Alpha-galactosidase mutants based on activity and thermostability engineering and use thereof
By mutating the amino acid sites of α-galactosidase, its hydrolytic activity and thermal stability were improved, solving the problem of insufficient hydrolytic efficiency of existing α-galactosidases in raffinose family oligosaccharides in legume products, and realizing efficient industrial application.
Patent Information
- Application Number
- CN202510057330.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2045-01-14
AI Technical Summary
The low thermal stability of existing α-galactosidases results in insufficient hydrolytic activity for removing raffinose family oligosaccharides (rfo) from legume products, limiting their practical application.
By rationally modifying the α-galactosidase derived from *Anoxybacillus vitaminiphilus*, and combining substrate binding pocket computational design and consensus sequence analysis, key amino acid sites such as N549, T550, and Y634F were mutated to improve its hydrolytic activity and thermal stability.
The catalytic efficiency of α-galactosidase was increased by 6.2 times and the thermal stability was increased by 2.0 times. It can efficiently hydrolyze raffinose and stachyose in soy milk, significantly reduce anti-nutritional effects, and has potential for industrial application.
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Figure CN120026009B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of functional gene modification, and particularly relates to a kind of alpha-galactosidase mutants and application thereof. BACKGROUND
[0002] Raffinose family oligosaccharides (rfo), such as raffinose and stachyose, are natural substrates of alpha-galactosidase, and exist in high concentrations in various legume seeds and vegetables. These rfo are known as anti-nutritional factors, causing intestinal gas and other gastrointestinal disorders in humans and monogastric animals lacking alpha-galactosidase. Hydrolysis and removal of rfo using alpha-galactosidase found in soy products is an important research area because soy milk has a rich protein profile and balanced amino acid content, and can be used as a substitute for cow's milk for lactose-intolerant patients.
[0003] However, the practical application of alpha-galactosidase is limited because of its low thermal stability and insufficient hydrolysis activity towards rfo, and therefore it is of great significance to improve the activity and thermal stability of alpha-galactosidase through rational modification. SUMMARY
[0004] The application significantly improves the hydrolysis activity of alpha-galactosidase derived from Anoxybacillus vitaminiphilus by computationally designing the substrate binding pocket, and then increases its stability by rationally designing conserved amino acid residues away from the active site, obtaining a combined variant N549Q / T550N / Y634F with improved catalytic efficiency and thermal stability, with a 6.2-fold increase in catalytic efficiency and a 2.0-fold increase in thermal stability.
[0005] The specific technical solutions of the application are as follows:
[0006] An alpha-galactosidase mutant has one or more of the following mutations: Y340, N549, T550, 124V, M309R, S489A or Y634F, based on the amino acid sequence shown in SEQ ID NO: 1 (alpha-galactosidase derived from Anoxybacillus vitaminiphilus).
[0007] The application is directed to the saturation mutation of the original amino acids at three positions Y340, N549, and T550 in the alpha-galactosidase amino acid sequence derived from Anoxybacillus vitaminiphilus into other 19 kinds of amino acids. The mutants obtained have different degrees of improvement in hydrolysis activity, and the threonine at position 550 is preferably mutated into asparagine. Further iteration saturation mutation of the amino acids at positions 549 and 340 is carried out on this basis. The mutant N549Q / T550N has the highest hydrolysis activity. On the other hand, there are 27 potential mutants away from the active site based on the amino acid sequence shown in SEQ ID NO: 1, including M394L, S132T, N305G, S489A, N309R, S143G, Y634F, F93L, C728V, Q613E, I124V, H342D, Y242D, S396D, Q236R, V212P, F678R, N695T, D7E, V719Q, A63E, F186R, R453E, H138E, K617I, wherein 124V, M309R, S489A, and Y634F have significantly improved thermal stability without loss of hydrolysis activity.
[0008] In a preferred embodiment of the application, the double mutant N549Q / T550N is combined with single-point mutations of 124V, M309R, S489A, or Y634F.
[0009] More preferably, the triple mutant N549Q / T550N / Y634F can increase the hydrolysis activity by 6.0 times and the residual activity by 2.0 times.
[0010] Another object of the application is to provide a DNA molecule encoding the alpha-galactosidase mutant.
[0011] Another object of the application is to provide an expression vector for the alpha-galactosidase mutant, which expresses the alpha-galactosidase mutant. The expression vector contains a DNA molecule encoding the alpha-galactosidase mutant. The expression vector is a plasmid, a bacteriophage, a virus, or a host cell.
[0012] The host cell is a prokaryotic cell or a eukaryotic cell, which can be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma, preferably Escherichia coli.
[0013] Another object of the application is to provide the use of the above-mentioned alpha-galactosidase mutant, its DNA molecule, or its expression vector in catalyzing the hydrolysis of oligosaccharides in the raffinose family. The oligosaccharides in the raffinose family include raffinose, stachyose, verbascose, and asparagoside.
[0014] A concrete example is the hydrolysis of raffinose and stachyose in soy milk using soy milk as a substrate.
