Carbonyl reductase mutant with high organic solvent tolerance and application thereof
By mutating amino acids at specific sites in the carbonyl reductase ZmCR, a ZmCR-M3 with high tolerance to organic solvents was constructed, solving the problems of poor enzyme tolerance and low substrate conversion in organic solvents, and realizing the efficient industrial application of the enzyme.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-09
- Publication Date
- 2026-04-03
AI Technical Summary
The existing carbonyl reductase ZmCR exhibits poor tolerance to organic solvents and low substrate conversion rates in the preparation of (S)-1-C-(β-D-xylopyranosyl)-propanol, which limits its widespread use in industrial applications.
By performing single or multiple mutations on the amino acid sequence of ZmCR, especially mutations at positions 197, 323, and 124, a carbonyl reductase mutant ZmCR-M3 with high organic solvent tolerance was constructed. Recombinant vectors and engineered bacteria were then constructed, and catalytic conditions were optimized to improve enzyme tolerance and conversion rate.
The mutant ZmCR-M3 significantly improves the tolerance to temperature and organic solvents, prolongs the half-life, enhances catalytic efficiency and substrate conversion, and expands its scope of industrial applications.
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Abstract
Description
(I) Technical Field
[0002] This invention belongs to the fields of enzyme engineering and genetic engineering technology, specifically relating to a carbonyl reductase mutant with high organic solvent tolerance and its applications. (II) Background Technology
[0004] Chiral alcohols, as important building blocks for chiral compounds, are often used in the synthesis of chiral drugs, such as the anticoagulant warfarin, the antidepressant sertraline, the beta-blocker propranolol, and the antibacterial drug levofloxacin. S )-1-C-(β-D-xylanopyranosyl)-propanol(( S Pro-Xylane is a bioactive C-glycoside compound and an important active ingredient in cosmetics. S 1-C-(β-D-xylanosyl)-propanol can bind to glycosaminoglycan precursors in the skin's extracellular matrix via glycosidic bonds, thereby stimulating the synthesis of glycosaminoglycans in the dermis and improving skin elasticity and hydration. Compared to traditional chemical synthesis methods, biocatalysis is a green alternative for the synthesis of chiral alcohols.
[0005] Carbonyl reductases are a class of enzymes that catalyze the asymmetric reduction of prochiral ketones to chiral alcohols in a cofactor-dependent manner. Currently, carbonyl reductases are considered key biocatalysts in the asymmetric synthesis and kinetic resolution of chiral alcohols. These enzymes have become important tools in the industrial production of pharmaceutical intermediates, pesticides, and fine chemicals, including chiral alcohols, chiral diols, and chiral hydroxy acids.
[0006] In industrial biocatalysis, using higher concentrations of organic solvents helps improve substrate solubility, reduce the risk of microbial contamination, and can enhance reaction selectivity to some extent. Despite these advantages, organic solvent systems still have limitations in practical applications, particularly the poor tolerance and low catalytic efficiency of most natural enzymes in organic solvents, which restricts their widespread industrial application. Therefore, improving the organic solvent tolerance of enzymes through targeted molecular design strategies is of great significance for expanding the application range of enzymes in practical industrial catalysis.
[0007] carbonyl reductase Change CR with NAD(P) + As a coenzyme, it performs a coenzyme cycle, oxidizing the cosubstrate isopropanol to acetone, thereby reducing the intermediate 1-C-(β-D-xylanosyl)-acetone to ( S )-1-C-(β-D-xylanopyranosyl)-propanol, reaction formula as follows Figure 1As shown, isopropanol, as an organic solvent, inhibits enzyme activity at certain concentrations; simultaneously, acetone accumulated during the reaction also has an inhibitory effect. Both factors together restrict the reaction process, leading to a decrease in substrate conversion. (III) Summary of the Invention
[0009] The purpose of this invention is to target existing carbonyl reductases. Change CR in preparation ( S To address the issues of poor organic solvent tolerance and low substrate conversion during the 1-C-(β-D-xylanosyl)-propanol process, a carbonyl reductase is proposed. Change CR mutant, encoding gene, engineered bacteria and its asymmetric reduction of 1-C-(β-D-xylanopyranosyl)-acetone ( S The application of 1-C-(β-D-xylanosyl)-propanol effectively improved the organic solvent tolerance and substrate conversion rate of carbonyl reductase mutants.
