Epoxide hydrolase mutant and construction method and application of co-immobilized enzyme of epoxide hydrolase mutant

Abstract

The invention discloses an epoxide hydrolase mutant and a construction method and application of a co-immobilized enzyme of the epoxide hydrolase mutant. The inventor firstly constructs a recombinant expression vector of epoxide hydrolysis MdEH, then constructs a recombinant expression vector of point mutation MdEH-K71I on the basis, then constructs an MdEH-K71I recombinant expression vector suitable for being used in bacillus subtilis, and then replaces a promoter and a signal peptide in the expression vector. The modified recombinant expression vector is used for transforming bacillus subtilis, so that efficient secretory expression of the epoxide hydrolase in a bacillus subtilis expression system is realized. The process conditions of co-immobilization of epoxide hydrolase and halohydrin dehalogenase are further explored, ECR8285 resin is selected as an immobilization carrier, and the conversion rate of glycerin generated by hydrolysis finally reaches 80.29%.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering, and in particular to a construction method and application of an epoxide hydrolase mutant and a co-immobilized enzyme thereof. Background Art

[0002] Chloropropanols (CPs), a common food contaminant, have been detected in soy sauce, cereal products, maltose products, and smoked foods produced from acid-hydrolyzed vegetable proteins. These pollutants can pollute the environment and, if present in food, can be toxic to humans and even cause genotoxicity. Physical and chemical treatments for CPs in food are inefficient and prone to secondary contamination. Enzymatic degradation of CPs offers advantages such as environmental friendliness, mild reaction conditions, low energy consumption, and high efficiency.

[0003] Epoxide hydrolases (EHs) are key enzymes in the biocatalytic synthesis of chiral chemicals. They specifically hydrolyze epoxides to efficiently produce optically pure epoxides, β-substituted alcohols, and other high-value-added products. However, naturally derived EHs face significant challenges in their industrial application. First, thermal stability is a significant issue. Many epoxide hydrolases have a half-life of less than 2 hours at 40°C, while industrial bioreactors often require temperatures of 50–60°C to inhibit bacterial contamination and maximize reaction rates. Second, substrate adaptability is limited. Natural enzymes generally have low catalytic efficiency for non-natural substrates, making them difficult to meet the demands of complex industrial substrate systems. Third, scalable production is a bottleneck. Existing expression systems suffer from low enzyme secretion efficiency and extensive fermentation processes, resulting in catalyst costs accounting for over 40% of total production costs. These challenges force most companies to rely on chemical synthesis, which increases energy consumption and leads to the use of large amounts of toxic solvents and heavy metal catalysts, contradicting the concept of green manufacturing. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an epoxide hydrolase mutant.

[0005] Another object of the present invention is to provide an engineered bacterium that expresses the epoxide hydrolase mutant.

[0006] Another object of the present invention is to provide an immobilized enzyme obtained by co-immobilizing the epoxide hydrolase mutant and the halohydrin dehalogenase.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An epoxide hydrolase mutant is a protein obtained by performing mutation on the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation in the sequence is at least one of H11E, H12F, K71I and N94R.

[0009] The nucleotide sequence of the gene encoding the epoxide hydrolase mutant is obtained according to the codon coding rules.

[0010] The gene encoding the epoxide hydrolase mutant is a nucleotide sequence obtained by mutation using mutation primers based on the nucleotide sequence shown in SEQ ID NO. 2.

[0011] The invention discloses a recombinant vector for expressing an epoxide hydrolase mutant, which carries an epoxide hydrolase mutant expression cassette.

[0012] A method for constructing an engineered bacterium expressing an epoxide hydrolase mutant comprises the following steps:

[0013] After the recombinant vector carrying the epoxide hydrolase mutant expression box is transferred into the engineering bacteria, the engineering bacteria expressing the epoxide hydrolase mutant are obtained.

[0014] The vector skeleton of the recombinant vector carrying the epoxide hydrolase mutant gene is pET-28b(+) or pTH1469.

[0015] The epoxide hydrolase mutant expression box comprises a promoter, a signal peptide, a coding gene of the epoxide hydrolase mutant and a terminator.

[0016] The promoter includes at least one of glvA, srfA, ydzA, fusA, and gsiB.

[0017] The signal peptide includes at least one of phoD, phrC, peI, amyE, and ydbN.

