Algoriella xinjiangensis source enzyme AxSDR gene, recombinant bacterium containing gene and application of recombinant bacterium

Abstract

The invention discloses an AxSDR (Adenosine Diphosphate Dehydrogenase Regulator) gene of an Algaea xinjiangensis source enzyme, a recombinant bacterium containing the gene and an application of the recombinant bacterium and the AxSDR gene of the Algaea xinjiangensis source enzyme. The nucleotide sequence of the enzyme AxSDR gene is as shown in SEQ ID NO. 1; the amino acid sequence of the enzyme protein coded by the enzyme AxSDR gene is SEQ ID NO. 2. The AxSDR gene is used for constructing a recombinant plasmid, and then the recombinant plasmid is introduced into E.coli BL21 (DE3) to obtain a gene recombinant bacterium containing the gene. Enzyme protein coded by the AxSDR gene or recombinant bacteria containing the gene can catalytically reduce 1500 mmol / L of m-trifluoromethyl acetophenone to prepare a chiral compound (R)-1-(3-trifluoromethyl phenyl) ethanol, conversion of a high-concentration (282g / L) substrate is achieved, the reaction lasts for 11 hours, the product yield is 99.6%, and the enantiomer excess value is 99% or above.

Description

Technical Field

[0001] The present invention relates to genetic engineering, specifically to the AxSDR gene of the enzyme from Algoriella xinjiangensis, a recombinant bacterium containing this gene, and its applications. Background Art

[0002] The technology for the efficient synthesis of chiral compounds is a core development direction in the chemical industry and the pharmaceutical field, and its technical level directly reflects the intensification ability of a country's high-end manufacturing industry and its economic development level. Chiral aromatic alcohols (characterized by a chiral carbon atom connected to a hydroxyl group and an aromatic ring substituent simultaneously) are key intermediates for constructing chiral active molecules such as neuroprotective agents, antiviral drugs, and anticancer drugs. For example, (R)-1-(3-trifluoromethylphenyl)ethanol ( Figure 1 (shown in red) is the core chiral building block for synthesizing the neuroprotective agent (R)-3-(1-(3-(tris(trifluoromethyl)phenyl)ethoxy)azetidine-1-carboxamide (such as Figure 1 (shown), and its efficient preparation technology has important industrialization value.

[0003] Currently, industrial production still mainly relies on chemical asymmetric catalysis, but there are obvious limitations as follows:

[0004] (1) The cost of the catalytic system is high: Ruthenium / rhodium-based chiral complexes (such as BINAP-Ru catalysts) need to be used, and the cost of the catalyst in a single batch accounts for 40%-60%;

[0005] (2) The process efficiency is low: The substrate loading is only 0.1 g / L, and the enantiomeric excess value (e.e.) is often lower than 95%;

[0006] (3) The environmental compatibility is poor: Strongly acidic reaction media (such as concentrated sulfuric acid) lead to problems of equipment corrosion and waste liquid treatment.

[0007] In recent years, biocatalysis technology has shown significant advantages by asymmetrically reducing prochiral ketones to chiral alcohols through enzymatic reactions. Using Microbacterium oxydans C3 whole cells, the ketoreductase (KRED) from Kluyveromyces thermotolerans, and the mutant enzyme (LXCAR-S154Y) from Leifsonia xyli HS0904 as catalysts, the biosynthesis of (R)-1-(3-trifluoromethylphenyl)ethanol has been achieved. However, there are still the following key bottlenecks in the existing biocatalytic systems:

[0008] (1) Insufficient substrate tolerance: The highest substrate loading is only 38 g / L, far lower than the industrial requirement (≥100 g / L);

[0009] (2) Limited enzyme stability: Free enzymes are easily inactivated during continuous reactions (half-life < 24 h);

[0010] (3) High cost of product separation: The aqueous reaction system leads to an increase in the energy consumption for product extraction (accounting for 30%-40% of the total cost).

