cell lysis supernatant; Lane 13: E. coli BL21 / pET28a-MeADH I156F Cell lysis supernatant; Lane 14: E. coli BL21 / pET28a-MeADH Q157M Cell lysis supernatant; Lane 15: E. coli BL21 / pET28a-MeADH S187R Cell lysis supernatant; Lane 16: E. coli BL21 / pET28a-MeADH K234Q Cell lysis supernatant; Lane 17: E. coli BL21 / pET28a-MeADH T295M Cell lysis supernatant; Lane 18: E. coli BL21 / pET28a-MeADH T296P Cell disruption supernatant.

[0028] Figure 2 Comparison of enzyme activities between wild-type and mutant strains.

[0029] Figure 3 Comparison of thermal stability between wild type and mutant.

[0030] Figure 4 Comparison of thermal stability between wild type and mutant after incubation at 37℃ for 24 hours.

[0031] Figure 5 Results of optimization of the two-enzyme system conditions.

[0032] Figure 6 : 4H2B synthesis results under the bottom logistics plus strategy. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] The culture media involved in the following examples are as follows:

[0035] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0036] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L.

[0037] The detection methods involved in the following embodiments are as follows:

[0038] Assay for alcohol dehydrogenase activity: Reaction system: The reaction solution was 0.1 mol / L Gly-NaOH buffer (pH 10), containing 200 mmol / L 1,3-butanediol (1,3-BDO) solution and 1 mmol / L NADP. + Add 20 μL of the diluted enzyme solution to be tested, and use a UV spectrophotometer to detect the change in absorbance at 340 nm.

[0039] Enzyme activity unit (U) is defined as: 1 μmol of NADP consumed in 1 minute. + The required amount of enzyme is 1 unit of enzyme activity (U).

[0040] The enzyme activity is the enzyme activity of 1 mg of wet cells, and the unit is U / mg.

[0041] Determination of the thermostability of alcohol dehydrogenase: The enzyme was incubated in a 60℃ water bath for 24 hours, with samples taken at regular intervals. Under optimal reaction conditions, the residual enzyme activity was measured after incubation for a certain period. The relative enzyme activity of each sample was calculated with the initial enzyme activity as 100%. The temperature stability of alcohol dehydrogenase was then investigated.

[0042] Detection methods for 4-hydroxy-2-butanone and 1,3-butanediol: Both 4-hydroxy-2-butanone and 1,3-butanediol require gas chromatography (GC) and the detection methods are identical. Before detection, the sample is extracted with ethyl acetate, dehydrated with anhydrous sodium sulfate, and filtered. The treated sample is then analyzed by GC using an HP-Innowax (60m × 0.25mm × 0.5μm) column. Detection conditions are set as follows: initial temperature 50℃, hold for 1 min, then increase to 230℃ at a rate of 10℃ / min, hold for 12 min. Injector temperature: 250℃. Carrier gas: helium (purity ≥99.999%), flow rate 1 mL / min. Injection method: pulsed splitless injection; injection pulse pressure: 5 kPa; time: 2.00 min; injection volume: 1 μL.

[0043] Example 1: Construction and expression of wild-type alcohol dehydrogenase

[0044] The specific steps are as follows:

[0045] (1) Chemically synthesized nucleotide sequences as shown in SEQ ID NO.4 for the alcohol dehydrogenase gene MeADH.

[0046] (2) The expression plasmid pET28a was digested with restriction endonuclease BamH I / EcoR I to obtain a linearized vector. The gene MeADH digested in the same way and the linearized pET28a were ligated to obtain the recombinant plasmid pET28a-MeADH.