[0018] The engineered bacteria is Bacillus subtilis WB600.

[0019] An engineered bacterium expressing an epoxide hydrolase mutant is constructed by the above construction method.

[0020] A co-immobilized enzyme for cascade catalytic degradation of 3-chloropropanol, the preparation method comprising the following steps:

[0021] A buffer solution is added to the resin for pre-equilibration treatment, and then an enzyme solution obtained by mixing the epoxide hydrolase mutant and the halohydrin dehalogenase is added, followed by adsorption, washing, and drying to obtain an immobilized enzyme obtained by co-immobilizing the epoxide hydrolase mutant and the halohydrin dehalogenase.

[0022] The resin includes at least one of ECR8404M, ECR8285, ECR1030M, ECR1090M, and PCG900C.

[0023] The buffer solution is a phosphate buffer solution with a pH value of 6.0 to 8.0, preferably a phosphate buffer solution with a pH value of 8.0.

[0024] The mass ratio of the epoxide hydrolase mutant to the halohydrin dehalogenase in the enzyme solution obtained by mixing the epoxide hydrolase mutant and the halohydrin dehalogenase is 1:5.

[0025] The halohydrin dehalogenase is halohydrin dehalogenase HheA.

[0026] The adsorption conditions are 30-50° C., 150-200 rpm, and 6-10 hours.

[0027] Application of the epoxide hydrolase mutant in catalyzing the hydrolysis of epoxides.

[0028] The application of the co-immobilized enzyme for cascade catalytic degradation of 3-chloropropanol in catalytic degradation of 3-chloropropanol.

[0029] The present invention has the following advantages and effects compared to the prior art:

[0030] (1) In the present invention, the inventors first constructed a recombinant expression vector for epoxide hydrolysis MdEH, and then constructed a recombinant expression vector for the point mutation MdEH-K71I. The modified recombinant expression vector was transformed into Escherichia coli, and the thermal stability parameters of the mutant were characterized. Then, a recombinant expression vector for MdEH-K71I suitable for Bacillus subtilis was constructed, and the promoter and signal peptide in the expression vector were replaced. The modified recombinant expression vector was transformed into Bacillus subtilis. Using the recombinant Bacillus subtilis strain, the efficient secretory expression of epoxide hydrolase was achieved in the Bacillus subtilis expression system.

[0031] (2) The present invention further explored the process conditions for producing epoxide hydrolase by recombinant Bacillus subtilis. When fermented in a 7L fermenter, when the induction temperature was 34°C and the induction pH was 7.0, the enzyme activity in the supernatant of the fermentation broth reached 792.86 U / mL.

[0032] (3) The present invention further explored the process conditions for co-immobilization of epoxide hydrolase and halohydrin dehalogenase. ECR8285 resin was selected as the immobilization support, and the immobilization buffer was kept at pH 8.0 and oscillated at 30°C for 8 h. 10 mM 3-MCPD and 0.3 g immobilized enzyme were added to 1 mL of the reaction system. The reaction was carried out at 35°C for 12 h, and the final glycerol conversion rate produced by hydrolysis reached 80.29%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Comparison of enzyme activities of the MdEH mutants in Example 1 of the present invention.

[0034] Figure 2 The optimal temperature curve (A) of the epoxide hydrolase MdEH and its mutants in Example 1 of the present invention and the fitting calculation of the inactivation rate constant at 70°C (B) are shown.

[0035] Figure 3 Effects of different promoters on the expression of MdEH-K7I in Example 2 of the present invention (A) Cell wet weight and OD600 (B) Enzyme activity and protein concentration 1: Pgrac; 2: PglvA; 3: PsrfA; 4: PydzA; 5: PfusA; 6: PgsiB

[0036] Figure 4 The effects of different signal peptides on the expression of MdEH-K71I in Example 2 of the present invention (A) wet weight of bacteria and OD600 (B) enzyme activity and protein concentration 1: SPamyQ; 2: SPphoD; 3: SPphrC; 4: SPpeI; 5: SPamyE; 6: SPydbN.

[0037] Figure 5 This is the effect of different resin supports on the co-immobilized enzyme in Example 3 of the present invention.

[0038] Figure 6 This is the effect of different immobilization temperatures on the co-immobilized enzyme in Example 3 of the present invention.