[0011] Typical cases show that when using immobilized LXCAR-S154Y to catalyze the reduction of 3-trifluoromethylacetophenone, although a product yield of 91.5% and an e.e. greater than 99% can be obtained, its substrate loading (38 g / L) can only meet the requirements of laboratory scale. This gap highlights the urgency of improving enzyme catalytic efficiency, developing engineering strains resistant to high concentrations of substrates, and optimizing reaction processes. Summary of the Invention

[0012] Aiming at the technical problems existing in the asymmetric reduction of chiral compounds (such as (R)-1-(3-trifluoromethylphenyl)ethanol) by most enzyme catalysts in the prior art, such as insufficient catalytic activity, poor substrate tolerance, and low initial substrate concentration, the present invention provides an enzyme gene from Algoriella xinjiangensis (named AxSDR gene), a recombinant bacterium containing the AxSDR gene, and an enzyme protein encoded by the AxSDR gene, or the application of a recombinant bacterium containing the AxSDR gene in the catalytic reduction for preparing chiral compounds. The enzyme protein encoded by the AxSDR gene exhibits obvious advantages in catalytic performance, such as significantly enhanced catalytic activity, excellent substrate tolerance, high substrate concentration adaptability, and reduced coenzyme dependence. In particular, AxSDR enzyme shows excellent performance in the synthesis of (R)-1-(3-trifluoromethylphenyl)ethanol. Experiments show that this enzyme not only achieves complete conversion of high-concentration (above 282 g / L) substrates, but also significantly improves the reaction efficiency and product optical purity, providing a new technical route for the green and efficient synthesis of chiral compounds.

[0013] The technical solution for the present invention to achieve the above-mentioned invention objectives is as follows:

[0014] The AxSDR gene of the enzyme from Algoriella xinjiangensis, its nucleotide sequence is SEQ ID NO.1. Identified by whole-genome sequencing, its open reading frame (ORF) is 753 bp in full length (coordinates: ATG to TAA), encoding 250 amino acids, and the amino acid sequence is SEQ ID NO:2. This gene has the following molecular characteristics that are significantly different from known members of the SDR family:

[0015] (1) The GC content (37.0%) is 18% lower than the average GC content (about 45%) of the SDR family (p < 0.01), suggesting its unique evolutionary trajectory;

[0016] (2) Encoding structure prediction shows that the core catalytic domain (Rossmann fold) is highly conserved, but the cofactor-binding region presents genus-specific mutations in Algoriella.

[0017] SEQ ID NO.1:

[0018]

[0019] SEQ ID NO.2:

[0020]

[0021] The recombinant bacterium containing the above AxSDR gene, with the AxSDR gene as the functional core, uses the pET-28a(+) expression vector as the backbone, and is directionally cloned by double digestion of the multiple cloning site of the vector with NdeⅠ and HindⅢ restriction endonucleases, and then transformed into the E. coli BL21(DE3) host bacterium. The recombinant bacterium is cultured on an LB plate containing 50 μg / mL kanamycin at 37 °C for 12 - 18 hours, and after antibiotic screening, single colonies are picked and stored frozen after verification by bidirectional Sanger sequencing (sequence identity 100%).

[0022] Furthermore, E. coli BL21(DE3)-AxSDR is cultured by two-stage gradient induction as follows:

[0023] (1) Pre-culture: Inoculate the recombinant bacterium E. coli BL21(DE3)-AxSDR into the LB medium (50 mL / 250 mL shake flask) at an inoculation amount of 1% - 6%, and culture at 37 °C, 150 - 250 rpm (preferably 200 rpm) for 8 - 12 h until the mid-log phase;

[0024] (2) Induced expression: Transfer the culture medium to fresh medium at a ratio of 1:80 - 120 (preferably 1:100), culture at 37°C, 150 - 250 rpm (preferably 200 rpm) for 1 - 2 h (preferably 1.5 h). Then, under sterile conditions, add IPTG with a concentration of 0.1 - 0.5 mmol / L (preferably 0.15 mmol / L) to the shake flask, and culture at 17 - 37°C (preferably 30°C), 150 - 250 rpm (preferably 200 rpm) for 6 - 20 h (preferably 14 h). Centrifuge the culture medium to obtain wet recombinant bacteria. Suspend it by adding buffer at a volume 40 times the weight of the bacteria, and then perform ice-bath ultrasonic disruption (preferred conditions: work for 3 s, stop for 3 s, for a total of 15 min). Centrifuge at 4°C and 10,000 r / min for 5 min to collect the supernatant crude enzyme solution and cell debris. Detect the expression results of the enzyme in the crude enzyme solution and cell debris by SDS-PAGE electrophoresis, and calculate the specific enzyme activity of the crude enzyme solution by detecting the absorbance change of NADH at 340 nm. The results show that the enzyme protein encoded by the AxSDR gene is soluble expressed, and the proportion of soluble protein reaches 45 - 55% (conventional 37°C induction system <15%), and the specific enzyme activity of the enzyme is 25.6 U / mg (the amount of enzyme required to catalyze the oxidation of 1 μmol NADH per minute is defined as one enzyme activity (U), and the enzyme activity per mg of enzyme protein is the specific enzyme activity).

