Amide hydrolase mutant, biological material, screening method, catalyst and application

Abstract

The invention discloses an amide hydrolase mutant, a biological material, a screening method, a catalyst and application, and compared with wild type amide hydrolase, the amide hydrolase mutant has amino acid residue mutation at the 317th site, or amino acid residue mutation at the 317th site and amino acid residue mutation at the 63rd site, or amino acid residue mutation at the 317th site, amino acid residue mutation at the 63rd site and amino acid residue mutation at the 65th site. The catalytic efficiency of the mutant can reach 16 times as high as that of wild type amide hydrolase, the yield of R-isobutylglutaric acid monoamide generated by catalyzing isobutylglutaric imide reaches 99%, the stereoselectivity ee value is greater than 99%, the reaction condition is mild, the reaction can be carried out only at 20-45 DEG C, and the mutant has high industrial application value.

Description

Technical Field

[0001] This invention relates to amide hydrolases, and more particularly to an amide hydrolase mutant, biomaterial, screening method, catalyst, and application. Background Technology

[0002] Pregabalin is a gamma-aminobutyric acid (GABA) analogue with antiepileptic, analgesic, and anti-anxiety activities. Due to its high safety profile in clinical use and effective pain relief, it is widely used to treat neuropathic pain, epilepsy, and other diseases. In 2021, pregabalin was included in the fourth batch of national centralized drug procurement due to its large usage. According to Data Bridge Market Research, the global pregabalin market size was approximately US$820 million in 2023 and is projected to reach US$1.09 billion by 2031, with a CAGR of 3.53%. With the continuous increase in market demand, the efficient synthesis of pregabalin has gradually become a research hotspot.

[0003] Currently, the main methods for synthesizing pregabalin include chemical and enzymatic methods. The chemical method is relatively mature; for example, it involves reacting 2-cyanoacetamide with isovaleraldehyde to generate a cyclic imine, followed by alkaline hydrolysis to obtain an R / S-racemic monoamide. This R-monoamide is then chirally resolved in chloroform using R-(+)-1-phenylethylamine, and finally, S-pregabalin is synthesized via the Hofmann rearrangement. However, this method has significant drawbacks, including the use of large amounts of organic solvents, severe environmental pollution, and the product being a racemic mixture requiring chiral resolution, leading to further reduced yields. Therefore, the direct and efficient synthesis of chiral intermediates has become a pressing technical challenge.

[0004] Compared to chemical methods, enzymatic methods have attracted widespread attention due to their high chiral selectivity and environmental friendliness. Enzymatic methods can precisely catalyze the synthesis of the chiral intermediate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, a precursor of pregabalin, avoiding the inefficiency of the chiral resolution step in traditional chemical methods and aligning with the demands of green and low-carbon development. Therefore, the synthesis technology of chiral drug intermediates based on bioenzymes is considered a core development direction for the efficient synthesis of pregabalin, possessing significant application value and promising prospects. However, the relatively high reaction temperatures of existing enzymatic methods somewhat limit their application. Summary of the Invention

[0005] Objectives of the invention: The first objective is to provide an amide hydrolase mutant with mild reaction conditions; the second objective is to provide biomaterials and screening methods related to the mutant; and the third objective is to provide the application of the mutant in the catalytic synthesis of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0006] Technical solution: The amide hydrolase mutant of the present invention is a protein with a mutation at amino acid residue 317, or a mutation at amino acid residues 317 and 63, or a mutation at amino acid residues 317, 63 and 65, based on the amino acid sequence of the wild-type amide hydrolase shown in SEQ ID NO: 1.

[0007] Preferably, the amide hydrolase mutant comprises: Proteins with a mutation at amino acid position 317 are obtained by mutating a cysteine ​​residue to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine. Proteins with mutations at amino acid residues 317 and 63 are obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue, and mutating the methionine residue at position 63 to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine. Proteins with mutations at amino acid residues 317, 63, and 65 are obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue, the methionine residue at position 63 to a tyrosine residue, and the phenylalanine residue at position 65 to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine.

