Beta-glycosidase Bgl1269 mutant and application thereof in catalytic hydrolysis of soybean isoflavone glycoside

Abstract

The invention relates to a beta-glycosidase Bgl1269 mutant and application of the beta-glycosidase Bgl1269 mutant in catalytic hydrolysis of soybean isoflavone glycoside. The beta-glycosidase Bgl1269 mutant is obtained by mutating threonine at the 47th site of an amino acid sequence as shown in SEQ ID No.1 into alanine. Based on a semi-rational design of a sequence and a structure and a site-directed mutagenesis method, excellent variants are screened through experiments to improve the thermal stability of the beta-glucoside hydrolase Bgl1269, the optimal variant enzyme is T47A, the optimal temperature Tm is 45 DEG C, the semi-inactivation temperature T50 is 51.36 DEG C, and the optimal temperature Tm and the semi-inactivation temperature T50 are respectively improved by 5 DEG C and 4.15 DEG C compared with those of a wild enzyme. The half-life period T1 / 2 at 45 DEG C is 1.64 + / -0.03 h, which is 4.6 times that of the wild enzyme; data show that the T47A has higher capability of combining daidzin and genistin and higher catalytic capability than wild enzyme, and the catalytic efficiency kcat / Km is increased by 44.38% and 39.01% respectively.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to a beta-glucosidase Bgl1269 mutant and application thereof in catalyzing the hydrolysis of soybean isoflavone glycosides. Background Art

[0002] Soy isoflavone glycosides are compounds formed by the binding of soy isoflavones with glucose. They belong to the plant flavonoid class and are the primary form of soy isoflavones, accounting for 97% to 98% of the total isoflavone content in soy. While soy isoflavone glycosides are poorly absorbed by the human body, the soy isoflavone aglycones produced by hydrolysis are highly absorbed and exert physiological effects such as antioxidant, immunomodulatory, and osteoporosis prevention. Aglycones bind to estrogen receptors and can exert either estrogen-like or anti-estrogen effects. When estrogen levels are low, they exhibit estrogen-like effects, while when estrogen levels are high, they exhibit anti-estrogen effects. Glycoside isoflavones require hydrolysis to convert into their corresponding aglycones. Soy isoflavones belong to the class of oxyglycosides, and hydrolysis methods primarily include acid hydrolysis and enzymatic hydrolysis.

[0003] The main drawbacks of the acid hydrolysis method are the high reaction temperature, demanding equipment, and high investment. Yu Liying et al. studied a composite organic acid catalytic process using a 4:3 ratio of malic acid to citric acid, maintaining a constant temperature of 135°C for 160 minutes, achieving a hydrolysis rate exceeding 97.0%. Enzymatic hydrolysis is currently the most widely studied method internationally, with advantages such as mild hydrolysis conditions, stable products, and high purity. Enzymatic hydrolysis of soy isoflavone glycosides is the most promising process.

[0004] β-Glucosidase is an important enzyme that hydrolyzes soy isoflavone glycosides to produce soy isoflavone aglycones. Bgl1269, a β-glucosidase derived from an unculturable microorganism, exhibits excellent hydrolytic activity and has great potential for application in the hydrolysis of soy isoflavone glycosides. During the hydrolysis process, glucose accumulation readily inhibits β-glucosidase activity. Its glucose tolerance ensures that Bgl1269 maintains high hydrolytic activity even when glucose accumulates in the product. Bgl1269 has the highest inhibition constant (Ki) (4.28 M) of any β-glucosidase reported to date, demonstrating greater resistance to glucose inhibition than any known β-glucosidase. Bgl1269 exhibits high thermal stability at 40°C, retaining over 60% of its activity after incubation at 40°C for one hour. When incubated at 45°C, Bgl1269 exhibits a half-life of less than half an hour. Bgl1269 has excellent catalytic activity as an enzyme for hydrolyzing soybean isoflavone glycosides, but its current thermal stability is not sufficient to meet the needs of industrial production. Summary of the Invention

[0005] The present invention addresses the poor thermal stability of Bgl1269 by providing a thermostable β-glucosidase Bgl1269 mutant and its application in catalyzing the hydrolysis of soy isoflavone glycosides. The present invention utilizes a semi-rational design approach to modify Bgl1269 to obtain thermostable variants T47A and T47A&T374S for the hydrolysis of soy isoflavone glycosides.

[0006] The technical solution adopted by the present invention to solve at least one of the above technical problems is:

[0007] A beta-glycosidase Bgl1269 mutant is provided, wherein the threonine at position 47 of the amino acid sequence shown in SEQ ID No. 1 is mutated to alanine.

[0008] Preferably, the β-glycosidase Bgl1269 mutant is a mutant in which the threonine at position 47 of the amino acid sequence shown in SEQ ID No. 1 is mutated to alanine and the threonine at position 374 is mutated to serine.

[0009] A nucleic acid encoding the above-mentioned β-glycosidase Bgl1269 mutant.

[0010] A recombinant expression vector comprising the above nucleic acid sequence.

[0011] A recombinant expression transformant comprising the above recombinant expression vector.