[0039] Figure 7 This is the effect of different immobilization buffer pH on the co-immobilized enzyme in Example 3 of the present invention.

[0040] Figure 8 This is the effect of different reaction temperatures on the co-immobilized enzyme in Example 3 of the present invention.

[0041] Figure 9 This is the effect of different enzyme addition amounts on the co-immobilized enzyme in Example 3 of the present invention.

[0042] Figure 10 This is the effect of different reaction times on the co-immobilized enzyme in Example 3 of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0044] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0045] In the following examples, the detection methods involved are as follows:

[0046] 1. Wet weight determination: Take three 2 mL centrifuge tubes and accurately weigh their total weight M. Accurately pipette 2 mL of fermentation broth into each 2 mL centrifuge tube, centrifuge at 12000 rpm for 3 min, carefully discard the supernatant, and accurately weigh the total weight M'. Calculate the cell wet weight (g / L) = (M'-M) / 6×1000

[0047] 2. OD 600 Determination: Use pure water as a blank and dilute the fermentation liquid with pure water a certain number of times so that the OD value measured at a wavelength of 600nm is between 0.2 and 0.8. Then multiply this value by the dilution multiple to obtain the cell OD value in the fermentation liquid.

[0048] 3. Determination of protein content in fermentation supernatant

[0049] The protein concentration of the fermentation supernatant was determined using a Bradford protein concentration kit.

[0050] Example 1 Construction of epoxide hydrolase mutants

[0051] 1.1 Construction of single-point mutation fragments

[0052] To improve the thermostability and enzymatic activity of the epoxide hydrolase MdEH (whose nucleotide sequence is disclosed in Chinese invention patent CN118272455A, referred to as SEQ ID NO. 3 herein), four mutants, H11E, H12F, K71I, and N94R, were designed by sequence and structure analysis. Corresponding primers were designed for these mutants. Overlap extension PCR was performed using the pET-28b(+)-MdEH plasmid as the amplification template to obtain recombinant plasmids of these mutants. Amplification was verified by electrophoresis on a 1% agarose (TAE) gel. Successful PCR products were then digested at 37°C for at least 1 hour to remove the template DNA. The products were then purified according to the instructions of the PCR product purification kit.

[0053] Table 1 Single point mutation primers

[0054]

[0055] 1.2 Transformation of single-point mutants

[0056] The pET-28b(+) plasmid carrying the mutant gene obtained in 1.1 was linearized using restriction endonuclease Dpn I and transformed into Escherichia coli BL21(DE3). Positive clones were screened and sequenced to obtain a recombinant strain carrying the epoxide hydrolase MdEH single-point mutant.

[0057] 1.3 Expression of recombinant strains

[0058] The recombinant strain obtained in 1.2 was inoculated into a shake flask for induction culture. After the culture was completed, the bacteria were isolated to obtain the epoxide hydrolase MdEH mutant. The positive recombinant was transferred to 5 mL containing 100 μg mL -1 Incubate in LB tube containing kanamycin at 37℃ and 200rpm for 16-20h to prepare the first-stage seed solution. Inoculate the first-stage seed solution at 4% to 50mL of 100μg·mL -1 Culture in LB medium containing kanamycin at 37°C and 200 rpm for 12 hours to prepare the secondary seed solution. Inoculate 500 mL of LB medium with a 4% inoculum of the secondary seed solution and incubate at 37°C and 200 rpm for 2 hours. Induce with 0.5 mM IPTG to a final concentration of 0.5 mM. Incubate at 25°C and 200 rpm for 18 hours. Remove the cells by centrifugation at 7000 rpm at 4°C for 25 minutes. Collect the supernatant and store at 4°C for later use.

[0059] 1.4 Enzyme activity detection

[0060] (1) Definition of enzyme activity: One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the conversion of substrate glycidol to 1 μmol of product glycerol within 1 min under certain reaction conditions.

[0061] (2) Principle of enzyme activity assay: Epoxide hydrolase can hydrolyze glycidol into corresponding sugar alcohols, while acidic periodate can oxidize sugar alcohols to produce formaldehyde, which then reacts with formaldehyde in the presence of Nash reagent to form a yellow compound with the maximum absorption peak detected at 412 nm.