[0008] Preferably, the amide hydrolase mutant comprises: Proteins with a mutation at amino acid position 317 are obtained by mutating a cysteine ​​residue to a tyrosine residue. Proteins with mutations in amino acid residues at positions 317 and 63 are obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue and the methionine residue at position 63 to a tyrosine residue. Proteins with mutations at amino acid residues 317, 63, and 65 are obtained by mutating cysteine ​​residue 317 to tyrosine residue, methionine residue 63 to tyrosine residue, and phenylalanine residue 65 to serine residue. The amino acid sequences are shown in SEQ ID NO: 3.

[0009] The nucleic acid molecule described in this invention is based on the wild-type amide hydrolase nucleic acid molecule shown in SEQ ID NO: 2, and contains corresponding base mutations that encode the amino acid sequence of the aforementioned amide hydrolase mutant.

[0010] The recombinant vector of the present invention comprises the aforementioned nucleic acid molecules.

[0011] The recombinant microorganisms described in this invention comprise the aforementioned nucleic acid molecules or the aforementioned recombinant vectors. The screening method for amide hydrolase mutants of the present invention includes: (1) Using the three-dimensional structure of wild-type amide hydrolase as a template, a visualization analysis was performed to screen key sites that affect enzyme activity and stereoselectivity; (2) Saturation mutations were performed on the key sites obtained from the screening to select the preferred mutation sites; (3) Using the preferred mutation sites obtained in step 2 as fixed mutation sites, saturate mutations are performed on the remaining key sites obtained in step 1 to screen out preferred mutation sites. (4) Using the preferred mutation sites obtained in steps 2 and 3 as fixed mutation sites, saturate mutations are performed on the remaining key sites obtained in step 1 to screen out preferred mutation sites, which are then used as amide hydrolase mutants.

[0012] Preferably, the key sites affecting enzyme activity and stereoselectivity in step 1 include amino acid residues at positions 63, 65, 67, 92, 94, 125, 151, 158, 181, 315, and 317.

[0013] The catalyst described in this invention includes the aforementioned amide hydrolase mutant.

[0014] The amide hydrolase mutant or catalyst described in this invention is used to synthesize (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0015] Preferably, the reaction temperature of the application is 20-45°C, and the reaction pH is 7-9.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The mutant catalyzes the synthesis of pregabalin chiral drug intermediates with high efficiency, which is 6.8 times that of the wild type; 2. High selectivity, with the ee value of the chiral product >99%; 3. Mild reaction conditions, which can be achieved at 20-45℃; 4. The screening method modifies the key amino acids of the enzyme active site through directed evolution and semi-rational design, which is highly efficient and effective. Attached Figure Description

[0017] Figure 1 The relative activity of iterative saturated mutant amide hydrolase mutant in catalyzing IBI to produce IBM; Figure 2 The following are liquid chromatograms of IBI standard, IBM standard and dilution reaction solution, where (a) is IBI standard, (b) is IBM standard and (c) is dilution reaction solution; Figure 3 SFC-ELSD chromatograms of racemic IBM standard and reaction catalytic product, where (a) is racemic IBM standard and (b) is reaction catalytic product; Figure 4 Generate IBM yield plots for wild-type and mutant strains under different pH conditions; Figure 5 Generate IBM yield plots for wild-type and mutant strains under different temperature conditions; Figure 6 SDS-PAGE images of protein yield at different fermentation induction temperatures and inducer concentrations are shown. 1 represents an induction temperature of 21℃ with a final IPTG concentration of 0.5 mM; 2 represents an induction temperature of 27℃ with a final IPTG concentration of 0.5 mM; 3 represents an induction temperature of 24℃ with a final IPTG concentration of 0.2 mM; 4 represents an induction temperature of 24℃ with a final IPTG concentration of 0.4 mM; and 5 represents an induction temperature of 24℃ with a final IPTG concentration of 0.6 mM. Figure 7 Kinetic curves for the production of R-IBM from IBI catalyzed by the mutant M63Y / F65S / C31Y. Detailed Implementation

[0018] The technical solution of the present invention will be further described below.