[0010] The technical solution adopted in this invention is:
[0011] This invention provides a strain derived from Marx's diploid bacteria. Zygophabospora marxiana carbonyl reductase Change CR mutant, the carbonyl reductase Change The CR mutant is obtained by making single or multiple mutations at positions 197, 323, and 124 of the amino acid sequence shown in SEQ ID NO.1.
[0012] Furthermore, the carbonyl reductase Change The CR mutant is formed by mutating one of the following amino acid sequences shown in SEQ ID NO.1: (1) phenylalanine at position 197 is mutated to glycine (F197G); (2) proline at position 323 is mutated to arginine (P323R); (3) tyrosine at position 124 is mutated to valine (Y124V); (4) a mutant F197G / P323R / Y124V is constructed, in which phenylalanine at position 197 is mutated to glycine, proline at position 323 is mutated to arginine, and tyrosine at position 124 is mutated to valine, denoted as F197G / P323R / Y124V. Change CR-M3 (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4).
[0013] This invention also relates to the carbonyl reductase. Change The coding gene of the CR mutant, the recombinant vector containing the coding gene, and the engineered bacteria constructed from the recombinant vector were selected. The recombinant vector was pET28a(+) as the base vector, and the engineered bacteria were preferably constructed using pET28a(+) as the base vector. E. coli BL21(DE3) is the host bacterium.
[0014] The present invention also provides the carbonyl reductase described above. Change CR mutant was prepared by asymmetric reduction of 1-C-(β-D-xylanosyl)-acetone. S Applications of 1-C-(β-D-xylanosyl)-propanol.
[0015] Furthermore, the method of application is as follows: [The text abruptly shifts to a seemingly unrelated topic about a carbonyl reductase.] Change The engineered bacteria encoding the CR mutant gene were induced and cultured using wet bacterial cells obtained by centrifugation as a catalyst, with 1-C-(β-D-xylanosyl)-acetone as the substrate, isopropanol as the co-substrate, and NAD(P) as the catalyst. + As a coenzyme, a conversion system was constructed using a pH 6-8 buffer solution (preferably pH 7.0, 100 mM sodium phosphate buffer) as the reaction medium. The reaction was carried out at 20-40℃ and 1000-3000 rpm (preferably 30℃, 1500 rpm for 24 h). After the reaction was completed, the reaction solution was separated and purified to obtain ( S )-1-C-(β-D-xylanosyl)-propanol.
[0016] Furthermore, in the conversion system, the substrate is added at a final concentration of 100-2000 mM (preferably 1000 mM), isopropanol is added at a volume concentration of 5-15% (preferably 10%), and the co-substrate is NAD(P). + Add to a final concentration of 0.1-1.0 mM (preferably 0.5 mM), and the amount of catalyst used is 5-20 g / L (preferably 20 g / L) based on wet cells.
[0017] Furthermore, the wet bacterial cells are prepared by the following method: [The method involves] preparing a carbonyl reductase-containing... Change Engineered bacteria encoding the CR mutant gene were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C and 200 rpm for 12 h. Then, at a volume ratio of 2.0% (V / V), they were inoculated into sterilized LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C and 200 rpm for 2 h. IPTG was then added to the culture medium to a final concentration of 0.10 mM, and the culture was incubated at 16 °C for 12 h. Finally, the culture was centrifuged at 4 °C and 8000 rpm for 10 min to obtain the carbonyl reductase-containing bacteria. Change Wet bacterial cells of the CR mutant.