[0062] (3) Enzyme activity determination method

[0063] Add glycidol to a final concentration of 10 mM and a certain amount of epoxide hydrolase to a 2 mL EP tube. Phosphate buffer (0.1 M, pH 8) is added to a 1 mL reaction volume. The reaction is then placed in a metal bath at 800 rpm for 10 minutes at the appropriate temperature. After completion, 0.125 mL of the reaction solution is quickly removed from the reaction system and transferred to a 2 mL EP tube containing 0.125 mL of sodium periodate solution. Mix thoroughly and incubate at 30°C for 10 minutes to terminate the enzymatic reaction and complete the oxidation reaction. Subsequently, 0.25 mL of L-rhamnose and 0.5 mL of Nash are added and mixed thoroughly. After incubating in a 53°C water bath for 15 minutes, the reaction solution is removed and allowed to cool at room temperature for 10 minutes. 200 μL of the reaction solution is then aspirated and the absorbance is measured at 412 nm. The product yield is calculated using the absorbance / sugar alcohol concentration curve, and the corresponding enzyme activity is calculated using the enzyme activity formula.

[0064] The experimental results showed that the enzyme activities of wild type and mutants were determined at 70°C using sodium periodate-Nash reagent colorimetric method with glycidol as substrate. The enzyme activities of H11E, H12F, K71I, and N94R were 100.36%, 94.96%, 129.22%, and 75.28% of the wild type, respectively. Figure 1 The experimental results showed that the enzyme activity of mutant K71I was improved compared with that of the wild type, while the enzyme activities of mutants H12F and N94R were reduced compared with that of the wild type.

[0065] 1.5 Determination of the optimal reaction temperature of mutants

[0066] Using glycidol as substrate, the epoxide hydrolase activity was measured at 30-80°C (10°C per temperature gradient). Three parallel experiments were performed, and the highest epoxide hydrolase activity was set as 100%. The temperature-relative enzyme activity curve was plotted.

[0067] The optimal reaction temperature of all mutants was the same as that of wild-type epoxide hydrolase, which was 70℃ ( Figure 2 A), indicating that the point mutation did not change the optimal reaction temperature of epoxide hydrolase MdEH. Furthermore, the hydrolysis activity of mutant H12F remained high within the temperature range of 30°C to 80°C, reaching over 60% of the optimal enzyme activity.

[0068] 1.6 Determination of mutant half-life

[0069] Half-life (t 1 / 2) is the time it takes for the enzyme activity to lose half its value under certain conditions. The enzyme solution was incubated at 70°C for 60 minutes, and samples were taken every 10 minutes to measure the residual enzyme activity of the samples under the optimal reaction conditions. Three parallel experiments were performed, and the hydrolysis activity of the unincubated sample was taken as 100%. A ln (residual activity)-time line graph was drawn. The slope of the linear fit is the first-order inactivation constant (kT) of the epoxide hydrolase at that temperature. Based on kT, t 1 / 2 .

[0070]

[0071] The experimental results are as follows Figure 2 As shown in B, the first-order inactivation constant of WT was 0.0143h -1 (R 2 =0.9665), linear correlation coefficient R 2 >95%, indicating that it conforms to the first-order inactivation equation. The half-life was calculated to be 48.47 min by formula 2-2. The mutant was incubated at 70°C for 1 hour and compared with the residual activity of WT incubated under the same conditions. It was found that the residual activity of the single-point mutation H12F was lower than that of WT, indicating that its thermal stability was reduced, while the mutants H11E, K71I and N94R all showed better thermal stability. By fitting, it was found that the linear correlation coefficient R 2 All were greater than 95%. The half-life of mutant H12F was 39.38 min, while the half-lives of mutants H11E, K71I and N94R were 71.46 min, 82.52 min and 77.02 min, respectively, which were 0.47 times, 0.70 times and 0.59 times longer than that of WT.

[0072] Based on the above results, the K71I mutation with better thermal stability and highest enzyme activity was selected for subsequent experiments.

[0073] Example 2 Optimization of epoxide hydrolase mutants

[0074] 2.1 Construction of pTH1469 vector

[0075] The PCR product obtained in 1.1 of Reference Example 1 was transformed into Escherichia coli TOP 10 competent cells, positive clones were screened, and sequencing was performed to obtain a pET-28b(+) plasmid carrying the mutant gene. Using this plasmid as a template, primers were used to amplify the mutant gene fragment, which was then ligated into the pTH1469 vector to obtain the pTH1469 vector carrying the mutant gene.