[0012] A recombinant β-glycosidase Bgl1269 mutant catalyst, wherein the recombinant β-glycosidase Bgl1269 mutant catalyst is any one of the following forms:

[0013] (1) culturing the recombinant expression transformant according to claim 5, and isolating transformant cells containing the β-glucosidase Bgl1269 mutant;

[0014] (2) culturing the recombinant expression transformant according to claim 5 and isolating a crude enzyme solution containing the β-glucosidase Bgl1269 mutant;

[0015] (3) Cultivating the recombinant expression transformant according to claim 5, isolating the crude enzyme solution containing the β-glucosidase Bgl1269 mutant, and drying the prepared crude enzyme powder or purifying the prepared pure enzyme.

[0016] The invention relates to an application of the beta-glucosidase mutant in catalyzing the hydrolysis of soybean isoflavone glycosides.

[0017] Preferably, the soy isoflavone glycoside is daidzein.

[0018] Preferably, the soybean isoflavone glycoside is genistin.

[0019] Compared with the existing technology, the advantages of the present invention are: based on semi-rational design of sequence and structure and using site-directed mutagenesis, the present invention screened for excellent variants to improve the thermal stability of β-glucosidase hydrolase Bgl1269 through experiments. Among them, the best variant enzyme is T47A, whose optimal temperature Tm is 45°C and half-inactivation temperature T50 is 51.36°C, which are 5°C and 4.15°C higher than those of the wild-type enzyme, respectively. The half-life T1 / 2 at 45°C is 1.64±0.03h, which is 4.6 times that of the wild-type enzyme.

[0020] In terms of kinetic parameters, when the substrate was daidzein, the variant T47A had a Km of 0.175±0.01mM and a kcat of 2.563±0.16s-1. When the substrate was genistin, the Km and kcat of the variant T47A were 0.0248±0.01mM and 1.977±0.12s-1, respectively. In contrast, the Km and kcat of the wild-type enzyme were 0.178±0.01mM and 0.033±0.01mM, 1.804±0.04s-1, and 1.376±0.05s-1, respectively. The data showed that T47A had a higher ability to bind to daidzein and genistin and a higher catalytic capacity than the wild-type enzyme, with the catalytic efficiency kcat / Km increased by 44.38% and 39.01%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the gel electrophoresis diagram of the target gene DNA after single-point mutation in Example 3 of the present invention;

[0022] Figure 2 This is the SDS-PAGE electrophoresis diagram of the recombinant protein in the supernatant of the cell lysate in Example 4 of the present invention;

[0023] Figure 3 This is the SDS-PAGE electrophoresis diagram of the recombinant protein after nickel column purification in Example 4 of the present invention;

[0024] Figure 4 The results of the relative enzyme activities of the wild type and mutants at various temperatures in Example 5 of the present invention are shown in FIG.

[0025] Figure 5 This is a graph showing the specific activity of the wild type and mutants in Example 5 of the present invention;

[0026] Figure 6 Graphs showing thermal inactivation curves of the wild type and variants at 40° C. (A) and 45° C. (B) in Example 5 of the present invention;

[0027] Figure 7This is a graph showing the effect of temperature on the stability of the wild type, variants T47A, and T374S (50 mM sodium citrate buffer, pH 6.0, incubation time 15 minutes) in Example 5 of the present invention;

[0028] Figure 8 Gel electrophoresis images of the native plasmid (A) and restriction enzyme-digested plasmid (B) of the combined mutations T47A & T374S in Example 6 of the present invention;

[0029] Figure 9 This is an SDS-PAGE electrophoresis diagram of the supernatant of the cell lysate and the purified T47A & T374S in Example 6 of the present invention; wherein M is a standard protein molecular weight marker (TaKaRa); lane 1 is the unpurified recombinant protein from the cell lysate supernatant; lane 2 is the recombinant protein purified by affinity chromatography;

[0030] Figure 10 Figure 7 (A) shows the effect of temperature on the enzyme activity of variants T47A & T374S and a Michaelis-Menten curve (B) in Example 7 of the present invention;

[0031] Figure 11 Graph showing the effect of pH on enzyme activity of the wild type (A) and mutants T47A (B), T374S (C), and T47A & T374S (D) in Example 8 of the present invention;

[0032] Figure 12 This is a graph showing the effect of pH on enzyme stability in Example 8 of the present invention;

[0033] Figure 13 is a Michaelis-Menten curve diagram when daidzein is used as a substrate in Example 8 of the present invention;

[0034] Figure 14 is a Michaelis-Menten curve diagram when genistin is used as a substrate in Example 8 of the present invention;

[0035] Figure 15 This is a Dixon plot of the inhibitory effect of glucose on the hydrolysis of pNPG by the purified wild-type enzyme (A), T47A (B), T374S (C), and T47A & T374S (D) in Example 8 of the present invention; wherein the pNPG concentrations used are 10 mM and 15 mM;

[0036] Figure 16 This is a molecular dynamics simulation analysis diagram of the wild type and mutant in Example 8 of the present invention. DETAILED DESCRIPTION

[0037] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0038] Example 1 Semi-rational design to find mutation sites