[0018] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in:
[0019] (1) The carbonyl reductase constructed in this invention Change CR mutants exhibit significantly enhanced tolerance to temperature and organic solubility: compared to carbonyl reductases Change Compared to CR, mutant Change CR-M3T 50 15 Increase by 2.7 °C; carbonyl reductase mutant Change The half-life of CR-M3 in sodium phosphate buffer (100 mM, pH 7.0) containing 10% (v / v) isopropanol was 6.80 h, while that of the wild type was only 0.80 h, representing an 8.50-fold increase. (Carbonyl reductase mutant) Change The half-life of CR-M3 in sodium phosphate buffer (100 mM, pH 7.0) containing 15% (v / v) acetone was 8.20 h, while that of the wild type was only 1.03 h, which is 7.96 times longer than that of the wild type.
[0020] (2) Carbonyl reductase constructed in this invention Change The CR mutant significantly improves the concentration and efficiency of the catalytic substrate: mutant Change CR-M3 significantly improves catalytic efficiency; when it acts on 1000 mM 1-C-(β-D-xylanosyl)-acetone, it generates ( ) within 24 hours. S The conversion rate of 1-C-(β-D-xylanosyl)-propanol was much higher than that of ZmCR.
[0021] The carbonyl reductase mutant obtained by this invention has improved tolerance to organic solvents, namely isopropanol and acetone, which helps to expand the scope of industrial applications and has great application prospects. (iv) Description of the attached drawings
[0023] Figure 1 It is a carbonyl reductase Change Preparation of CR and mutants by asymmetric reduction of 1-C-(β-D-xylanopyranosyl)-acetone ( S The reaction formula for )-1-C-(β-D-xylanosyl)-propanol.
[0024] Figure 2 It is a carbonyl reductase Change Residual enzyme activity of CR and mutant after incubation at 10% (v / v) isopropanol concentration for 10 h.
[0025] Figure 3 It is a carbonyl reductase Change Residual enzyme activity of CR and mutant after incubation in 15% (v / v) acetone for 10 h.
[0026] Figure 4 It is a carbonyl reductase Change CR and mutants catalyze the preparation of 1000 mM substrate 1-C-(β-D-xylanopyranosyl)-acetone within 24 h. S Conversion rate of 1-C-(β-D-xylanosyl)-propanol. (V) Detailed Implementation Methods
[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0029] Example 1: Change Construction of CR-M3 combined mutant
[0030] 1. Recombinant plasmid pET28a(+)- Change CR
[0031] Artificially synthesized from Marx's butter Zygophabospora marxiana carbonyl reductase Change The sequence of CR (GenBank: BAP72132.1) (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence encoding the protein as shown in SEQ ID NO.1), inserted into plasmid pET28a(+) Where I restriction site and Xho Between the I restriction enzyme sites, construct the recombinant plasmid pET28a(+)- Change CR, and then transferred to E. coli E.coli BL21(DE3) competent cells were plated onto LB agar plates containing a final concentration of 50 mg / L kanamycin resistance. After incubation at 37 °C for 12 h, positive clones were screened to obtain wild-type carbonyl reductase engineered bacteria. E.coli BL21(DE3)-pET28a(+)- Change CR-WT, and extract plasmid pET28a(+)- Change CR-WT.
[0032] SEQ ID NO.1
[0033] MSYTVITGANGFIAQHIIKILLSNGHKVIGTVRTQEKADTIANLFDNENLILEIVPDLNAVEAFDPLFKKYNTQIKYVLHTASPVDSTLTDLQNDFINTAITGTLSVFNAIKKYAADSVESVVYTSSSVAGANFKTFLDPTAVINEESWNPDEREDVVDILSAYSVSKKLAE KAAWNFKEENKDVIKFRLSTVNPFFVTGPQAFDETAKGKLNTTASYFEQILASKPDGNLAPFIGSPAVDVRDLAKAHYLALTEPRFDGQRIFLDGENYTTQQWYDVIHEKFPELNGKIAKGQPGTNKFQEGSMAKMDFSKSKKLLGFDQIPLKQTITDGMGQLLRVRGIPQN
[0034] 2. Construction and screening of mutants
[0035] Wild-type carbonyl reductase constructed using AlphaFold3 Change The CR-WT structural model was used, with the substrate β-pyruvate paired into the active site. Mutations were performed at sites Y124, F197, and P323, which are close to the substrate, to investigate the effect of the active site sites on the enzyme's tolerance to organic solvents. The recombinant plasmid pET28a(+)- Change Using CR-WT as a template, mutations were performed using the primers in Table 1 (N represents A / T / G / C, K represents G / T). The mutations were introduced via PCR, and the PCR product was treated with DpnI at 37 ℃ for 3 h, followed by transformation to... E.coli In the BL21(DE3) host bacterium, the cells were plated on LB agar plates containing a final concentration of 50 mg / L kanamycin resistance and incubated at 37 °C for 12 h. Single colonies were randomly selected for sequencing analysis to obtain a mutant library. Mutant activity was determined according to the method in Example 3, and dominant mutants were screened. The results are shown in Table 2, namely, Y mutation at position 124 becomes V, F mutation at position 197 becomes G, and P mutation at position 323 becomes R.