[0019] Example 1: Construction and expression of pET22b(+)-Gs-D-HYDase plasmid Source Geobacillus stearothermophilusThe Gs-D-HYDase (Uniprotentry Q45515) gene, an aminohydrolase, was synthesized by Genewiz (Suzhou) Co., Ltd. and constructed into the pET22b(+) vector. The constructed plasmid was transformed into competent E. coli DH5α cells. The transformed mixture was evenly spread on LB agar plates and incubated upside down at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of sterile LB liquid medium and incubated at 37°C and 150 rpm for 8–12 h. The pET22b(+)-Gs-D-HYDase plasmid was then extracted from E. coli DH5α cells using a commercial column-based plasmid extraction kit as a template for iterative saturation mutagenesis. Using the three-dimensional structure of Gs-D-HYDase as a template, visualization analysis was performed using PyMOL (v1.9). It was found that positions 63, 65, 67, 92, 94, 125, 151, 158, 181, 315, and 317 are key sites affecting enzyme activity and stereoselectivity.

[0020] Example 2: Construction of a site-directed saturated library of amide hydrolases Based on the conclusion of Example 1, primers were designed based on the gene sequence of wild-type amide hydrolase Gs-D-HYDase recorded in Uniprot (nucleotide sequence shown in SEQ ID NO: 2, amino acid sequence shown in SEQ ID NO: 1) (see Table 1). Site-directed saturation mutagenesis was performed on the parental Gs-D-HYDase gene (nucleotide sequence SEQ ID NO: 2) using primers M63X-F / M63X-R, F65X-F / F65X-R, G67X-F / G67X-R, F92X-F / F92X-R, L94X-F / L94X-R, M125X-F / M125X-R, V151X-F / V151X-R, V158X-F / V158X-R, M181X-F / M181X-R, D315X-F / D315X-R, and C317X-F / C317X-R, respectively. Mutant plasmids carrying the target gene were obtained using pET-22b(+) as the expression vector, and these mutant plasmids were then transformed into E. coli. Mutants of recombinant bacteria containing amide hydrolase mutant genes were obtained from C43(DE3), namely E. coli C43(DE3)-M63X (denoted as mutant M63X), E. coli C43(DE3)-F65X (denoted as mutant F65X), E. coli C43(DE3)-G67X (denoted as mutant G67X), E. coli C43(DE3)-F92X (denoted as mutant F92X), E. coli C43(DE3)-L94X (denoted as mutant L94X), E. coli C43(DE3)-M125X (denoted as mutant M125X), E. coli C43(DE3)-V151X (denoted as mutant V151X), and E. coli C43(DE3)- V158X (denoted as mutant V158X), E. coli C43(DE3)-M181X (denoted as mutant M181X), E. coli C43(DE3)-D315X (denoted as mutant D315X), E. coli C43(DE3)-C317X (denoted as mutant C317X).

[0021] Table 1 Primer design for constructing site-directed saturation mutant libraries of amide hydrolases

[0022] Table 2 PCR reaction system

[0023] The PCR reaction system was constructed according to Table 2. The reaction conditions were as follows: pre-denaturation at 95℃ for 3 min, followed by temperature cycling at 95℃ for 20 s, 55℃ for 10 s, and 72℃ for 30 s for a total of 30 cycles, with a final extension at 72℃ for 10 min, and a termination temperature of 4℃. After verification by 1% agarose gel electrophoresis, 1 µL of DpnI and 5 µL of buffer were added to the PCR product, and the template plasmid DNA was removed by digestion at 37℃ for 2 h. After inactivation at 65℃ for 10 min, the product was purified using a PCR cleanup kit and transformed into E. coli C43(DE3) competent cells. The cells were plated on LB plates containing ampicillin (100 µg / mL) and cultured overnight at 37℃ to obtain a mutant library of amidolytic enzymes. Single colonies grown on LB plates were used for subsequent screening of mutant libraries.

[0024] The parental strain was constructed using the same method: E. coli C43(DE3)-Gs-D-HYDase.