[0047] (3) The recombinant plasmid pET28a-MeADH was transformed into Escherichia coli BL21(DE3) using chemical transformation. The transformed plasmid was plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Randomly selected clones were then subjected to colony PCR identification and sequencing verification. The results showed that the recombinant plasmid pET28a-MeADH had been successfully transformed into Escherichia coli BL21(DE3). The recombinant plasmid was extracted from the bacterial culture that confirmed the successful mutation and stored at -20°C. Sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0048] (4) The prepared recombinant strain E. coli BL21 / pET28a-MeADH was inoculated into 10 mL of LB liquid medium and cultured at 37℃ and 200 r / min for 10 h to prepare seed culture; the prepared seed culture was transferred into 50 mL of LB liquid medium at an inoculation rate of 1% (v / v) and cultured at 37℃ and 200 r / min for 2 h. When OD 600When the concentration of β-D-thiogalactoside was approximately 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to bring the final concentration to 0.5 mM. Alcohol dehydrogenase expression was then induced at 16°C and 200 rpm. After 12 h of culture, the bacterial culture was collected, cells were washed with PBS, centrifuged, and then collected. The cells were diluted with PBS to OD0.05. 600 =15 minutes, then ultrasonically disrupted: disrupt for 1 second, pause for 3 seconds, for 15 minutes. After disruption, centrifuge at 4℃ and 12000 rpm for 20 minutes to separate the supernatant and precipitate. The supernatant is the crude enzyme solution, and the enzyme activity of the crude enzyme solution is 6.94 U ± 3.6.

[0049] (5) The crude enzyme was purified by Ni-NTA affinity chromatography to obtain a pure enzyme solution of wild-type alcohol dehydrogenase MeADH. The specific activity of the pure enzyme was 1.87 U / mg ± 2.7.

[0050] Example 2: Construction and expression of alcohol dehydrogenase mutant

[0051] The specific steps are as follows:

[0052] Mutation primers were designed to construct MeADH single mutant recombinant plasmids. The relevant primers and their functions are shown in Table 1.

[0053] Using the plasmid pET28a-MeADH constructed in Example 1 as a template, a single mutant plasmid was constructed using the primers described in Table 1. Taking the construction of the plasmid expressing the single mutant T296P as an example, the wild-type plasmid pET28a-MeADH was used as a template, and P23 / P24 were used as primers to construct the plasmid pET28a-MeADH expressing the single mutant T296P by PCR. T296P .

[0054] Based on this, using a single mutant recombinant plasmid as a template, double mutant plasmids and triple mutant plasmids were further constructed using the primers listed in Table 1. Taking the construction of the plasmid expressing the double mutant T296P / E246P as an example, and the construction of the mutant plasmid pET28a-MeADH expressing the single mutant as an example... T296P Using P19 / P20 as primers, the plasmid pET28a-MeADH expressing the double mutant T296P / E246P was constructed by PCR. T296P / E246P .

[0055] Table 1: Mutant primers and their functions

[0056]

[0057]

[0058] The mutant recombinant plasmid constructed above was expressed in *E. coli* BL21(DE3) using the same method as in Example 1, thus obtaining the corresponding mutant recombinant bacterium. The recombinant bacterium expressing the double mutant T296P / E246P was named *E. coli* BL21 / pET28a-MeADH. T296P / E246P .

[0059] Following the method described in Example 1, each of the above-mentioned mutant recombinant strains was inoculated into 10 mL of LB medium and cultured at 37°C and 200 rpm for 10 h to prepare seed culture. The prepared seed culture was then transferred to 50 mL of LB medium at an inoculation rate of 1% (v / v) and cultured at 37°C and 200 rpm for 2 h. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and alcohol dehydrogenase expression was performed at 16°C and 200 rpm. After culturing for 12 h, the bacterial culture was collected, cells were washed with PBS, centrifuged, and the cells were collected. The cells were diluted with PBS to OD0.05. 600 =15, perform ultrasonic disruption: disrupt for 1 second, pause for 3 seconds, for 15 minutes. After disruption, centrifuge at 4℃ and 12000 rpm for 20 minutes to separate the cell wall supernatant and cell wall precipitate; the supernatant is the crude enzyme solution.

[0060] SDS-PAGE validation of wild-type and mutant crude enzymes yielded the following results: Figure 1 As shown in the figure, the results indicate that both wild-type and mutant strains can be successfully expressed.

[0061] Example 3: Comparison of the thermostability of wild-type enzymes and mutant enzymes

[0062] The specific steps are as follows:

[0063] 1. Comparison of enzyme activities among mutants

[0064] The crude enzyme obtained in Example 2 was added with 10% glycerol and stored at 4°C for later use. First, the mutants were preliminarily screened, and the OD values ​​of wild-type and mutant alcohol dehydrogenase cells were experimentally determined. 600 The enzyme activity of the crude enzyme solution obtained under the condition of 15 was compared when reacted with 1,3-butanediol at 60℃ and pH 10.0, excluding mutants with significantly reduced activity compared to the wild type. The results are as follows: Figure 2 As shown.