[0036] PCR amplification system (25 μL): 0.5 μL forward primer (100 μM), 0.5 μL reverse primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 0.5 μL plasmid template, 0.5 μL DNA polymerase and 10 μL ultrapure water.
[0037] PCR reaction procedure: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 10 s, 55 °C annealing for 5 s, 72 °C extension for 3 min, repeat for 25 cycles; 72 °C extension for 10 min.
[0038] Table 1. Mutant Primer Design
[0039]
[0040] Table 2. Mutant enzyme activity
[0041]
[0042] Further combined mutations were performed using the primers in Table 1 to obtain... Change Engineered bacteria of CR-M3 (Y mutation at position 124 becomes V, F mutation at position 197 becomes G, and P mutation at position 323 becomes R). E.coli BL21(DE3)-pET28a(+)- Change CR-M3, its vitality compared to Change CR-WT increased by 8.5 times.
[0043] Example 2: Carbonyl reductase Change Protein purification of CR and mutants
[0044] (1) The engineered bacteria constructed in Example 1 E.coli BL21(DE3)-pET28a(+)- Change CR and E.coli BL21(DE3)-pET28a(+)- Change CR-M3 was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37 ℃ and 200 rpm for 12 h. Then, it was inoculated into sterilized LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume ratio of 2.0% (V / V) and cultured at 37 ℃ and 200 rpm for 2 h. IPTG was then added to the culture medium to a final concentration of 0.10 mM, and the medium was cultured at 16 ℃ for 12 h. Finally, the medium was centrifuged at 4 ℃ and 8000 rpm for 10 min to obtain carbonyl reductase. Change Wet bacterial cells of the CR mutant.
[0045] (2) The wet bacterial cells were resuspended in sodium phosphate buffer (pH 7.0, 100 mM) at a concentration of 50 g / L, placed in an ice-water mixture, and sonicated for 10 min. The disruption conditions were: power of 250 W, disruption for 1 s, pause for 2 s, to obtain crude enzyme solution.
[0046] (3) The crude enzyme solution was centrifuged at 4 ℃ and 8000 rpm for 10 min to remove the precipitate. The supernatant was filtered through a 0.22 μm microfiltration membrane, and the filtrate was used as the loading solution to purify the enzyme protein using a nickel affinity chromatography column (40×12.6 mm, Bio-Rad, USA). The enzyme protein purification procedure is as follows: First, rinse the tubing with ultrapure water to remove impurities and air, and remove 20% ethanol from the nickel column; Equilibrate the baseline: Equilibrate the nickel column with 5-10 column volumes of binding buffer (pH 7.0, 100 mM sodium phosphate buffer containing 0.5 M NaCl) to achieve baseline equilibrium; Sample loading: Load the previously collected filtrate at a flow rate of 0.25 mL / min, with a total loading volume of 10 mL; Elute contaminating proteins: Elute contaminating proteins with 5-7 column volumes of washing buffer (pH 7.0, 10 mM imidazole, 100 mM sodium phosphate buffer containing 0.5 M NaCl) at a flow rate of 1 mL / min until baseline equilibrium is achieved, ensuring complete elution of impurities; Elute the target protein: Elute the target protein with elution buffer (pH 7.0, 100 mM sodium phosphate buffer containing 0.5 M NaCl, 150 mM imidazole, 100 mM sodium phosphate buffer containing 0.5 M NaCl) at a flow rate of 1 mL / min. Collect 15 mL of the target protein flow-through solution based on the absorption peak at 280 nm wavelength at baseline and store it on ice.