[0025] Example 3: Screening of Amide Hydrolase Mutant Library Screening of the amide hydrolase mutant library was performed using wild-type amide hydrolase Gs-D-HYDase as a reference. Single colony clones (from the mutant library constructed in Example 2) were picked and cultured in 1 mL deep 96-well plates. 400 µL of LB medium containing 100 µg / mL ampicillin was added beforehand. Two parental strains were also picked as controls in the last two wells of the 96-well plate. The 1 mL 96-well plate was incubated at 37°C for 8 h as seed culture. Then, 100 µL of the seed culture was added to a new 2 mL deep 48-well plate and cultured in sterile TB medium containing 100 µg / mL ampicillin beforehand. After incubation at 37°C for 8 h, IPTG was added at a final concentration of 0.4 mM, and expression was induced at 24°C for 16 h. The plates were then centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the wet cells were collected for further screening.

[0026] A 500 µL reaction mixture (400 mM Tris-HCl buffer, 400 mM 3-isobutylglutarimide (IBI), and 5 mM MnSO4) was added to each well. The bacterial cells were resuspended and reacted at 37 °C and 400 rpm for 24 h. 500 µL of methanol was added to precipitate the protein, followed by centrifugation at 12000 rpm for 1 min. 100 µL of the supernatant was added to 400 µL of methanol and mixed thoroughly. The yield of isobutylglutarimide monoamide (IBM) was determined by liquid chromatography, and the optical purity of IBM was determined using supercritical fluid chromatography with a tandem evaporative light scattering detector.

[0027] Liquid chromatography analysis conditions: Agilent-1260 HPLC and Agilent TC-C18 column, column oven 35 ℃, mobile phase A: 0.1% TFA water, mobile phase B: acetonitrile, flow rate 1 mL / min, detection wavelength: 210 nm, injection volume 5 μL, and the mobile phase gradient is shown in Table 3. Table 3 Mobile phase gradient

[0028] The retention time is Rt(IBM) = 8.55 min, Rt(IBI) = 15.95 min.

[0029] The liquid chromatograms of IBI standards, IBM standards, and diluted reaction solutions are shown below. Figure 2 As shown.

[0030] Methods for determining the optical purity of catalytic products IBM: Supercritical fluid chromatography-evaporative light scattering detector (SFC-ELSD) conditions: Column: CHIRALPAK® AD-H SFC; Mobile phase: 5% methanol, 95% CO2; Flow rate: 1 mL / min; Injection volume: 5 μL; Column temperature: 35℃; ELSD evaporation temperature: 35℃; Run time: 30 min SFC-ELSD chromatograms of the racemic IBM standard and the reaction catalytic product are shown below. Figure 3 As shown.

[0031] The amount and ee value of IBM produced by the parental strain of E. coli C43(DE3)-Gs-D-HYDase were used as controls. A superior mutant strain was obtained, with the mutation site C317Y, whose relative activity was 2.5 times that of the wild type. Figure 1 Its ee value is greater than 97%.

[0032] Example 4: Construction of an iterative saturated mutant library of amide hydrolases Primers were designed based on the gene sequence of the amide hydrolase Gs-D-HYDase (see Table 4). Using primers M63X-F and M63X-R, F65X-F and F65X-R, respectively, and the preferred mutant C317Y at position 317 as a template, iterative saturation mutagenesis was performed at positions 65 and 63.

[0033] The 50 µL PCR reaction system is the same as in Example 3.

[0034] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 3 min, followed by temperature cycling at 95℃ for 20 s, holding at a suitable annealing temperature for 10 s, then at 72℃ for 30 s, for a total of 30 cycles, and finally extension at 72℃ for 10 min, with a termination temperature of 4℃. After verification by agarose gel electrophoresis, the PCR product was purified using a PCR cleanup kit. After positive PCR analysis by 0.9% agarose gel electrophoresis, 20 µL of the PCR solution was taken, 1 µL of LDpnI was added, and the template plasmid DNA was removed by digestion at 37℃ for 2 h. The DNA was then inactivated at 65℃ for 10 min, and the solution was transformed into competent E. coli C43 (DE3) cells and plated on LB agar plates containing ampicillin (100 mg / L).

[0035] Table 4 Primer design for the second round of iterations of saturated mutant library construction of amide hydrolase

[0036] The screening of the amide hydrolase mutant library used the superior mutant C317Y obtained from the first round of screening of Gs-D-HYDase as a reference, and the second round of iterative screening of the saturated mutant library was carried out using the method in Example 3. The yield of IBM was detected by liquid chromatography, and the optical purity of IBM was detected by supercritical fluid chromatography with a tandem evaporative light scattering detector. The amount and ee value of IBM produced by the parent strain were used as controls. A superior mutant strain was obtained, with the mutation site C317Y / M63Y, and its relative activity was 4.3 times that of the wild type. Figure 1 Its ee value is greater than 99%.