[0039] (1) Amino acid sequence analysis: The amino acid sequence of β-glucosidase Bgl1269 was downloaded from the NCBI database (GenBank: JQ957567.1), see SEQ ID No. 1. The sequence of SEQ ID No. 1 is as follows: MEWQFP PNFHWGSTTAAYQVEGNNVNSDFWAEEHAEGSPYKDKSGDTIDHYRLYREDIALMASLGLNTYRFSIEWSRVEPEPGQYSRSAIEHYRDVLETCYKHGITPMVAMHHFSSPKWLMRLGGWASPEVPDRFAKYCEVVFREIGHLIPHVLTMNEINLPVMLREIFSRIGFIPPVGIDRDAWTAPKWRESAARLCGTTTQNYFTFHMISDE FSIHNLKETHRKAREAIKRIQPNTKVGFSMALSDIQSIPGGEEEADRKWNSYFRQYLDMLEGDDFFGLQNYTREVYGPEGQVKPAEGAELTQMGYEFYPKALSN VIRKVAKDISIPIIIITEHGIATDEDERRVAFIREALEGVQDCLSEGLDVRGYLHWTTFDNFEWQSGYALKFGLIEVDRSTQERKVKESGRYLGRLAQQESLASK.

[0040] The web-based automated program Consensus Finder (http: / / kazlab.umn.edu) was used to search for conserved amino acids. Consensus Finder performed a multiple sequence alignment of 1,000 reported β-glucosidases of the same family and screened out nine conserved amino acid sites using an 80% threshold. These nine conserved amino acid sites were predicted as potential mutation sites. The results are shown in Table 1.

[0041] Table 1 Consensus search results with 80% as the threshold

[0042]

[0043] (2) AlphaFold modeling: We used Google Colab to run AphaFold2 to perform protein modeling on the β-glucosidase Bgl1269. We identified the substrate binding pocket of Bgl1269 and found that the predicted potential mutation sites did not overlap with the active site, so we could proceed to the next step of screening. Among them, T374S is very close to the active pocket and may affect the enzyme activity. This needs further experimental verification.

[0044] (3) Calculate the single-point mutation free energy of the predicted potential mutation site: Use the repair function of FoldX to reduce the energy content of the wild type to a minimum by rearranging the side chains to minimize the protein structure model, and then use the BuildMode l function to introduce mutations and optimize the structure of the new protein variant. The energy function of FoldX can thus calculate the energy difference between the wild type and the protein variant. The stability change evaluation criteria are ΔΔG < -1.0, which means that the point mutation has a stabilizing effect on the protein; ΔΔG > 1.0, which means that the point mutation has an unstable effect on the protein. The results are shown in Table 2 below. The ΔΔG of the five mutants Y18H, D42E, T47A, I146L and T374S is less than -0, among which the ΔΔG values of T47A and T374S are less than -1 kcal·mol -1 , indicating that variants T47A and T374S bring a stabilizing effect to the protein.

[0045] Table 2 FoldX calculation results

[0046]

[0047] Example 2 Construction and expression of wild-type β-glucosidase Bgl1269 recombinant engineering bacteria

[0048]

[0049] Transform the commercially synthesized pET32a(+)-Bgl1269 plasmid into competent E. coli DH5α for amplification and storage. Select positive clones by plating, culture in vials, and prepare glycerol stocks for storage at -80°C. Recombinant plasmids were extracted from E. coli DH5α using a kit and transformed into competent Escherichia coli BL21(DE3) for expression of the target protein.

[0050] Example 3 Construction of mutant recombinant plasmids and engineered bacteria

[0051] (1) Primer design: Use Vayme online primer design ( https: / / crm.vazyme.com / cetool / singlepoint t.html), A primer was designed at the 5' and 3' ends of the mutation site, respectively, so that the primer sequences included the mutation site and the primers partially overlapped. The design results are shown in Table 3.

[0052] Table 3 Primer sequences required for single point mutation

[0053]

[0054] (2) Construction of mutation vector: Using a pair of primers containing the mutation site, after annealing to the template plasmid pet32a(+)-Bgl1269 (length 7120 bp), DNA polymerase amplification was performed. The obtained amplified fragment was phosphorylated at the 5' end and circularized by DNA ligase, while the original plasmid was digested by Dpn I to obtain the mutant plasmid. According to Table 4 below, the following reagents were mixed in sequence, and after mixing, centrifuged for 5 seconds for PCR amplification. The PCR amplification program was set as follows: 94℃ pre-denaturation for 2 minutes, 98℃ denaturation for 10 seconds, 68℃ extension for 4 minutes, and 35 cycles. After the last cycle, the reaction was kept at 72℃ for 5 minutes to fully amplify the reaction product.

[0055] Table 4 PCR reaction solution components and their respective amounts

[0056]

[0057] The PCR products were analyzed by 0.7% agarose DNA gel electrophoresis. Figure 1 The DNA length of the mutated product was as expected, and no other bands were observed. The original plasmid in the PCR product was then digested with Dpn I to obtain the mutant plasmid to be transformed.