[0047] The above 15 mL of target protein flow-through solution was loaded into an ultrafiltration tube (10 kDa) and centrifuged at 4 ℃ and 3500 rpm for 15 min to concentrate it to 2 mL. Then, 100 mM sodium phosphate buffer (pH 7.0) was added to the ultrafiltration tube to make up to 15 mL. The tube was then centrifuged at 4 ℃ and 3500 rpm for 15 min for 3 rounds of centrifugation and replacement. The retentate after the replacement was completed was the purified enzyme, which was aliquoted and stored.
[0048] Example 3: Carbonyl reductase Change Determination of the reducing activity of CR and mutants
[0049] The reducing activity assay reaction system (200 μL) consisted of 1 mM 1-C-(β-D-xylopyranosyl)-acetone, 0.5 mM NAD(P)H, 0.01 mg / mL (based on protein content) of the purified enzyme solution prepared in Example 2, and 100 mM sodium phosphate buffer (pH 7.0) to a final volume of 200 μL. The reaction was carried out at 30 °C for 5 min using a microplate reader. The reducing activity of the enzyme was calculated by measuring the decrease in the absorbance of NAD(P)H at 340 nm.
[0050] Enzyme activity definition: Under the above conditions, the amount of enzyme required to oxidize 1 μmol of NAD(P)H per minute is defined as one enzyme activity unit (U).
[0051] Example 4: Carbonyl reductase Change Determination of the thermal stability of CR and mutants
[0052] The purified enzyme prepared in Example 2 was incubated at a certain temperature gradient for 15 min, and then tested under the enzyme activity detection standard conditions of Example 3. The temperature at which the enzyme activity dropped to half of its initial activity was considered the optimal temperature. T 50 15 Assay method: The purified enzyme was diluted to the same protein concentration (0.1 mg / mL) with 100 mM sodium phosphate buffer (pH 7.0). The diluted purified enzyme was incubated at a specific temperature gradient (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃) for 15 min, and then immediately cooled on ice. The residual enzyme activity at each temperature was measured using the method in Example 3. The enzyme activity after incubation at 30 ℃ for 15 min was defined as 100%. The analysis and calculation were performed using Origin 9.1 software, and nonlinear fitting was performed using Sigmoidal Boltzmann to obtain the results. T 50 15 The results are shown in Table 3. (Compared with...) Change Compared to CR, Change CR-M3 T 50 15 Increasing the temperature by 2.7 °C and introducing the mutant F197G / P323R / Y124V enhanced the activity of carbonyl reductase. Change Thermal stability of CR.
[0053] Table 3 Carbonyl reductases Change Thermal stability determination of CR and its mutants
[0054]
[0055] Example 5: Carbonyl reductase Change Isopropanol tolerance assay for CR and mutants
[0056] Half-life (t) 1 / 2 This refers to the solution prepared at 37 °C in a 100 mM sodium phosphate buffer solution (pH 7.0) containing 10% (v / v) isopropanol. ChangeThe time required for the residual activity of CR and its mutants to decrease to 50% was determined. The enzyme half-life was measured as follows: The purified enzyme solution prepared according to the method in Example 2 was incubated at 37 °C in 100 mM sodium phosphate buffer (pH 7.0) containing 10% (v / v) isopropanol for 0-10 h, maintaining the concentration of the purified enzyme protein in the incubation system at 0.1 mg / mL. Subsequently, the incubation solution was collected at incubation times of 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.5 h, 7.0 h, and 10.0 h, and the residual enzyme activity was measured using the method in Example 3. The half-life (t) corresponding to the enzyme activity decreasing to 50% was obtained by plotting using Origin 9.1 software and performing nonlinear fitting using Exponential. 1 / 2 Carbonyl reductase Change The residual enzyme activities of CR and mutants after incubation at 10% (v / v) isopropanol for 10 h are as follows: Figure 2 As shown, Change CR and mutants Change The half-life of CR-M3 in 100 mM sodium phosphate buffer (pH 7.0) containing 10% (v / v) isopropanol at 37 °C is shown in Table 4. Change The half-life of CR-M3 is 6.80 h, which is Change 8.5 times that of CR.