[0076] 2.2 Transformation of recombinant strains

[0077] The pTH1469 plasmid carrying the mutant gene obtained in 1.1 was linearized using restriction endonuclease Dpn I and transformed into Bacillus subtilis WB600. Positive clones were screened and sequenced to obtain a recombinant strain carrying the epoxide hydrolase MdEH single-point mutant.

[0078] 2.3 Replacing the promoter to improve expression efficiency

[0079] To further improve the expression efficiency of the epoxide hydrolase mutant, the original promoter on the vector was replaced with glvA / srfA / ydzA / fusA / gsiB. The specific steps are as follows:

[0080] Using the B. subtilis WB600 genome as a template, primers were used to amplify the glvA / srfA / ydzA / fusA / gsiB promoter. Using the pTH1469 plasmid carrying the mutant gene obtained in 2.1 as a template, a linearized fragment with the promoter removed was amplified. The PCR products were purified and recovered, and then recombined using a seamless cloning experiment. The PCR products were purified and recovered, then transformed into the cloning strain Escherichia coli TOP 10. Transformants were sequenced, and positive transformants were screened for recombinant plasmids.

[0081] 2.4 Expression verification of recombinant plasmid with replaced promoter

[0082] The recombinant plasmid with the replaced promoter was transferred into Bacillus subtilis WB600 for expression verification and enzyme activity detection according to steps 1.2 to 1.4 of Reference Example 1. The shake flask fermentation method of Bacillus subtilis was similar to that of step 1.3 of Reference Example 1, except that the corresponding inducer was added or the temperature was directly lowered to 30°C for induction, and the induction culture was performed for 48 hours to harvest the bacteria.

[0083] The experimental results are as follows Figure 3 As shown, in which the promoter P glvA Under these conditions, the highest enzyme activity of epoxide hydrolase MdEH-K71I was 9.25 U / mL, which was 1.41 times that of the control group; followed by promoter P fusA 、P ydzA Under these conditions, the enzyme activities were 8.64 U / mL and 8.34 U / mL, respectively, which were 1.32 times and 1.28 times that of the control group; while P srfA 、P gsiB Compared with the control group P grac The enzyme activities under the same conditions were similar, all around 6.5 U / mL. fusA The highest value under the conditions was 0.15 mg / mL, which was 1.55 times the protein content of the control group; glvAUnder the same conditions, the protein content was the lowest, at 0.07 mg / mL; the other groups were similar to the control group, all around 0.1 mg / mL. Figure 3 A), in the promoter P glvA Under the same conditions, the wet weight of the bacteria and OD 600 The highest values ​​were 45 g / L and 6.89 respectively, and the rest were not much different from the control group. The possible reason is that P glvA The group added maltose during induction, which promoted the growth of the bacteria as an exogenous carbon source, resulting in a higher accumulation of biomass than other groups. glvA It was the best promoter for subsequent experiments.

[0084] The promoters are all derived from the Bacillus subtilis genome, wherein: the position of the nucleotide sequence of the promoter glvA on the chromosome is NC_000964.3: 889757-890021 bp; the position of the nucleotide sequence of the promoter srfA on the chromosome is NC_000964.3: 376362-376967 bp; the position of the nucleotide sequence of the promoter ydzA on the chromosome is NC_000964.3: 475875-476239 bp; the position of the nucleotide sequence of the promoter fusA on the chromosome is NC_000964.3: 130332-130686 bp; and the position of the nucleotide sequence of the promoter gsiB on the chromosome is NC_000964.3: 494200-494505 bp.

[0085] 2.5 Replacing the signal peptide to improve expression efficiency

[0086] Using the B. subtilis WB600 genome as a template, primers were used to amplify the phoD / phrC / peI / amyE / ydbN signal peptide. Using the recombinant plasmid obtained in 2.1, in which the promoter was replaced with glvA, a linearized fragment without the signal peptide was amplified. The PCR products were purified and recovered, and then recombined using a seamless cloning experiment. The PCR products were purified and recovered, then transformed into the cloning strain Escherichia coli TOP 10. Transformants were sequenced, and positive transformants were screened for extraction of the recombinant plasmid.