[0037] Example 5: Combined Mutation and Activity Screening of Dominant Strains of Amide Hydrolase Primers were designed based on the gene sequence of the amide hydrolase Gs-D-HYDase (see Table 3). Using primers F65X-F and F65X-R, a combined mutation at position 65 was performed on the plasmid C317Y / M63Y (Example 4), a preferred mutant, with combined mutations at positions 317 and 63. The 50 µL PCR reaction system was the same as in Example 4. The PCR program was: 95℃ pre-denaturation for 3 min, 30 cycles: 95℃ for 20 s, 55℃ for 10 s, 72℃ for 30 s, and a final extension at 72℃ for 10 min. After verification by agarose gel electrophoresis, the PCR products were purified using a PCR cleanup kit. After PCR was confirmed to be positive by 0.9% agarose gel electrophoresis, 20 µL of PCR solution was taken, 1 µL of DpnI was added, and the DNA of the template plasmid was removed by enzyme digestion at 37℃ for 2 h. The plasmid was then inactivated at 65℃ for 10 min and transformed into competent E. coli C43 (DE3) cells. The cells were then plated on LB plates containing ampicillin resistance (100 mg / L).

[0038] Table 5 Primer design for the third round of iterative construction of the saturated mutant library of amide hydrolase.

[0039] The screening of the amide hydrolase mutant library used the superior mutant C317Y / M63Y obtained from the second round of screening of Gs-D-HYDase as a reference, and the second round of iterative screening of the saturated mutant library was carried out using the method in Example 3. The yield of IBM was detected by liquid chromatography, and the optical purity of IBM was detected by supercritical fluid chromatography with a tandem evaporative light scattering detector. The amount and ee value of IBM produced by the parent strain were used as controls. A superior mutant strain was obtained, with the mutation site C317Y / M63Y / F65S, whose relative activity was 6.8 times that of the wild type. Figure 1 Its ee value is greater than 99%.

[0040] Example 6: Optimal pH screening of amide hydrolase mutants C317Y / M63Y / F65S Based on the conclusion of Example 5, the mutant E. coli BL21(DE3)-C317Y / M63Y / F65S (amino acid sequence of SEQ ID NO: 3, nucleotide sequence of SEQ ID NO: 4) obtained above was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 100 µg / mL ampicillin. The tubes were cultured at 37 °C and 150 rpm for 6-8 h in a shaker. Then, 2% of the inoculum was added to 2 L Erlenmeyer flasks for culture. 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin was added beforehand. After culturing at 37 °C for 24 h, IPTG with a final concentration of 0.4 mM was added. Expression was induced at 24 °C for 22 h. After centrifugation at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. Resuspend the bacterial cells in 100 mL of 400 mM Tris-HCl (pH=8.5) buffer. Add 1 mL of the resuspended bacterial solution to each of four 2 mL sterile centrifuge tubes, centrifuge at 4000 rpm for 10 min at 4 ℃, discard the supernatant, and add 1 mL of buffer with different pH values ​​(pH=6 disodium hydrogen phosphate-citrate buffer, pH=7.4 disodium hydrogen phosphate-citrate buffer, pH=8 Tris-HCl buffer, and pH=8.5 Tris-HCl buffer) to each of the four centrifuge tubes to resuspend the bacterial solution. Take 500 μL of the supernatant and add it to each of four 5 mL glass reaction flasks. Then add IBI to a final concentration of 400 mM and MnSO4 to a final concentration of 5 mM, respectively. Mix well with a magnetic stirrer and react at 37 ℃ and 400 rpm for 20 h. Take 500 μL of reaction solution from each reaction flask, add 500 µL of methanol, mix well, centrifuge at 12000 rpm for 1 min, take 100 µL of supernatant and add it to 400 µL of methanol, detect the IBM yield by liquid chromatography, and detect the optical purity of IBM by supercritical fluid chromatography with tandem evaporative light scattering detector.