[0057] Table 4 Carbonyl reductases Change Isopropanol tolerance assay of CR and its mutants
[0058]
[0059] Example 6: Carbonyl reductase Change Acetone tolerance assay for CR and mutants
[0060] Half-life (t) 1 / 2 This refers to the solution prepared in 100 mM sodium phosphate buffer (pH 7.0) containing 15% (v / v) acetone at 37 °C. ChangeThe time required for the residual activity of CR and its mutants to decrease to 50% was determined. The enzyme half-life was measured as follows: The purified enzyme solution prepared according to the method in Example 2 was incubated at 37 °C in 100 mM sodium phosphate buffer (pH 7.0) containing 15% (v / v) acetone for 0-10 h, maintaining the concentration of the purified enzyme protein in the incubation system at 0.1 mg / mL. Subsequently, the incubation solution was collected at incubation times of 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.5 h, 7.0 h, and 10.0 h, and the residual enzyme activity was measured using the method in Example 3. The half-life (t) corresponding to the enzyme activity decreasing to 50% was obtained by plotting using Origin 9.1 software and performing nonlinear fitting using Exponential. 1 / 2 Carbonyl reductase Change The residual enzyme activities of CR and mutants after incubation in 15% (v / v) acetone for 10 h are as follows: Figure 3 As shown; Change CR and mutants Change The half-life of CR-M3 in 100 mM sodium phosphate buffer (pH 7.0) containing 15% (v / v) acetone at 37 °C is shown in Table 5. Change The half-life of CR-M3 is 8.20 h, which is Change 7.96 times that of CR.
[0061] Table 5 Carbonyl reductases Change Acetone tolerance assay of CR and its mutants
[0062]
[0063] Example 7: Carbonyl reductase Change Preparation of CR and mutant asymmetric reduction ( S )-1-C-(β-D-xylanopyranosyl)-propanol
[0064] The final concentration composition of the 20 mL reaction system was: 1000 mM 1-C-(β-D-xylanopyranosyl)-acetone, 0.5 mM NAD(P). +20 g / L of wet cells prepared according to the method in Example 2, 10% (v / v) isopropanol, and 100 mM sodium phosphate buffer (pH 7.0) were added to a final volume of 20 mL. The mixture was reacted at 30 °C and 1500 rpm for 24 h. Samples were taken every 1 h during the reaction, and the substrate content was determined by HPLC to calculate the substrate conversion rate. HPLC detection conditions: Agilent 1260 Infinity II instrument (Agilent Technologies, USA), Ultimate C18 column (4.6 × 250 mm, 5 μm), differential detector, ultrapure water as mobile phase, 10 μL injection volume, 1 mL / min flow rate, and column temperature 30 °C. Results are shown below. Figure 4 .
[0065] The results are as follows Figure 4 It can be seen that carbonyl reductase Change CR and mutants Change CR-M3 can be prepared by asymmetric reduction of 1000 mM 1-C-(β-D-xylanosyl)-acetone. S )-1-C-(β-D-xylanopyranosyl)-propanol, carbonyl reductase Change The conversion rate of CR was 46.7%, and the mutant... Change CR-M3 conversion rate can reach 87.5%, which is higher than... Change The CR increased by 40.8%.
[0066] Example 8: Carbonyl Reductase Change CR and mutants were prepared at different substrate concentrations ( S )-1-C-(β-D-xylanosyl)-propanol (abbreviated as ( S Comparison of β-Bosonic
[0067] The final concentration composition of the 20 mL reaction system is as follows: 500-2000 mM 1-C-(β-D-xylanopyranosyl)-acetone (abbreviated as β-acetone xylose), 0.1 mM NADP. + 5-20 g / L of wet cells prepared according to the method in Example 2, 10% (v / v) isopropanol, and 100 mM potassium phosphate buffer (pH 7.0) were added to a final volume of 20 mL. The reaction was carried out at 30°C and 200 rpm for 24 h, and the substrate conversion rate was determined using the method in Example 7.