[0025] The above application of the AxSDR gene uses the enzyme protein encoded by the AxSDR gene or the recombinant bacteria containing the AxSDR gene in the preparation of chiral compounds. The chiral compound is preferably (R)-1-(3-trifluoromethylphenyl)ethanol.

[0026] Furthermore, the application specifically is: Using meta-trifluoromethylacetophenone as the substrate, and using the enzyme protein encoded by the AxSDR gene or the wet recombinant bacteria obtained by the above method as the enzyme source, in 100 - 2100 mmol / L of the substrate meta-trifluoromethylacetophenone and a buffer solution or distilled water with a pH of 6.0 - 8.0, add isopropanol with a volume fraction of 1 - 50% (preferably 25 - 50%), 0.1 - 4 mmol / L (preferably 0.2 - 1.5 mmol / L) of NAD + , 50 - 400 g / L (preferably 100 - 300 g / L) of wet recombinant bacteria or the enzyme protein encoded by the AxSDR gene, and carry out the reaction at 25 - 55°C. After the reaction is completed, add ethyl acetate or n-hexane to the reaction solution for extraction for 10 - 30 min, and then centrifuge to collect the upper ethyl acetate extraction solution. The concentrations of the product and the unreacted substrate in the extraction solution are analyzed by gas chromatography and quantified by the internal standard method.

[0027] The experimental results show that it can catalyze high-concentration 1,3,5-trifluoromethylacetophenone above 1.5 mol / L, with a substrate conversion rate > 95%, breaking through the traditional enzyme-catalyzed substrate loading limit (conventional < 0.5 mol / L); only 0.2 - 1.5 mmol / L NAD + (conventional system requires 2 - 4 mmol / L) is needed to achieve coenzyme autologous recycling without an external regeneration system.

[0028] Aiming at the technical problems in the field of biocatalysis of chiral compounds, such as low catalytic efficiency, poor substrate tolerance, and insufficient optical purity, through the exploration of new enzyme genes and process innovation, the following beneficial effects are obtained:

[0029] (1) Enhanced high catalytic activity, extreme substrate tolerance, and stereoselectivity: The specific activity of the AxSDR enzyme reaches 25.6 U / mg, which is 194% higher than the reported SDR enzyme (the highest in the typical representative such as CN113025635A is 8.7 U / mg); it can catalyze high-concentration 1,3,5-trifluoromethylacetophenone at 1.5 mol / L (282 g / L), and the substrate loading is 3 times that of the traditional biocatalytic system (usually ≤ 0.5 mol / L), with a space-time yield of 613 g / L / d, significantly higher than the existing technology (such as 47 g / L / d in the above typical case LXCAR-S154Y), and the e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol reaches 99.8%.

[0030] (2) Strong applicability of the application process: Only 0.2 - 1.5 mmol / L NAD + (conventional system requires 2 - 4 mmol / L) is needed to achieve coenzyme self-sustaining recycling through endogenous metabolism, and the coenzyme cost is significantly reduced; the catalytic temperature is adapted to the temperature control conditions of industrial reactors, and the energy consumption of low-temperature-dependent enzymes is significantly reduced.

[0031] (3) The application value is prominent: It provides a green synthesis route with an optical purity > 99.5% for key chiral intermediates of drugs such as the neuroprotective agent (R)-3-(1-(3-(tris(trifluoromethyl)phenyl)ethoxy)azetidine-1-carboxamide), meeting the pharmacopoeial quality standards. The whole-cell catalytic system directly uses wet cells without the enzyme purification step, and the production cost is significantly reduced compared with the free enzyme method. In addition, there are no heavy metal catalysts and organic solvent residues in the reaction system, meeting the ICH Q3C drug impurity control guidelines. Description of the Drawings

[0032] Figure 1 Schematic diagram for the biocatalytic preparation of the chiral intermediate (R)-1-(3-trifluoromethylphenyl)ethanol of neuroprotective agent drugs.