[0065] Table 2 Comparison of enzyme activity between wild-type and mutant strains

[0066] Wild type / mutant Enzyme activity U Enzyme activity U / mg wild type 6.94±3.6 1.87±2.7 <![CDATA[MeADH D51P ]]> 5.54±1.2 1.49±0.92 <![CDATA[MeADH D97R ]]> 5.52±2.9 1.52±2.2 <![CDATA[MeADH N117D ]]> 7.29±3.8 1.96±2.5 <![CDATA[MeADH I156F ]]> 6.04±3.1 1.63±2.3 <![CDATA[MeADH Q157M ]]> 6.17±3.2 1.66±2.6 <![CDATA[MeADH S187R ]]> 6.11±3.2 1.65±2.5 <![CDATA[MeADH K201P ]]> 5.21±2.7 1.40±0.88 <![CDATA[MeADH G222D ]]> 5.41±2.8 1.46±0.89 <![CDATA[MeADH K234Q ]]> 4.93±2.5 1.33±0.76 <![CDATA[MeADH E246P ]]> 6.18±3.3 1.84±2.4 <![CDATA[MeADH G292T ]]> 4.72±2.4 1.27±0.55 <![CDATA[MeADH T295M ]]> 2.77±1.4 0.75±0.22 <![CDATA[MeADH T296P ]]> 5.01±2.6 1.35±0.78 <![CDATA[MeADH A307R ]]> 6.31±3.3 1.71±1.7 <![CDATA[MeADH T310K ]]> 5.27±2.7 1.42±0.82 <![CDATA[MeADH T296P / E246P ]]> 4.33±2.7 1.17±0.35 <![CDATA[MeADH T296P / G292T ]]> 2.08±1.1 0.56±0.12 <![CDATA[MeADH E246P / G292T ]]> 2.77±1.4 0.75±0.24 <![CDATA[MeADH T296P / E246P / G292T ]]> 2.43±1.3 0.65±0.23

[0067] 2. Comparison of thermal stability

[0068] Thermal stability at 60℃:

[0069] After incubation at 60℃ for 48 hours, the residual enzyme activity of each mutant was compared with the initial enzyme activity. The results are as follows: Figure 3 As shown, the results indicate that the mutant MeADH E246P MeADH G292T MeADH T296P All had higher residual enzyme activities than the wild type, among which the mutant MeADH T296P After incubation at 60℃ for 48 hours, 34.55% of the enzyme activity remained; the mutant MeADHE 246P / T296P After incubation at 60℃ for 48 hours, the mutant MeADHE still retained 40.27% enzyme activity, compared to the wild type (7.51% residual enzyme activity). 246P / T296P The residual enzyme activity increased by 5.36 times.

[0070] Thermal stability at 37℃:

[0071] Wild type and mutant MeADH E246P / T296P The thermal stability changes after incubation at 37℃ for 24 hours are as follows: Figure 4 As shown. The results showed that after incubation at 37℃ for 24 hours, the residual enzyme activity of the wild type was approximately 61.22% of the initial enzyme activity, while that of the mutant MeADH... E246P / T296P After incubation at 37°C for 24 hours, its residual enzyme activity was approximately 74.55% of the initial enzyme activity.

[0072] Example 4: E. coli BL21 / pET28a-MeADH T296P / E246P Coupled NADH oxidase mutant LrNox L179S Synthesis of 4-hydroxy-2-butanone

[0073] E. coli BL21 / pET28a-MeADH from Example 2 E246P / T296P The expressed mutant enzyme is coupled with LrNox L179S The reaction system for the synthesis of 4-hydroxy-2-butanone is as follows:

[0074] (1) E. coli BL21 / pET28a-MeADH T296P / E246P Preparation of mutant enzyme catalysts

[0075] Recombinant bacteria E. coli BL21 / pET28a-MeADH and E. coli BL21 / pET28a-MeADH were cultured according to the method described in Example 1. T296P / E246P The cells were broken down to obtain a crude enzyme solution.