[0068] The results are shown in Table 6. Change CR-M3 can catalyze substrate reactions up to 2000 mM, achieving a conversion rate of 80.3% after 24 h. However, its conversion rate is even higher than 99.9% at a substrate concentration of 500 mM, demonstrating significant potential for industrial applications. In contrast, the conversion rate before mutation was only 80.5% with the same amount of enzyme added. Change CR-WT can catalyze the complete conversion of a 100 mM substrate. The results indicate that the carbonyl reductase screened in this invention... Change Both CR and its mutants can catalyze the synthesis of high concentrations of β-pyruvone xyloside. S It contains 1-Bosein and exhibits high substrate conversion rate. Change The CR-M3 offers superior performance.
[0069] Table 6. Effects of substrate concentration on reduction reactions of wild-type carbonyl reductase and mutants.
[0070]
Claims
1. A carbonyl reductase with high tolerance to organic solvents ZmCR Mutant, characterized by, The carbonyl reductase ZmCR The mutant was obtained by performing multiple mutations on positions 197, 323, and 124 of the amino acid sequence shown in SEQ ID NO.
1.
2. The carbonyl reductase as described in claim 1 Zm CR mutant, characterized by, The carbonyl reductase Zm The CR mutant is formed by mutating one of the following amino acid sequences shown in SEQ ID NO.1: (1) mutating phenylalanine at position 197 to glycine; (2) mutating proline at position 323 to arginine; (3) mutating tyrosine at position 124 to valine; (4) constructing a mutant in which phenylalanine at position 197 is mutated to glycine, proline at position 323 is mutated to arginine, and tyrosine at position 124 is mutated to valine.
3. A carbonyl reductase according to claim 1 Zm Recombinant expression plasmids for the coding gene of CR mutants.
4. A genetically engineered bacterium containing the recombinant expression plasmid of claim 3.
5. A carbonyl reductase according to claim 1 Zm CR mutant prepared by asymmetric reduction of 1-C-(β-D-xylanosyl)-acetone ( S Applications of 1-C-(β-D-pyranoxyl)-propanol.
6. The application as described in claim 5, characterized in that, The method of application is as follows: using carbonyl reductase... Zm Using wet cells obtained from engineered bacteria encoding the CR mutant gene through induction culture as a catalyst, 1-C-(β-D-xylanosyl)-acetone as a substrate, isopropanol as a co-substrate, and NADP as a catalyst... + As a coenzyme, a conversion system was constructed using a buffer solution at pH 6-8 as the reaction medium, and the reaction was carried out at 20-40℃ and 1000-3000 rpm to obtain ( S )-1-C-(B-D-pyranoxyl)-propanol.
7. The application as described in claim 6, characterized in that, In the conversion system, the substrate is added at a final concentration of 100-2000 mM, isopropanol is added at a volume concentration of 5-15%, and the co-substrate is NAD(P). + Add to a final concentration of 0.1-1.0 mM, and the amount of catalyst used is 5-20 g / L based on wet bacterial cells.
8. The application as described in claim 6, characterized in that, The reaction was carried out at 30 °C and 1500 rpm for 24 h.
9. The application as described in claim 6, characterized in that, The wet bacterial cells were prepared according to the following method: a carbonyl reductase-containing enzyme was used... Zm Engineered bacteria encoding the CR mutant gene were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h. Then, at a volume ratio of 2.0%, the culture was inoculated into sterilized LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 2 h. IPTG was then added to the culture medium to a final concentration of 0.10 mM, and the culture was incubated at 16°C for 12 h. Finally, the culture was centrifuged at 4°C and 8000 rpm for 10 min to obtain the carbonyl reductase-containing bacteria. Zm Wet bacterial cells of the CR mutant.