[0058] (3) Construction of expression strain: Take out the stored DH5α competent cells from the -80℃ freezer and place the cells in an ice water bath to thaw for 5-10 minutes. Add about 10μL of the mutant plasmid to be transformed for every 100μL competent cells, gently tap the bottom of the tube about 3 times and immediately incubate on ice for 30 minutes. After heat shock treatment at 42℃ for 90s, add 900μL of LB liquid medium preheated at 37℃, invert several times to mix, and resuscitate on a 37℃ shaker at 220rpm for 45 minutes. After shaking, centrifuge at 5000×g for 1min at room temperature to precipitate the bacteria, remove 900μL of supernatant, resuspend the remaining 100μL of bacterial liquid, and spread it on an LB plate containing ammonia. Then, incubate inverted in a biochemical incubator for 12 hours. After overnight incubation, two single clones were selected for each mutant for colony PCR verification. Single clones showing the target band were placed in 10 mL of LB liquid medium and incubated at 37°C, 200 rpm for 12 hours. One mL of the culture was sent to Nanjing Qingke Biotechnology for sequencing to verify the sequence accuracy.

[0059] Use a plasmid extraction kit to extract the recombinant plasmid from 10 mL of overnight DH5α bacterial culture for protein expression. Remove the cryopreserved BL21 competent cells and thaw them on ice. Use a pipette to aspirate 10 μL of the recombinant product and add it to 100 μL of the thawed competent cells. Gently tap the tube. Let it stand on ice for 10 minutes before directly plating overnight to obtain engineered bacteria containing the mutant recombinant plasmid.

[0060] Example 4 Expression of mutant engineering bacteria

[0061] The recombinant E. coli BL21 strain containing the mutant recombinant plasmid was inoculated into a 250 mL flask containing 100 mL of LB (100 μg / mL ampicillin) and grown until the cell concentration (OD600) reached 1.3. Next, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.2 mM, and the culture was incubated at 25°C for 16 hours with shaking at 200 rpm. The cells were then harvested by centrifugation at 6000 × g for 5 minutes at 4°C. The cells were gently washed one to two times with Lysis Buffer to remove residual medium. The cells were then sonicated with Lysis Buffer at a ratio of 1:10 (w / v) and the supernatant was collected by centrifugation at 1000 rpm for 25 minutes at 4°C. The cell lysate was essentially transparent, indicating that the recombinant enzyme was highly soluble.

[0062] The recombinant enzyme was purified by Ni-NTA chromatography, and then the supernatant of cell debris and cell lysate and the purified protein were verified by SDS-PAGE to determine the molecular weight of the recombinant protein. The results are shown in Figure 2 and Figure 3 The calculated molecular weight of Bgl1269 is approximately 48.7 kDa. The molecular weight of the thioredoxin tag (TrxA) is 11.8 kDa, and the molecular weight of the S-tag epitope (S-Tag) is 1.7 kDa. Therefore, the total molecular weight of the recombinant protein should be approximately 62.2 kDa. A distinct protein band of approximately 60 kDa was observed, which is consistent with the predicted molecular weight.

[0063] Example 5 Screening of excellent single-point mutants using pNPG as a substrate

[0064] One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of pNP per minute. The enzymatic reaction system, with a reaction volume of 150 μL, consisted of 25 μL of 0.1 M pNPG solution, 50 μL of citric acid-sodium hydrogen phosphate (pH 6.5) buffer, and 25 μL of appropriately diluted pure enzyme solution. The reaction was carried out at 45°C for 10 minutes, then terminated by adding 50 μL of 1 M Na2CO3 solution. The sample absorbance was measured on a microplate reader.

[0065] (1) Optimal reaction temperature of pNPG enzyme

[0066] The enzyme activity was measured using 10mM pNPG as a substrate, and the unit enzyme was defined as the amount of enzyme required to produce 1μmol of pNP per minute. The reaction consisted of 25μL pNPG and 25μL purified enzyme reacting at 40°C for 10 minutes under 0.05M Mcllvaine Bffer, where the concentration of each pure enzyme was approximately 0.07mg / mL (final concentration was 0.0175mg / mL). The enzyme-catalyzed reaction was measured under different temperature conditions ranging from 30°C to 55°C (every 5°C) to determine the optimal reaction temperature of the enzyme, that is, to determine the effect of reaction temperature on enzyme activity. The maximum enzyme activity measured in the above-mentioned temperature range was taken as 100%, and the ratio of enzyme activity to the maximum enzyme activity under other temperature reaction conditions was calculated as relative enzyme activity (%). The graph was plotted with the relative enzyme activity ratio (%) as the vertical axis and the reaction temperature (°C) as the horizontal axis. The results are shown in the figure. Figure 4 The optimal temperature for the wild type, Y18H, D42E, and I146L enzymes was 40°C, while that for T47A and T374S was 45°C. Since the activity at 40°C and 45°C was not much different, for ease of comparison, all reactions were performed at 40°C to compare enzyme activity.

[0067] The experimental results are as follows Figure 5As shown, mutant Y18H exhibited a significantly lower specific enzyme activity and was therefore not considered for subsequent experiments. Of the five mutants, only T47A exhibited higher enzyme activity than the wild type, at nearly double that of the wild type. D42E and I146L, however, had comparable specific enzyme activities to the WT. T374S exhibited a slightly lower specific enzyme activity than the WT, at approximately 70% of that.