[0041] Experimental results are as follows Figure 4 As shown, the optimal pH for the mutant C317Y / M63Y / F65S is 8.5. Above 8.5, the substrate IBI spontaneously hydrolyzes, and the optical purity of the product IBM is significantly reduced. It can maintain good activity in the pH range of 7.4 to 8.5, and the activity decreases significantly when pH < 7.

[0042] Example 7: Screening for the optimal temperature of amide hydrolase mutant C317Y / M63Y / F65S Based on the conclusions of Example 5, the mutant E. coli BL21(DE3)-C317Y / M63Y / F65S obtained above was inoculated into 10 mL of sterile LB medium containing 100 µg / mL ampicillin. The culture was incubated at 37 °C and 150 rpm for 6-8 h. Then, 2% of the inoculum was added to a 2 L Erlenmeyer flask, which was incubated with 1 L of sterile TB medium containing 100 µg / mL ampicillin. After incubation at 37 °C for 12 h, IPTG (0.4 mM) was added, and expression was induced at 24 °C for 22 h. The flask was then centrifuged at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. The wet cells were resuspended in 100 mL of 400 mM Tris-HCl (pH=8.5) buffer. Take 1 mL of the resuspended bacterial culture into three 5 mL glass reaction flasks. Add IBI to a final concentration of 400 mM and MnSO4 to a final concentration of 5 mM. Place a magnetic stir bar in each flask and mix thoroughly. React at 20 ℃, 37 ℃, and 45 ℃ at 400 rpm for 20 h, respectively. Take 500 μL of the reaction solution, add 500 µL of methanol, mix well, and centrifuge at 12000 rpm for 1 min. Take 100 µL of the supernatant and add it to 400 µL of methanol. Detect the yield of IBM using liquid chromatography and the optical purity of IBM using supercritical fluid chromatography with tandem evaporative light scattering detector.

[0043] Experimental results are as follows Figure 5 As shown, the C317Y / M63Y / F65S mutant exhibits the highest activity for the substrate IBI at 45℃, and good activity is observed in the range of 20℃ to 37℃. Therefore, the C317Y / M63Y / F65S mutant can efficiently hydrolyze IBI under mild temperature conditions, saving energy and demonstrating green and low-carbon characteristics.

[0044] Example 8: Determination of the Michaelis kinetic constant of the aminohydrolase mutant C317Y / M63Y / F65S Based on the conclusions of Example 5, the mutant E. coli BL21(DE3)-C317Y / M63Y / F65S and wild-type E. coli BL21(DE3)-Gs-D-HYDase obtained above were inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 100 µg / mL ampicillin, respectively. The tubes were cultured at 37 ℃ and 150 rpm for 6-8 h in a shaker. Then, 2% of the inoculum was added to 2 L Erlenmeyer flasks, which were then cultured in 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. After culturing at 37 ℃ for 12 h, IPTG with a final concentration of 0.4 mM was added, and expression was induced at 24 ℃ for 22 h. The cells were then centrifuged at 4000 rpm for 30 min at 4 ℃, the supernatant was discarded, and the wet cells were collected. The wet cells were resuspended in 35 mL of 400 mM Tris-HCl (pH=8.5) buffer. The protein was sonicated at 4 ℃ for 10 min with 65% sonication power, and the sonication cycle was 2 s on and 6 s off. It was then centrifuged at 12000 rpm at 4 ℃ for 15 min. The supernatant was collected, and the protein was purified by affinity chromatography. The absorbance was measured using an A280 UV spectrophotometer to calculate the protein concentration. The protein was diluted to a concentration of 2 μM with 400 mM Tris-HCl (pH=8.5) buffer. 1 mL of the diluted mutant C317Y / M63Y / F65S protein solution was placed in five 5 mL glass reaction flasks. IBI was added to each flask at final concentrations of 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, and 100 mM, respectively. The reaction mixture was magnetically stirred at 400 rpm at 37 ℃ for 60 min. 1 mL of diluted wild-type Gs-D-HYDase protein solution was placed into five 5 mL glass reaction flasks. IBI was added to each flask at final concentrations of 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, and 100 mM, respectively. The mixtures were magnetically stirred at 37 ℃ and 400 rpm for 60 min. 500 μL of the reaction solution from each flask was added to 500 µL of methanol, mixed thoroughly, and centrifuged at 12000 rpm for 1 min. 100 µL of the supernatant was added to 400 µL of methanol, and the IBM yield was determined by liquid chromatography.