[0087] The signal peptides are all derived from the Bacillus subtilis genome, wherein: the position of the nucleotide sequence of the signal peptide phoD on the chromosome is NC_000964.3: 284011~284163bp; the position of the nucleotide sequence of the signal peptide phrC on the chromosome is NC_000964.3: 429963~430028bp; the position of the nucleotide sequence of the signal peptide peI on the chromosome is NC_000964.3: 827993~828055bp; the position of the nucleotide sequence of the signal peptide amyE on the chromosome is NC_000964.3: 327618~327716bp; and the position of the nucleotide sequence of the signal peptide ydbN on the chromosome is NC_000964.3: 224858~224932bp.

[0088] 2.6 Verification of expression of recombinant plasmid with replaced signal peptide

[0089] Refer to the method in 2.4 to transform and express the recombinant plasmid with the replaced signal peptide. The recombinant strain was shake-flask fermented for 48 hours and then the relevant data were measured. The experimental results are as follows: Figure 4 As shown, the signal peptide SP phoD Under these conditions, the highest activity of epoxide hydrolase MdEH-K71I was 10.61 U / mL, which was higher than that of the control group SP amyQ The enzyme activity was 1.16 times higher than that of the signal peptide SP amyE Under these conditions, the enzyme activity was 10 U / mL, which was 1.1 times that of the control group; while SP phrC 、SP peI and SP ydbN The enzyme activity under these conditions was 0.97 times, 0.63 times and 0.66 times lower than that of the control group. Figure 4 A), in the signal peptide SP phoD Under the same conditions, the wet weight of the bacteria and OD 600 were the highest, 42.3 g / L and 4.39 respectively; while in SP amyE Under the same conditions, the wet weight of the bacteria and OD 600 The lowest are 34.8g / L and 3.84 respectively. In summary, choose SP phoD It is the optimal signal peptide for subsequent experiments.

[0090] 2.5 Results and Discussion

[0091] P glvA It is an inducible promoter that uses chemical inducers to achieve on-off control of gene expression, avoiding the host metabolic burden caused by the continuous activity of constitutive promoters, thereby achieving better expression.

[0092] SP phoDThe Tat pathway is used to secrete proteins. In Bacillus subtilis, the advantage of the Tat secretion pathway over the Sec pathway is that its unique twin-arginine transport mechanism can mediate the transmembrane transport of partially folded substrate proteins, significantly improving the secretion efficiency of complex enzymes by maintaining the correct conformation of the protein.

[0093] Based on the above experimental results, select the P with the best comprehensive effect glvA -SP phoD -MdEH-K71I expression cassette (the complete sequence of the expression cassette is shown in SEQ ID NO. 11) was used for subsequent expression and experiments.

[0094] Example 3 Preparation of co-immobilized carrier

[0095] The haloalcohol dehalogenase HheA and the epoxide hydrolase MdEH-K71I, both of which have similar optimal reaction conditions, were co-immobilized. This co-immobilization allows for the cascade catalytic degradation of 3-chloropropanol to glycerol. Co-immobilization also facilitates the subsequent separation and purification of the enzyme and substrate. (The haloalcohol dehalogenase HheA has been previously published; its preparation method can be found in De, Jong, René, et al. The X-Ray Structure of the Haloalcohol Dehalogenase HheA from Arthrobacter sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family. [J]. Journal of Bacteriology, 2006. DOI: 10.1128 / JB.01866-05.)

[0096] Both were cultured in 7L fermenters for 54 hours, after which the fermentation broths were removed and immediately centrifuged at 7000 rpm for 20 minutes at 4°C to obtain the fermentation supernatants, which were then stored at 4°C until further use. The protein concentrations of the HheA and MdEH-K71I enzyme solutions were measured. During preparation, the mixed enzyme solutions were mixed at a protein-to-protein ratio of 5:1. The crude enzyme activities before dilution were 793.4 U / mL for MdEH-K71I and 22.3 U / mL for HheA.

[0097] 3.1 Optimization of co-immobilization carrier matrix

[0098] Prepare a mixed enzyme solution with a total protein content of 60 mg and record the volume. Weigh 2 g of each of ECR8404M, ECR8285, ECR1030M, ECR1090M, and PCG900C into a 250 mL Erlenmeyer flask. Add 0.1 M phosphate buffer (pH 6.0) at a 1:1 volume ratio. Mix the resin thoroughly and let it stand at room temperature for pre-equilibration. Then, add the enzyme solution at a loading of 30 mg enzyme protein / g wet resin. Transfer the resin to a 30°C constant-temperature shaker and allow dynamic adsorption to proceed at 180 rpm for 8 h. After removal and settling, collect 1 mL of the supernatant to determine the protein concentration. Filter the mixture, rinse the filter residue with buffer, and dry the immobilized enzyme in a drying oven for 8 h.