[0068] (2) pNPG determination of enzyme half-life

[0069] Because Bgl1269 will be inactivated in less than 30 minutes at a reaction temperature of 45°C, the present invention measures the time T when the enzyme loses half of its activity at 45°C. (1 / 2,45℃) The time T for the enzyme to lose half of its activity at 40°C (1 / 2,40℃) . T (1 / 2,45℃) The determination process is as follows: incubate the purified enzyme solution at 45°C, and take out part of the enzyme solution for measuring the residual enzyme activity at intervals of 30 minutes each time. The specific experimental steps are: immediately place the enzyme solution in an ice bath for 2 minutes after taking it out, and then use the above method to measure the relative residual activity of the enzyme. Assuming that the activity of the enzyme stored at 4°C in the optimal reaction system is measured as the initial enzyme activity, which is set to 100%, calculate the residual ratio (%) of the activity of the enzyme incubated at 45°C compared to the initial enzyme activity, and plot the value (%) as the vertical axis and the incubation time (min) as the horizontal axis. T (1 / 2,40℃) The determination process is similar.

[0070] The relative enzyme activity at each temperature was calculated, and the temperature corresponding to the highest enzyme activity was considered the optimal temperature. To determine the thermal stability of the enzyme, the purified protein solution was placed in a water bath at different temperatures, and then the residual activity was measured under the optimal reaction conditions. The inactivation half-life of the enzyme (t 1 / 2 ) is calculated as follows:

[0071]

[0072] where k d is the enzyme inactivation constant.

[0073] The experimental results are as follows Figure 6 As shown, the half-life of the wild enzyme at 40°C is more than 1 hour, but the enzyme activity drops below 40% after 30 minutes at 45°C. The thermal stability needs to be improved. The thermal stability of D42E is not as good as that of the wild type, and I146L is on par with the wild type. The half-lives of T47A and T374S at 40°C and 45°C are both about 3 times that of WT.

[0074] (3) pNPG determination of enzyme half-inactivation temperature

[0075] The purified enzyme solution was incubated at 40°C, 42°C, 45°C, 45.8°C, 47.8°C, 50°C, 51°C, 53.1°C, 55°C and 60°C for 15 minutes, and then placed on ice for 2 minutes. The relative residual activity of the enzyme at different incubation temperatures was determined using the above method. The activity of the enzyme stored at 4°C in the optimal reaction system was taken as the initial enzyme activity, which was set as 100%. The relative specific activity under each temperature condition was calculated. The temperature corresponding to the relative residual enzyme activity dropping to 50% is the half-inactivation temperature T 50 The results are as follows Figure 7 As shown, the half-inactivation temperature T50 of mutant T47A was 51.36±0.13℃, which was 3.18℃ higher than that of the wild type.

[0076] The experimental results of Example 5 showed that only two mutant proteases, T47A and T374S, exhibited better thermal performance. Among them, the optimal mutant T47A had an optimal reaction temperature of 45°C, which was 5°C higher than the wild-type enzyme. At 45°C, its half-life was 1.64±0.03h, which was 4.6 times that of the wild-type enzyme. Its half-inactivation temperature T50 was 51.36±0.13°C, which was 3.18°C higher than that of the wild-type enzyme.

[0077] Example 6 Construction of combined mutation mutant recombinant plasmid and engineering bacteria and expression

[0078] Combinatorial mutagenesis was performed at the dominant mutation sites to attempt to obtain beneficial mutants. A DH5α glycerol stock carrying the pet-32a(+)-T47A recombinant plasmid was removed from a -80°C freezer and streaked to extract the plasmid. Using the pet-32a(+)-T47A recombinant plasmid as a template, the upstream primer sequence was 5'-GAACGTGcTCCAATGCAGGTAACCACGA ACAT-3', and the downstream primer sequence was 5'-TGCATTGGAgCACGTTCGATAACTTTGAATGGC-3'. PCR products were transformed into DH5α and BL21 competent cells for amplification and expression. Combinatorial mutants were analyzed by DNA and protein electrophoresis.

[0079] The recombinant plasmid obtained after the combined mutation was transformed and introduced into DH5α for amplification and preservation. After overnight culture of the successfully transformed E. coli DH5α vial, the combined mutated plasmid was extracted from the bacterial solution for verification, and 500 μL of the bacterial solution was taken for sequencing verification. DNA electrophoresis was performed on the combined mutated plasmid, and the results were as follows Figure 8 As shown, Figure 8 The helical DNA band in A is bright and has a high content, with very little broken and unwound DNA. The extracted plasmid is of good quality and Figure 8 The target band length in B is around 1280 bp, which is in line with the estimated range.

[0080] The combined mutated plasmid was further transformed into BL21 competent cells to express the protein. The protein electrophoresis results were as follows: Figure 9 The target protein bands were very bright in the protein supernatant electrophoresis, indicating that the T47A & T374S combined mutant enzyme was highly soluble, and the purified enzyme bands were distinct without other contaminating protein bands.