[0045] The enzyme-catalyzed reaction rate was measured, and Michaelis kinetic parameters were calculated by plotting a double reciprocal curve based on the reaction rate and the reciprocal of the substrate concentration. The results showed that the mutant's K... cat / K m It is 16 times that of the wild type, meaning that the catalytic efficiency of the amide hydrolase is significantly improved.

[0046] Example 9: Optimization of fermentation conditions for the aminohydrolase mutant C317Y / M63Y / F65S Based on the conclusions of Example 5, the mutant E. coli BL21(DE3)-C317Y / M63Y / F65S obtained above was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 100 µg / mL ampicillin. The tubes were cultured at 37 ℃ and 150 rpm in a shaker for 6-8 h. Then, 1% of the inoculum was added to 250 mL Erlenmeyer flasks, which contained 50 mL of sterile LB medium with a final concentration of 100 µg / mL ampicillin. After culturing at 37 ℃ for 12 h, the culture was transferred to five sterile 1 L parallel fermenters. Each fermenter contained 300 mL of sterile TB medium with a final concentration of 100 µg / mL ampicillin. The pH was controlled at 7 using ammonia, the fermentation temperature was maintained at 37 ℃, the stirring speed at 400 rpm, and the dissolved oxygen concentration (DO) at 30%. Feeding was started after approximately 6-8 h of culture. When the OD600 reached 40, IPTG was added as an inducer, the induction temperature was adjusted, and the culture was continued for 22 h. The cells were then centrifuged at 4000 rpm for 30 min at 4 °C, the supernatant was discarded, and the wet cells were collected. The induction conditions for the five parallel fermenters were as follows: 1-Induction temperature 21 °C, final IPTG concentration 0.5 mM; 2-Induction temperature 27 °C, final IPTG concentration 0.5 mM; 3-Induction temperature 24 °C, final IPTG concentration 0.2 mM; 4-Induction temperature 24 °C, final IPTG concentration 0.4 mM; 5-Induction temperature 24 °C, final IPTG concentration 0.6 mM. The cells were resuspended in 100 mL of 400 mM Tris-HCl (pH=8.5) buffer. The cells were then sonicated at 4 °C for 10 min at 65% power, with the sonication cycle on for 2 s and off for 6 s. The sample was then centrifuged at 12000 rpm for 15 min at 4 ℃. The supernatant was collected, diluted 10-fold with 400 mM Tris-HCl (pH=8.5), and subjected to SDS-PAGE electrophoresis. The results were compared with protein markers. The experimental results are as follows: Figure 6 As shown in the figure. The results indicate that the highest protein yield was achieved at an induction temperature of 24 °C and an IPTG concentration of 0.4 mM.

[0047] Example 10: Kinetic curve of catalytic reaction of amide hydrolase mutant C317Y / M63Y / F65S Based on the conclusions of Example 9, scale-up fermentation was carried out under the optimal fermentation conditions selected above. The mutant E. coli BL21(DE3)-C317Y / M63Y / F65S obtained above was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 100 µg / mL ampicillin. The tubes were cultured at 37 °C and 150 rpm in a shaker for 6-8 h. Then, 1% of the inoculum was added to a 1 L Erlenmeyer flask and cultured with 250 mL of sterile LB medium containing a final concentration of 100 µg / mL ampicillin. After culturing at 37 °C for 12 h, the flask was transferred to a 5 L fermenter. The fermenter contained 2 L of sterile TB medium with a final concentration of 100 µg / mL ampicillin. The pH was controlled at 7 using ammonia water, the fermentation temperature was controlled at 37 °C, the stirring speed was 400 rpm, and the DO was controlled at 30%. Feeding was started after about 6-8 h of culture. When the OD600 reaches 40, add IPTG to a final concentration of 0.4 mM, adjust the induction temperature to 24 ℃, and continue culturing for 24 h. Centrifuge at 4000 rpm for 30 min at 4 ℃, discard the supernatant, and collect the wet bacterial cells. Resuspend the bacterial cells in 1 L of 400 mM Tris-HCl (pH=8.5) buffer. Take 300 mL of the resuspended bacterial culture into a 1 L fermenter, add IBI to a final concentration of 1 M and MnSO4 to a final concentration of 5 mM, control the pH of the reaction with 5% ammonia water to 8.5, and react for 30 h at 37 ℃ and 400 rpm. Take 500 μL of the reaction solution at 0 h, 2.5 h, 4 h, 6 h, 18 h, 24 h, and 30 h, respectively, add 500 µL of methanol, mix well, centrifuge at 12000 rpm for 1 min, take 100 µL of the supernatant and add it to 400 µL of methanol, detect the yield of IBM by liquid chromatography, and detect the optical purity of IBM by supercritical fluid chromatography with tandem evaporative light scattering detector.