[0099] The steps for testing the efficiency of co-immobilized enzyme are as follows:

[0100] Add 3-chloropropanol (3-MCPD) with a final concentration of 10 mM and a certain amount of co-immobilized enzyme into a 2 mL EP tube, make up the reaction system to 1 mL with phosphate buffer solution (0.1 M pH = 8), place it in a metal bath at 40°C and 800 rpm for 30 min, add 1 mL of ethyl acetate solution, shake and extract for 5 min, dry with sodium sulfate, and centrifuge at 11000 rpm for 3 min. Aspirate the supernatant and filter it with a 0.22 μm filter membrane, then transfer it to a 2 mL gas chromatography bottle for gas chromatography analysis.

[0101] The gas chromatography system used was an Agilent 8890B (Agilent Technologies, Palo Alto, CA, USA), and the column used was an HP-5 (30 m × 0.25 mm, 0.25 μm). The specific analytical method and procedure were as follows: initial temperature was 100°C, held for 1 minute, then heated to 240°C at a rate of 20°C / min and held for 1 minute, for a total run time of 9 minutes. A standard curve of concentration versus peak area was constructed based on the gas chromatographic retention time (RT) of glycerol obtained from the standard test results. The peak area of ​​the sample to be measured was then substituted into the standard curve for quantitative analysis.

[0102] The experimental results are as follows Figure 5 As shown in the figure, the five resins exhibited significant differences in their adsorption capacity for the MdEH-K71I and HheA enzyme mixture and their enzymatic activity after co-immobilization. PCG900C and ECR8285 exhibited the highest protein adsorption capacities, reaching 16.01 mg / g and 15.96 mg / g, respectively. ECR8285 and PCG900C also exhibited high specific enzyme activities, at 5.17 U / mg and 4.87 U / mg, respectively. Therefore, considering all factors, ECR8285 was selected as the optimal support for co-immobilizing the MdEH-K71I and HheA enzymes.

[0103] 3.2 Fixed temperature

[0104] The optimal carrier resin ECR8285 was selected for immobilization according to the steps in 3.1, except that the temperature of the dynamic adsorption process was adjusted to 30, 35, 40, 45 and 50 °C.

[0105] The experimental results are as follows Figure 6 As shown in the figure, it can be seen that the protein adsorption of the co-immobilized enzymes increases with increasing immobilization temperature. At 30°C, the lowest protein adsorption was 14.79 mg / g and the highest specific enzyme activity was 5.29 U / mg. Therefore, 30°C was selected as the optimal immobilization temperature for the co-immobilized enzymes MdEH-K71I and HheA to reduce the impact of temperature on the decline in enzyme activity.

[0106] 3.3 Immobilization buffer pH

[0107] The optimal carrier resin ECR8285 was selected for immobilization according to the steps in 3.2, except that the pH of the buffer solution during the dynamic adsorption process was adjusted to pH 6.0, 6.5, 7.0, 7.5, and 8.0.

[0108] The experimental results are as follows Figure 7 As shown in the figure, the protein adsorption and specific enzyme activity of the co-immobilized enzymes increased with increasing pH in the immobilization buffer. The highest protein adsorption and specific enzyme activity were achieved at pH 8.0, reaching 14.54 mg / g and 7.48 U / mg, respectively. Therefore, based on comprehensive considerations, 8.0 was selected as the optimal immobilization buffer pH for the co-immobilized enzymes MdEH-K71I and HheA.

[0109] 3.4 Reaction temperature

[0110] Refer to the steps in 3.3 to select the most suitable carrier resin ECR8285 for immobilization, except that the temperature of the reaction process in the cascade catalytic efficiency detection step is adjusted to 30, 35, 40, 45 and 50°C.

[0111] The experimental results are as follows Figure 8 As shown in the figure, it can be seen that as the temperature continues to rise, the conversion of the co-immobilized enzyme first increases and then decreases. The highest conversion rate reaches 7.59% at 35°C.