[0033] Figure 2SDS-PAGE analysis of the soluble expression of the AxSDR gene in E. coli BL21(DE3) (M: standard protein molecular weight; Lane 1: recombinant bacterial cells; Lane 2: crude enzyme solution; Lane 3: cell debris).

[0034] Figure 3 Gas chromatogram of the extraction solution of the bioreduction reaction of the recombinant bacterium containing the AxSDR gene. Specific implementation mode

[0035] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0036] Example 1

[0037] Based on gene mining technology, 8 genes were screened as candidate genes by sequence alignment from the GenBank database. Through literature search, the 8 candidate genes were screened, and 4 enzyme genes with unknown functions were determined as target genes. Then, based on conservative sequence analysis, 4 new enzyme genes (named SDR01, SDR02, SDR03, and AxSDR respectively) were designed from the 4 screened enzyme genes, and NdeⅠ and HindⅢ restriction enzyme sites were added to their 5' and 3' ends respectively. Then, GeneCreate BioTech Co., Ltd. was commissioned to complete the gene total synthesis and the construction of the pET-28a(+) recombinant expression vector. The function of the 4 constructed recombinant bacteria for asymmetric reduction of m-trifluoromethylacetophenone (substrate concentration: 300 mmol / L) was verified: the 4 recombinant strains were induced to express in LB medium (containing 50 μg / mL kanamycin) to obtain whole-cell catalysts. The reaction was carried out in a standard reaction system (300 mmol / L m-trifluoromethylacetophenone, 25% (v / v) isopropanol, 35 °C, pH 7.4) for 3 h. Negative result: No substrate conversion was detected in the recombinant bacteria of SDR01, SDR02, and SDR03 (yield < 0.1%, detected by GC-FID). Positive result: The recombinant bacterium of AxSDR catalyzed the formation of (R)-1-(3-trifluoromethylphenyl)ethanol, with a yield of 85.1 ± 1.2% (n = 3), and the enantiomeric excess value (e.e.) reached 99.8 ± 0.1% (detected by GC-FID).

[0038] Example 2

[0039] Cultivation of recombinant bacterium E. coli BL21(DE3)-AxSDR containing AxSDR gene and obtaining of wet bacteria: Take the recombinant bacterium E. coli BL21(DE3)-AxSDR with an inoculation amount of 5% and add it to a 250 mL shake flask containing 50 mL of LB medium. Cultivate at 37 °C and 200 rpm for 12 h; then take 1 mL of the culture solution and transfer it to a 500 mL shake flask containing 100 mL of fresh LB medium. After culturing at 37 °C and 200 rpm for 1.5 h, add the inducer IPTG with a final concentration of 0.15 mmol / L to the shake flask under sterile conditions. Continue to culture at 30 °C and 200 rpm for 14 h. Centrifuge the culture solution, and wash the obtained precipitate with phosphate buffer to obtain wet bacteria, which is the enzyme source.

[0040] Suspend with a buffer at a volume 40 times the weight of the wet bacteria, and then perform ice bath ultrasonic disruption (disruption conditions: work for 3 s, stop for 3 s, for a total of 15 min). Centrifuge at 4 °C and 10,000 r / min for 5 min to collect the crude enzyme solution in the supernatant and cell debris. Detect the expression results of the enzyme in the crude enzyme solution and cell debris by SDS-PAGE electrophoresis (as Figure 2 shown). Detect the change in the absorbance value of NADH at 340 nm to calculate the specific enzyme activity of the crude enzyme solution. The results show that the enzyme protein encoded by the AxSDR gene is expressed solubly, and the proportion of soluble protein reaches 45 - 55%. The specific enzyme activity of the enzyme is 25.6 U / mg (the amount of enzyme required to catalyze the oxidation of 1 μmol of NADH per minute is defined as one enzyme activity (U), and the enzyme activity per mg of enzyme protein is the specific enzyme activity).