[0081] Example 7 Functional Verification of T47A / T374S Double-Point Combination Mutation

[0082] The thermal stability of mutant T47A / T374S was tested according to the method of Example 5. T47A and T374S were very active at 40°C to 45°C, with the highest enzyme activity at 45°C and a sharp drop in activity after 45°C. The thermal stability test at 45°C showed the following results: Figure 10 As shown, it was found that the half-life of T47A & T374S was T 1 / 2 The relative enzyme activity after 15 min incubation at each temperature was recorded, and the data were fitted using the least squares method (Sigmoidal, 4PL, X is concentration) using GraphPad Prism. 50 The best fitting value is 51.67℃, R 2 The specific activity of the enzyme was 0.9973, a 3.57°C increase relative to the wild-type. Using pNPG as a substrate, the specific activity was lower than that of the wild-type but higher than that of T374S, reaching 80.46% of that of the wild-type. The results indicate that the mutation at position 47 improves the activity and thermal stability of T374S, but does not outperform the variant T47A.

[0083] The mutants were modeled in three dimensions and the free energy required for the mutation was calculated using FoldX. The simulation results are shown in Table 5, which are generally consistent with the experiment. This further confirms that when ΔΔG < -1.0, the point mutation has a stabilizing effect on the protein.

[0084] Table 5 dG change results of point mutations

[0085]

[0086] Example 8 Investigation of Enzymatic Properties of Mutants with Excellent Thermal Performance

[0087] (1) Effect of pH on enzyme activity: Enzyme activity at different pH values was measured at 45°C. McIlvaine (pH 3.0–8.0) and glycine-NaOH (pH 8.0–10.0) buffers were used. The enzyme activity at each pH was calculated relative to the highest activity (100%), and the pH value corresponding to the highest enzyme activity was the optimal pH value. The results are shown in Figure 2. Figure 11As shown, the activities of the wild-type and D47A, T374S, and T47A&T374S mutant enzymes varied with pH within the pH range of 4.5 to 10. The optimal pH range for both the wild-type and mutants was 6.5-7.0. Their activities remained above approximately 80% between pH 6.5 and 8.0. The activity of the D156E mutant, however, decreased significantly at pH levels below 6.5 and above 8.0. At pH levels below 5.5, the enzyme activities of both the wild-type and mutant enzymes were essentially lost.

[0088] To test the stability of the enzyme relative to pH, the purified enzyme was incubated in buffers of different pH values at 4°C for 12 hours, and the residual activity of the enzyme was determined under the optimal reaction conditions. Similarly, the enzyme activity was also measured at different temperatures. The highest enzyme activity was defined as 100%. The results of the effect of pH on enzyme stability are shown in Figure 2. Figure 12 As shown, the experimental results show that the wild-type and mutant enzymes almost completely lose their activity after being stored at 4°C for 12 hours at pH 4.5. However, within the pH range of 5-10, the wild-type and mutant enzymes still retain over 80% of their activity after being stored at 4°C for 12 hours, and the enzyme activity is very stable when stored between pH 6-9. Most β-glucosidases exhibit maximum activity in the acidic pH range and are unstable under alkaline conditions. T47A and T47A&T374S exhibit optimal activity at neutral pH but are stable under alkaline conditions. Alkaline tolerance is rare among β-glucosidases.

[0089] (2) Determination of Michaelis–Menten kinetic parameters: The Michaelis–Menten kinetic parameters of the purified enzyme activity were determined by substrate saturation assay using daidzein and genistin in the range of 0–2 μM as substrates. The maximum velocity (Vmax) and half-saturation coefficient (Km) were determined by plotting the relationship between substrate concentration and the initial velocity of each reaction and performing nonlinear regression analysis on the data. The initial reaction velocities of the wild-type Bgl1269 and mutants T47A, T374S, and T47A & T374S pure enzymes at different final concentrations of daidzein and genistin substrates were determined by curve fitting kinetic analysis. A 100 μL enzyme reaction system was established, including 25 μL of appropriately diluted enzyme solution and 75 μL of different concentrations of daidzein or genistin (pre-mixed with citric acid-sodium hydrogen phosphate buffer (pH = 6.5) and 200 mg / L daidzein solution in different proportions). After preheating at 45°C for 1 minute, add 25 μL of appropriately diluted enzyme solution and continue the reaction at 45°C. After 5 minutes, immediately heat the reaction mixture for inactivation. After extraction with 300 μL of 50% acetonitrile, centrifuge the reaction mixture to remove insoluble material. Filter 300 μL of the supernatant through a 0.22 μm organic syringe filter, and analyze the filtrate using liquid chromatography. Nonlinear fitting was performed using the Michaelis-Menten equation in Graphd Prism software, with the initial reaction velocity v as the horizontal axis and the substrate concentration [S] as the vertical axis.

[0090] When daidzein was used as substrate, the Michaelis-Menten curves of wild-type enzyme and mutant enzyme were shown in Figure 2. Figure 13 , wild enzyme K m was 0.178±0.001mM, and the K values of mutants T47A, T374S, and T47A&T374S were m The k of wild enzyme is 0.175±0.001mM, 0.175±0.001mM, and 0.174±0.001mM respectively. cat 1.80±0.04s -1 , mutants T47A, T374S and T47A&T374S k cat 2.56±0.16s respectively -1 and 1.67±0.03s -1 , 1.92±0.15s -1 The k values of mutants T47A and T47A&T374S were cat Compared with the wild enzyme, the k cat / K m 10.16s -1 ˙mM-1 , mutants T47A, T374S and T47A&T374S k cat / K m 14.67s respectively -1 ˙mM -1 and 9.59s -1 ˙mM -1 , 11:00s -1 ˙mM -1 Compared to the wild type, the overall catalytic efficiency of T47A and T47A&T374S increased by 44.38% and 8.26%, respectively. The catalytic efficiency of the variant T374S was 94.09% of that of the wild type, a decrease of 5.9%.