[0112] 3.5 Amount of enzyme added to the reaction

[0113] The optimal carrier resin ECR8285 was selected for immobilization according to the steps in 3.3, except that the amount of immobilized enzyme added during the reaction process of the cascade catalytic efficiency test step was adjusted to 0.1, 0.2, 0.3, 0.4, and 0.5 g.

[0114] The experimental results are as follows Figure 9 As shown, the conversion rate gradually increases with increasing enzyme dosage. When 0.5g of immobilized enzyme was added, the conversion rate reached a maximum of 16.23%. Overall, when 0.1-0.3g of immobilized enzyme was added, the conversion rate increased nearly linearly, but with subsequent additions, the rate of increase gradually slowed. Based on a comprehensive consideration of application and economic cost, an enzyme dosage of 0.3g was selected.

[0115] 3.6 Reaction Time

[0116] Refer to the steps in 3.3 to select the most suitable carrier resin ECR8285 for immobilization, except that the reaction time of the cascade catalytic efficiency detection step is adjusted to 10 min, 30 min, 1 h, 3 h, 6 h and 12 h.

[0117] The experimental results are as follows Figure 10 As shown in the figure, it can be seen that the conversion rate of catalytic hydrolysis of 3-MCPD gradually increases with the extension of time. After 12 hours of reaction, the conversion rate reaches a maximum of 80.29%.

[0118] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An epoxide hydrolase mutant, characterized in that: The protein is obtained by mutation based on the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation in the sequence is at least one of H11E, H12F, K71I, and N94R.

2. The epoxide hydrolase mutant according to claim 1, characterized in that: The gene encoding the epoxide hydrolase mutant is a nucleotide sequence obtained by mutation using mutation primers based on the nucleotide sequence shown in SEQ ID NO.

2.

3. A method for constructing an engineered bacterium expressing an epoxide hydrolase mutant, characterized in that The steps include: After the recombinant vector carrying the epoxide hydrolase mutant expression box is transferred into the engineering bacteria, the engineering bacteria expressing the epoxide hydrolase mutant are obtained.

4. The method for constructing an engineered bacterium expressing an epoxide hydrolase mutant according to claim 3, wherein: The epoxide hydrolase mutant expression box comprises a promoter, a signal peptide, a coding gene of the epoxide hydrolase mutant and a terminator.

5. The method for constructing an engineered bacterium expressing an epoxide hydrolase mutant according to claim 4, wherein: The promoter includes at least one of glvA, srfA, ydzA, fusA, and gsiB; The signal peptide includes at least one of phoD, phrC, peI, amyE, and ydbN; The engineered bacteria is Bacillus subtilis WB600.

6. A co-immobilized enzyme for cascade catalytic degradation of 3-chloropropanol, characterized in that The preparation method comprises the following steps: A buffer solution is added to the resin for pre-equilibration treatment, and then an enzyme solution obtained by mixing the epoxide hydrolase mutant and the halohydrin dehalogenase is added, followed by adsorption, washing, and drying to obtain an immobilized enzyme obtained by co-immobilizing the epoxide hydrolase mutant and the halohydrin dehalogenase.

7. The co-immobilized enzyme for cascade catalytic degradation of 3-chloropropanol according to claim 6, characterized in that: The resin includes at least one of ECR8404M, ECR8285, ECR1030M, ECR1090M, and PCG900C.

8. The co-immobilized enzyme for cascade catalytic degradation of 3-chloropropanol according to claim 6, characterized in that: The buffer solution is a phosphate buffer solution with a pH value of 6.0 to 8.0; The epoxide hydrolase mutant and the halohydrin dehalogenase are mixed to obtain an enzyme solution in which the protein concentration ratio of the epoxide hydrolase mutant to the halohydrin dehalogenase is 1:5; The halohydrin dehalogenase is a halohydrin dehalogenase HheA; The adsorption conditions are 30-50° C., 150-200 rpm, and 6-10 hours.

9. Use of the epoxide hydrolase mutant according to any one of claims 1 to 2 in catalyzing the hydrolysis of epoxides.

10. Use of the co-immobilized enzyme for cascade catalytic degradation of 3-chloropropanol according to any one of claims 6 to 8 in catalytic degradation of 3-chloropropanol.

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