[0076] (2)LrNox L179S Preparation of mutant enzyme catalysts

[0077] LrNox was prepared according to the method described in the literature "Switching the substrate specificity from NADH to NADPH by a single mutation of NADH oxidase from Lactobacillus rhamnosus". L179S The enzyme is mutated, specifically by modifying the LrNox gene. L179S The recombinant plasmid pET28a-LrNox was constructed by inserting the BamHI and EcoRI sites into the pET-28a plasmid. L179S The LrNox L179S The amino acid sequence is shown in SEQ ID NO.5, encoding the LrNox. L179S The nucleotide sequence is shown in SEQ ID NO.6, and SEQ ID NO.7 is LrNox containing the restriction enzyme site (shown in bold underline). L179S Nucleotide sequence.

[0078] SEQ ID NO.7:

[0079] GGATCC GAATTC.

[0080] The constructed pET28a-LrNox L179S E. coli BL21 / pET28a-LrNox was constructed by introducing the chemical transformation method into E. coli BL21 competent cells. L179S The recombinant bacteria were used to obtain a crude enzyme solution according to the method in Example 1. After purification using Ni-NTA affinity chromatography, LrNox was obtained. L179S The enzyme activity of the mutant enzyme fermentation broth was 3.02 U, and the specific enzyme activity was 6.3 U / mg ± 1.8.

[0081] (3) Preparation of 4H2B by a two-enzyme system reaction

[0082] Using 1,3-butanediol at a final concentration of 4 g / L as the substrate, in a 100 mL system (1,3-butanediol: 4 g / L, NADP...), + : 1mM, FAD: 0.1mM, DTT: 1mM, buffer pH 8.0 (Tris-HCl) Temperature controlled at 30–60℃, pH 7.0–10.0, with MeADH T296P / E246P With LrNox L179S The enzyme activity addition ratio is 2:3 (in the reaction system, MeADH... T296P / E246P The enzyme activity is 10U, LrNox L179S A conversion experiment was conducted using an enzyme with an activity of 15 U to explore the optimal reaction conditions for the two-enzyme system. After 96 hours of reaction, the relative concentration of the product in the reaction system was measured, and the results are as follows: Figure 5 As shown: Select the mutant MeADHE 246P / T296P Coupled with LrNox L179S After optimization of the conversion conditions for the synthesis of 4-hydroxy-2-butanone, the optimal temperature was 37℃ and the optimal pH was pH 8.0.

[0083] (4) Preparation of 4H2B

[0084] Using 1,3-butanediol at a final concentration of 4 g / L as the substrate, in a 100 mL system (1,3-butanediol: 4 g / L, NADP...), + The conversion experiment was conducted in a buffer solution (Tris-HCl) at 37°C and pH 8.0, with the concentrations: 1 mM FAD, 0.1 mM DTT, 1 mM, and pH 8.0. MeADH was used as the buffer. T296P / E246P With LrNox L179S The enzyme activity was added at a ratio of 2:3 for conversion (in the reaction system, MeADH... T296P / E246P The enzyme activity is 10U, LrNox L179SThe enzyme activity was 15 U. Every 12 hours, 1,3-butanediol and an equal amount of mixed enzyme were added to a final concentration of 2 g / L, maintaining a 2:3 ratio of the two enzyme activities to continue the reaction. The total volume of enzyme added was 20 mL. After 96 hours of reaction, 2.2 g / L of 4H₂B was finally obtained. The accumulation process of the product is as follows: Figure 6 As shown.

[0085] The results showed that the thermostability of the mutant enzyme was significantly improved, and it provided a foundation for the study of biosynthesis of 4-hydroxy-2-butanone and its derivatives.

[0086] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The mutant is formed by mutating glutamic acid at position 246 of the alcohol dehydrogenase shown in SEQ ID NO. 3 to proline, and threonine at position 296 of the alcohol dehydrogenase shown in SEQ ID NO. 3 to proline.

2. The gene encoding the alcohol dehydrogenase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the alcohol dehydrogenase mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells use bacteria as the expression host.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells used Escherichia coli as the expression host.

7. A method for preparing 4-hydroxy-2-butanone, characterized in that, The method involves using the alcohol dehydrogenase mutant of claim 1 with an enzyme activity addition ratio of 2:3 and LrNox. L179S Using 1,3-butanediol as a catalyst, 4-hydroxy-2-butanone was prepared by reaction; the LrNox L179S The amino acid sequence is shown in SEQ ID NO.

5.

8. The method according to claim 7, characterized in that, The reaction conditions are: react at 25-60℃ for at least 24 hours.

9. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant cell of any one of claims 4 to 6 in the preparation of 4-hydroxy-2-butanone.

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