[0041] Example 3

[0042] Gas-phase detection and analysis of the product: After centrifuging the reaction solution at the end of the reaction, take the supernatant and add an equal volume of ethyl acetate for extraction. The concentrations of the product and unreacted substrate in the extraction solution are analyzed by gas chromatography, and quantitative analysis is performed by the internal standard method (internal standard: dodecane, addition amount 2 μl / 1 ml of sample). Gas chromatography analysis conditions: Shimadzu GC-2014 gas chromatograph in Japan; Varian CP-Chirasil-Dex chiral capillary gas chromatography column in the United States (25 m × 0.25 mm × 0.25 μm). The carrier gas is high-purity nitrogen with a flow rate of 2 mL / min; the injection volume is 1 μL, and the split ratio is 15:1; the detector and injection port temperatures are both 250 °C; the column temperature is 80 - 150 °C; the heating rate is 8 °C / min; the detector is FID. The peak elution times of each substance (as Figure 3Approximately: 6.737 min for 3-(trifluoromethyl)acetophenone, 8.312 min for dodecane, 10.850 min for (S)-1-(3-(trifluoromethyl)phenyl)ethanol, and 10.516 min for (R)-1-(3-(trifluoromethyl)phenyl)ethanol, as shown. According to the gas chromatography detection chromatogram, the concentration, yield, and e.e. value of the product in the reaction solution were calculated using the relative correction factor method.

[0043] Example 4

[0044] The wet cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4 - KH2PO4, pH 7.4), and the wet cell concentration was adjusted to 200 g / L (wet weight); isopropanol with a final concentration of 25% (v / v) was added as a co-substrate, and 3-(trifluoromethyl)acetophenone with different concentrations (100, 300, 600, 900, 1200, 1500, 1800, 2100 mmol / L) was added as the reaction substrate, and the mixture was reacted in a shaker at 35 °C and 150 rpm for 3 h. After the reaction was completed, the yield and e.e. of the product were determined according to the method of Example 2.

[0045] The concentration, yield, and e.e. value of the product (R)-1-(3-(trifluoromethyl)phenyl)ethanol are shown in Table 1. It can be seen from Table 1 that when the concentration of 3-(trifluoromethyl)acetophenone is 1500 mmol / L, the concentration of the product (R)-1-(3-(trifluoromethyl)phenyl)ethanol is 511.5 mmol / L, the yield is 34.1% at this time, and the e.e. value is 99.8%. Continuing to increase the substrate concentration, the product concentration did not increase significantly, so the optimal substrate concentration was determined to be 1500 mmol / L.

[0046] Table 1 Effects of different substrate concentrations of 3-(trifluoromethyl)acetophenone on the concentration, yield, and e.e. value of the product

[0047]

[0048] Example 5

[0049] The wet cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4 - KH2PO4, pH 7.4), isopropanol with a final concentration of 25% (v / v) was added as a co-substrate, 1500 mmol / L of 3-trifluoromethylacetophenone was added as a reaction substrate, and wet cells with different concentrations (50, 100, 150, 200, 250, 300, 350, and 400 g / L) were added. The mixture was placed in a shaker at 35 °C and 150 rpm for 3 h. After the reaction, the yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 2. As can be seen from Table 2, when the wet cell concentration was 300 g / L, the yield of the product (R)-1-(3-trifluoromethylphenyl)ethanol was 38.5%, and the e.e. value was 99.8%. Continuing to increase the cell concentration, the product yield did not increase significantly. Therefore, the cell concentration was determined to be 300 g / L.

[0050] Table 2 Effects of wet cell concentration on yield and e.e. value

[0051]

[0052]

[0053] Example 6

[0054] The wet cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4 - KH2PO4, pH 7.4), and the wet cell concentration was adjusted to 300 g / L (wet weight); isopropanol with a final concentration of 25% (v / v) was added as a co-substrate, 1500 mmol / L of 3-trifluoromethylacetophenone was added as a reaction substrate, and NAD with concentrations of 0, 0.5, 1, 2, 3, and 4 mmol / L was added. + . The mixture was placed in a shaker at 35 °C and 150 rpm for 3 h. After the reaction, the yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 3. As can be seen from Table 3, when 1 mmol / L of NAD + was added to the reaction system, the yield of the product (R)-1-(3-trifluoromethylphenyl)ethanol was 41.9%. Continuing to increase the NAD + concentration, the product yield did not increase significantly. Therefore, the NAD + concentration was determined to be 1 mmol / L.