[0048] Experimental results are as follows Figure 7 As shown, the C317Y / M63Y / F65S mutant catalyzes the production of R-IBM from 1M IBI at 37℃ for 30 h with a yield of 99% and a stereoselectivity ee value greater than 99%, which has industrial application value.

Claims

1. An amide hydrolase mutant, characterized in that, Based on the amino acid sequence of the wild-type amide hydrolase shown in SEQ ID NO: 1, a protein having a mutation at amino acid residue 317, or a mutation at amino acid residues 317 and 63, or a mutation at amino acid residues 317, 63 and 65.

2. The amide hydrolase mutant according to claim 1, characterized in that, The protein with the mutated amino acid residue at position 317 is obtained by mutating a cysteine ​​residue to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine. The protein with the mutations at amino acid residues 317 and 63 is obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue, and mutating the methionine residue at position 63 to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine. The protein with mutations at amino acid residues 317, 63, and 65 is obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue, the methionine residue at position 63 to a tyrosine residue, and the phenylalanine residue at position 65 to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine.

3. The amide hydrolase mutant according to claim 2, characterized in that, The protein with the mutation at amino acid residue 317 is obtained by mutating a cysteine ​​residue to a tyrosine residue. The protein with the mutations at amino acid residues 317 and 63 is obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue and the methionine residue at position 63 to a tyrosine residue. The protein with mutations at amino acid residues 317, 63, and 65 was obtained by mutating the cysteine ​​residue at position 317 to a tyrosine residue, the methionine residue at position 63 to a tyrosine residue, and the phenylalanine residue at position 65 to a serine residue, with the amino acid sequence shown in SEQ ID NO:

3.

4. A nucleic acid molecule, characterized in that, Based on the wild-type amide hydrolase nucleic acid molecule shown in sequence SEQ ID NO: 2, containing the corresponding base mutation, the amino acid sequence encoding the amide hydrolase mutant of any one of claims 1-3 is provided.

5. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 4.

6. A recombinant microorganism, characterized in that, The recombinant microorganism comprises the nucleic acid molecule of claim 4 or the recombinant vector of claim 5.

7. A method for screening amide hydrolase mutants according to any one of claims 1-3, characterized in that, include: (1) Using the three-dimensional structure of wild-type amide hydrolase as a template, a visualization analysis was performed to screen key sites that affect enzyme activity and stereoselectivity; (2) Saturation mutations were performed on the key sites obtained from the screening to select the preferred mutation sites; (3) Using the preferred mutation sites obtained in step 2 as fixed mutation sites, saturate mutations are performed on the remaining key sites obtained in step 1 to screen out preferred mutation sites. (4) Using the preferred mutation sites obtained in steps 2 and 3 as fixed mutation sites, saturate mutations are performed on the remaining key sites obtained in step 1 to screen out preferred mutation sites, which are then used as amide hydrolase mutants.

8. A catalyst, characterized in that, The catalyst comprises the amide hydrolase mutant according to any one of claims 1-3.

9. The use of an amide hydrolase mutant according to any one of claims 1-3 or the catalyst according to claim 8 in the synthesis of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

10. The application according to claim 9, characterized in that, The reaction temperature for this application is 20-45℃, and the reaction pH is 7-9.

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