[0091] When genistein standard was used as substrate, the Michaelis-Menten curves of wild-type enzyme and mutant enzyme were shown as follows: Figure 14 , wild enzyme K m was 0.033±0.001mM, and the K values of mutants T47A, T374S, and T47A&T374S were 0.033±0.001mM. m The k of wild enzyme is 0.025±0.001mM, 0.034±0.001mM, and 0.031±0.001mM respectively. cat 1.38±0.05s -1 , mutants T47A, T374S and T47A&T374S k cat 1.98±0.12s respectively -1 and 1.34±0.04s -1 , 1.79±0.21s -1 The k values of mutants T47A and T47A&T374S were cat Compared with the wild enzyme, the k cat / K m 41.74s -1 ˙mM -1 , mutants T47A, T374S and T47A&T374S k cat / K m 58.06s respectively -1 ˙mM -1 、39.21s -1 ˙mM -1 and 58.0s -1 ˙mM -1 Compared with the wild type, the overall catalytic efficiency of T47A and T47A&T374S increased by 39.01% and 38.96%, respectively. The catalytic efficiency of the variant T374S was 93.94% of that of the wild type, a decrease of 6.06%.

[0092] (3) Effects of metal ions and chemical reagents on enzyme activity

[0093] 0.1 M EDTA, Mn 2+ Mg 2+ , Ca 2+ 、Cu 2+ 、Fe 3+ 、Fe 2+ 、Zn 2+ The solution was prepared by adding the above-mentioned ions to the reaction system with pNPG as the substrate, so that the final ion concentration in the reaction system was 1mM and 10mM, respectively. The enzyme activity of the wild-type enzyme and the mutant T47A and T47A&T374S enzymes was then measured in the optimal reaction system. The relative enzyme activity was calculated by taking the enzyme activity in the reaction system without the above-mentioned ions as 100%. The experimental results are shown in Table 6. The addition of EDTA affected the activity of the wild-type enzyme and the mutant enzyme, indicating that both the wild-type enzyme and the mutant enzyme are metal-dependent proteins. The addition of EDTA and Cu2+ inhibited the activity of these four enzymes, and the inhibitory effect became more and more obvious as the ion concentration increased. In addition, when added at a concentration of 1 or 10mM, SDS completely inhibited the enzymatic action. Most metal ions had a slight effect on the enzyme activity of T47A. Ca2+, Co2+, Mn2+, and Mg2+ all promoted the enzyme activity of WT, T374S, and T47A&T374S, while Zn2+ had a significant inhibitory effect on these three enzymes.

[0094] Table 6 Effects of metal ions and chemical reagents on enzyme activity

[0095]

[0096] (4) Effect of glucose on enzyme activity

[0097] 24 μL of glucose solutions of different concentrations (0.1, 0.3, 0.6, 1.0, 1.3, 1.6, 2.0, 2.3, 2.6, 3.0, 3.3 and 3.6 M) were pre-incubated with 1 μL of enzyme solution at room temperature for 1 hour. Then, each pre-incubation mixture was mixed with 25 μL of pNPG (10 mM) and 50 μL of sodium citrate buffer (50 mM, pH 6.5) containing the same concentration of glucose. The residual enzyme activity of the reaction mixture was determined under standard conditions. Relative activity was defined as the relative value of the control activity without glucose.

[0098] The glucose inhibition constant K was determined by fitting the Dixon plot using enzyme solution and various concentrations of glucose solution (0.1, 0.3, 0.6, 1.0, 1.3, 1.6, 2.0, 2.3, 2.6, 3.0, and 3.3 M) with two concentrations of pNPG (10 and 15 mM) as substrate. i . A mixed solution (25 μL) containing 1 μL of enzyme solution and 24 μL of glucose solution of different concentrations was first incubated at 25°C for 1 h. Each pre-incubated mixed solution was then mixed with 25 μL of pNPG (10 or 15 mM) and 50 μL of sodium citrate buffer (50 mM, pH 6.5) containing the same concentration of glucose. The absorbance (reaction rate) of the reaction mixture was measured at 405 nm, and the K was analyzed by plotting the relationship between the reaction rate and glucose concentration at each pNPG concentration. i Value, the result is Figure 15 As shown. The horizontal coordinate of the intersection of the two straight lines represents -K i The simulation shows that the wild enzyme K i The glucose inhibition constant K of T47A, T374S and T47A&T374S is 4.283 mM. i The concentrations of the two proteins were 3.723mM, 3.468mM and 3.825mM, and the mutation had little effect on glucose tolerance.