[0055] Table 3 Effects of NAD + concentration on yield and e.e. value

[0056]

[0057] Example 7

[0058] The wet cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4 - KH2PO4, pH 7.4), and the concentration of wet cells was adjusted to 300 g / L (wet weight); 1500 mmol / L of 3-trifluoromethylacetophenone was added as the substrate, and the final concentration of NAD was 1 mmol / L + , and then different concentrations (volume fraction) of isopropanol (v / v) (such as 0, 15, 20, 25, 30, 35, 40, 45 and 50%) were added. The mixture was placed in a shaker at 35 °C and 150 rpm for 3 h. After the reaction was completed, the yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 4.

[0059] Table 4 Effects of isopropanol addition amount on yield and e.e. value

[0060]

[0061] As can be seen from Table 4, when the addition amount of isopropanol was 40% (v / v), the yield of the product (R)-1-(3-trifluoromethylphenyl)ethanol was the highest, which was 52.6%, and the e.e. value was 99.7%.

[0062] Example 7

[0063] The wet cells obtained in Example 2 were suspended in phosphate buffer with different pH values (6.0, 6.4, 7.0, 7.4, 7.6, 8.0), and the concentration of wet cells was controlled to 300 g / L (wet weight); 40% (v / v) of isopropanol was added as the co-substrate, and the final concentration of NAD was 1 mmol / L + , and then 1500 mmol / L of 3-trifluoromethylacetophenone was added as the reaction substrate. The mixture was placed in a shaker at 35 °C and 150 rpm for 3 h. After the reaction was completed, the yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 5. As can be seen from Table 5, when the pH value of the buffer was 7.4, the yield of the product (R)-1-(3-trifluoromethylphenyl)ethanol was the highest, which was 52.1%, and the e.e. value was 99.8%.

[0064] Table 5 Effects of buffer pH value on yield and e.e. value

[0065]

[0066] Example 8

[0067] The wet cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4 - KH2PO4 buffer, pH 7.4), and the concentration of wet cells was controlled to be 300 g / L (wet weight); isopropanol with a final concentration of 40% (v / v) was added as a co-substrate, and NAD with a final concentration of 1 mmol / L + , and 1500 mmol / L of 3-trifluoromethylacetophenone was added as a reaction substrate, and then the mixture was placed in a shaker at different temperatures (25, 30, 35, 40, 45, 50, 55, and 60 °C) at 150 rpm for 3 h. After the reaction was completed, the yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 6. As can be seen from Table 6, when the temperature was 50 °C, the yield of the product (R)-1-(3-trifluoromethylphenyl)ethanol was the highest, 71.1%, and the e.e. value was 99.8%.

[0068] Table 6 Effects of reaction temperature on yield and e.e. value

[0069]

[0070] Example 9

[0071] The wet cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4 - KH2PO4, pH 7.4), and the concentration of wet cells was controlled to be 300 g / L (wet weight); the cells were treated in a shaker at different temperatures (25, 30, 35, 40, 45, 50, 55, and 60 °C) at 150 rpm for 3 h, and then isopropanol with a final concentration of 40% (v / v) was added as a co-substrate, 1500 mmol / L of 3-trifluoromethylacetophenone was added as a reaction substrate, and NAD with a final concentration of 1 mmol / L + , and the mixture was placed in a shaker at 50 °C and 150 rpm for 3 h. After the reaction was completed, the yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 7.

[0072] Table 7 Effects of treating cells at different temperatures on yield and e.e. value

[0073]

[0074] As can be seen from Table 7, when the cells were treated at a higher temperature, the enzyme was severely inactivated. When treated at 45 °C for 3 h, the relative yield was 71.1%. When treated at 50 °C for 3 h, the relative yield was 53.4%. When treated at 55 °C for 3 h, the relative yield was only 23.5%. Considering comprehensively, the reaction temperature was determined to be 45 °C.

[0075] Example 10

[0076] The wet bacterial cells obtained in Example 2 were suspended in phosphate buffer (50 mM K2HPO4-KH2PO4, pH 7.4), and the concentration of the wet bacterial cells was controlled to be 300 g / L (wet weight); isopropanol with a final concentration of 40% (v / v) was added as a co-substrate, and NAD with a final concentration of 1 mmol / L + , and 1,3,5-trifluoroacetophenone with a final concentration of 1500 mmol / L was used as the reaction substrate, and the reaction was carried out for different times (1, 2, 3, 5, 7, 9, 11, 13, and 15 h) in a shaker at 45 °C and 150 rpm. The yield and e.e. value of the product were determined according to the method of Example 2. The yield and e.e. value of the product (R)-1-(3-trifluoromethylphenyl)ethanol are shown in Table 8.

[0077] Table 8 Effect of reaction time on the yield

[0078]

[0079] As can be seen from Table 8, when the reaction time was 11 h, the product yield was 99.6%, and the e.e. value of the product was above 99%.

Claims

1. An Algoriella xinjiangensis derived enzyme AxSDR gene, characterized in that: The nucleotide sequence of the AxSDR gene is SEQ ID NO.

1. The ORF of the AxSDR gene is 753 bp in full length and has a G+C content of 37.0%.

2. An enzyme protein encoded by the AxSDR gene according to claim 1, characterized in that: The enzyme protein contains 250 amino acids, and the amino acid sequence is: SEQ ID NO.

2.

3. A recombinant bacterium containing the AxSDR gene according to claim 1, characterized in that: The AxSDR gene was used as the functional core and the pET-28a(+) expression vector was used as the backbone. The vector multiple cloning site was cloned using NdeⅠ and HindⅢ restriction endonucleases.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacterium is E. coli BL21 (DE3)-AxSDR, and its construction method is as follows: the AxSDR gene is directionally cloned into the pET-28a (+) vector by double restriction digestion with NdeⅠ and HindⅢ, and then transformed and introduced into the E. coli BL21 (DE3) host bacterium, and the recombinant bacteria are cultured at 37° C. for 14 hours on an LB plate containing 50 μg / mL kanamycin, and a single clone is picked for verification by bidirectional Sanger sequencing after antibiotic screening and then frozen.

5. The recombinant bacterium according to claim 4, characterized in that The E.coli BL21(DE3)-AxSDR was cultured using a two-stage gradient induction method, and the specific steps were as follows: (1) Pre-culture: Inoculate the recombinant E. coli BL21(DE3)-AxSDR into a 250 mL shake flask containing 50 mL LB medium at a 1% to 6% inoculum, and culture at 37°C, 150-250 rpm for 8-12 h until mid-logarithmic phase; (2) Induced expression: The culture medium was transferred to fresh medium at a ratio of 1:80-120, cultured at 37°C and 150-250 rpm for 1-2 h, and then the inducer IPTG at a concentration of 0.1-0.5 mmol / L was added to the shake flask under sterile conditions. The culture was induced at 17-37°C and 150-250 rpm for 6-20 h. The culture medium was centrifuged to obtain wet cells of the recombinant bacteria.

6. Use of the AxSDR gene according to claim 1, the enzyme protein according to claim 2, or the recombinant bacteria according to any one of claims 3 to 5, characterized in that: The AxSDR gene, enzyme protein or recombinant bacteria is used to prepare a chiral compound, and the chiral compound is (R)-1-(3-trifluoromethylphenyl)ethanol.

7. The use according to claim 6, characterized in that: The recombinant bacteria is E. coli BL21 (DE3)-AxSDR.

8. The use according to claim 6, characterized in that: The application is specifically as follows: using m-trifluoromethylacetophenone as a substrate, using the enzyme protein encoded by the AxSDR gene or the wet cells of the recombinant bacteria as an enzyme source, adding 1-50% isopropanol and 0.1-4 mmol / L NAD in a buffer solution or distilled water with a pH of 6.0-8.0 and a concentration of 100-2100 mmol / L of the substrate m-trifluoromethylacetophenone. + , 50-400g / L of recombinant bacterial wet cells or enzyme protein encoded by the AxSDR gene, react at 25-55°C. After the reaction, add ethyl acetate to the reaction solution for extraction for 10-30min, then centrifuge to collect the upper ethyl acetate extract, and analyze the concentrations of the product and unreacted substrate in the extract by gas chromatography and quantify by the internal standard method.

9. The use according to claim 8, characterized in that: The volume fraction of isopropanol is 25-50%, NAD + The concentration of the AxSDR gene is 0.2-1.5 mmol / L, and the concentration of the recombinant wet bacteria or the enzyme protein encoded by the AxSDR gene is 100-300 g / L.

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