[0099] (5) MD dynamics simulation

[0100] In recent years, many strategies have been developed to identify weak residues in enzymes as specific targets for mutation. Molecular dynamics (MD) simulations have proven to be a useful tool for understanding enzyme structure and behavior. Using MD simulations, it is possible to observe distinct enzyme motions at different temperatures. Molecular dynamics (MD) simulations can reveal the rigidity and flexibility of proteins at the atomic level. First, select SolutionBuilder in the input generator section of the CHARMM-GUI (https: / / charmmgui.org / ), solvate the protein, and generate a water box. The resulting file can be used to perform molecular dynamics simulations in an aqueous solvent environment using the NAMD program (https: / / www.ks.uiuc.edu / Research / namd / alpha / 3.0alpha / download / NAMD_3.0alpha13_Linux-x86_64-multicore-CUDA-SingleNode.tar.gz). AphaFold2 was run in Google Colab to model the point mutant protein. The corresponding protein sequence was imported into AphaFold2 to obtain the three-dimensional structure of the mutant. Using this structure as the starting point, molecular dynamics simulations were performed using NAMD with the CHARMM force field. The protein molecule was placed in a cubic water box. After the water molecules were filled with solvent and ions to neutralize the system, energy minimization was performed using the teepestdescent method. The system temperature was maintained at a constant 45°C using a velocity rescaling algorithm, and the pressure was controlled at 1 bar for 1 ns using a weak coupling method. After NVT model optimization, a 30 ns NPT equilibrium simulation was performed with a simulation time step of 2 fs. Coordinates were recorded every 5000 steps.

[0101] Select a rectangular water box with the size of the water box appropriate to the protein size and use the default margin of 10.0. Place the water box using the Monte-Carlo method for the addition of 0.05M sodium hydrogen phosphate. + and HPO4 2- The count ratio was 42:15, the force field was CH ARMM 36, energy minimization was performed under NVT, dynamics simulation was performed at 348.15K under NPT, and the trajectory files generated by the simulation were used to calculate the RSMD of the entire protein and the RSMF values of individual amino acid residues using VMD. The flexible region fluctuates significantly with increasing temperature. Analysis of the RSMF graph shows that the mutations at positions 47 and 374 cause fluctuations in the protein residues. Figure 16As can be seen, the variants exhibit lower RMSF values at most residues, while T47A exhibits higher RMSF values at residues 175-186, including the loop region and catalytic residue Val178. The wild-type enzyme's residues 175-179 are within 10 angstroms of the ligand. To facilitate observation of the specific RSMD magnitudes of each region, three regions were roughly divided based on RMSF values: 20-150, 150-200, and 200-400, for protein domain RSMD calculation. The results are shown in Table 7 below. At 45°C, the RSMD of the residues 150-200 was the largest among all enzymes. Therefore, it is hypothesized that the intense movement in this region affects the size of the active pocket, which may facilitate substrate regulation and turnover. In the 50-150 region, the variant enzymes are all more stable than the wild-type enzyme and are generally stable. The RSMD values in the 200-400 range indicate that both T47A and T374S are more stable than the wild-type enzyme in this region, while T47A and T374S are less stable in this region. Experimental results show that the order of thermal stability among the variants is T47A > T47A & T374S > T374S > wt, and the order of activity is T47A > T47A & T374S > wt > T374S. Overall, the relative increase in structural rigidity contributes to the improved thermal stability of the variants.

[0102] Table 7 RMSD values of protein structure regions of wild type and mutants

[0103]

[0104] The present invention successfully obtained mutants T47A and T47A&T374S with better activity and thermal stability through a semi-rational design method, among which T47A was the most excellent variant.

Claims

1. A β-glucosidase Bgl1269 mutant, characterized in that: The β-glycosidase Bgl1269 mutant is obtained by mutating the threonine at position 47 of the amino acid sequence shown in SEQ ID No. 1 to alanine.

2. The β-glucosidase Bgl1269 mutant according to claim 1, characterized in that: The β-glycosidase Bgl1269 mutant is obtained by mutating the threonine at position 47 of the amino acid sequence shown in SEQ ID No. 1 to alanine and the threonine at position 374 to serine.

3. A nucleic acid encoding the β-glycosidase Bgl1269 mutant according to claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid sequence according to claim 3.

5. A recombinant expression transformant comprising the recombinant expression vector according to claim 4.

6. A recombinant β-glucosidase Bgl1269 mutant catalyst, characterized in that: The recombinant β-glucosidase Bgl1269 mutant catalyst is any one of the following forms: (1) culturing the recombinant expression transformant according to claim 5, and isolating transformant cells containing the β-glucosidase Bgl1269 mutant; (2) culturing the recombinant expression transformant according to claim 5 and isolating a crude enzyme solution containing the β-glucosidase Bgl1269 mutant; (3) Cultivating the recombinant expression transformant according to claim 5, isolating the crude enzyme solution containing the β-glucosidase Bgl1269 mutant, and drying the prepared crude enzyme powder or purifying the prepared pure enzyme.

7. Use of the β-glucosidase mutant according to claim 1 or 2 in catalyzing the hydrolysis of soybean isoflavone glycosides.

8. The use according to claim 7, characterized in that: The soy isoflavone glycoside is daidzein.

9. The use according to claim 7, characterized in that: The soybean isoflavone glycoside is genistin.

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