[0015] The advantages of this invention are as follows:
[0016] This invention uses the α-galactosidase AgaV (nucleotide sequence shown in SEQ ID NO:2) from Anoxybacillus vitaminiphilus as a template. The three-dimensional structure of AgaV was predicted using the Alphafold3 algorithm, and then the substrate pNPG was docked to the active site using AutoDock 1.5.7 and default parameters. Figure 1 The active pocket is defined as the space between the substrate and the active pocket. The amino acid residues within the region were analyzed. A total of 20 residues were identified in this region: W119, N334, W336, E337, Y340, F341, D366, D367, R373, W411, E413, R443, K476, N480, C526, G528, G529, N549, T550, and E604. TurNup kcat was used to predict the hydrolytic activity of the virtual saturation mutant, revealing varying degrees of increased hydrolytic activity at residues N334, E337, Y340, F341, R373, E413, N549, and T550. Furthermore, fold X was used to predict the ΔG values before and after the mutation for the residues with increased activity; ΔG < 0, indicating minimal impact on enzyme stability. The results showed that the mutations of Y340, N549, and T550 were relatively stable. Saturation mutations were performed on Y340, N549, and T550. The mutant with the highest activity and the least loss of stability was selected to perform iterative saturation on the remaining two sites, and finally the N549Q / T550N combined mutation was obtained, which increased the activity by 6.0 times and the residual activity was only 0.7 times that of the wild type. To compensate for or further improve the thermal stability loss caused by increased activity, consensus sequence design was used to identify hotspots affecting the thermal stability of AgaV. This resulted in 27 potential mutations, including M394L, S132T, N305G, S489A, N309R, S143G, Y634F, F93L, C728V, Q613E, I124V, H342D, Y242D, S396D, Q236R, V212P, F678R, N695T, D7E, V719Q, A63E, F186R, R453E, H138E, K617I, V140E, and M45L. Among these, I124V, M309R, S489A, and Y634F showed 2–3 times the residual activity of wild-type AgaV at 65°C. Figure 5A). These mutants retained more than 70% of the catalytic activity of wild-type AgaV, and notably, the residual activity of mutant Y634F was increased by 2-fold with little loss of catalytic activity. Combining N549Q / T550N with the four thermostability mutants, these mutants exhibited minimal loss of catalytic activity Figure 5 B). The catalytic activity of triple mutant N549Q / T550N / Y634F was increased by 6-fold and the residual activity was increased by 2-fold compared to wild-type AgaV. This mutant retained both the enhanced catalytic activity of N549Q / T550N and the thermostability of Y634F. Thus, by employing a computational-aided dual-site mutagenesis strategy, the present application successfully modified residues in the active site pocket to improve catalytic efficiency while combining these mutations with distal mutations to enhance thermostability. Triple mutant N549Q / T550N / Y634F was far more efficient than wild-type in hydrolyzing soybean milk in practical applications, Figure 6 ), confirming the improvement of enzyme performance, effectively reducing the anti-nutritional effects of raffinose family oligosaccharides, and providing a robust and general strategy for simultaneously enhancing enzyme activity and thermostability, which has potential application prospects in industrial biocatalysis. BRIEF DESCRIPTION OF DRAWINGS
[0017] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The illustrations are given primarily for purposes of illustrating the illustrative embodiments of the application and are not to be construed as setting limits on the present application.
[0018] Figure 1 Amino acids identified by consensus sequence design for α-galactosidase AgaV.
[0019] Figure 2 Amino acids identified by consensus sequence design for α-galactosidase AgaV.
[0020] Figure 3 Relative enzyme activity and relative residual activity of mutant Y340, N549, T550 described in the present application.
[0021] Figure 4 Relative enzyme activity and relative residual activity of the iterative saturation mutants described in the present application.
[0022] Figure 5 Relative enzyme activity and relative residual activity of the consensus sequence mutants described in the present application (A); relative enzyme activity and relative residual activity of the active center and distal consensus sequence combination mutants described in the present application (B).
[0023] Figure 6TLC results (A) of soybean milk hydrolysis by the original enzyme and the mutant of the present application; concentration of reducing sugar in soybean milk hydrolysis by the original enzyme and the mutant of the present application (B).
[0024] Figure 7 Liquid chromatography of soybean milk hydrolysis by the original enzyme and the mutant of the present application. DETAILED DESCRIPTION
[0025] In order to make the technical personnel in the art better understand the present application scheme, the present application is further illustrated below in combination with the drawings and the implementation examples. It should be pointed out that the present embodiment is only used for explaining the present application, and is not limited to the scope of the present application. Obviously, the described embodiment is only a part of the embodiment of the present application, and is not all the embodiments. Based on the embodiment of the present application, all other embodiments obtained by the technical personnel in the art without making creative labor should belong to the protection scope of the present application.
[0026] Example 1 Determination of key amino acids for improving the hydrolysis activity and thermal stability of α-galactosidase AgaV and construction of α-galactosidase AgaV mutant.
[0027] 1. The α-galactosidase AgaV selected by the present application can hydrolyze raffinose family oligosaccharides (rofs) to generate galactose and sucrose. Studies have shown that due to the poor thermal stability of α-galactosidase and insufficient hydrolysis activity of rofs, the practical application of α-galactosidase in removing rofs in soybean products is limited. The present application adopts an integrated strategy to simultaneously improve the hydrolysis activity and thermal stability of α-galactosidase AgaV, which respectively improves the hydrolysis activity by modifying the amino acids in the active pocket region and improves the thermal stability by modifying the amino acids far from the active pocket. Figure 1 Amino acid modification strategy for the surface of α-galactosidase AgaV. Figure 2The amino acids around the AgaV substrate channel for alpha-galactosidase AgaV. The three-dimensional structure of AgaV was predicted by the Alphafold3 algorithm to determine the amino acids of the active center, and then the hydrolytic activity of virtual saturation mutation was predicted by TurNup kcat to determine that the hydrolytic activity of the 8 amino acid sites N334, E337, Y340, F341, R373, E413, N549, and T550 was improved to different degrees. To avoid the influence of enzyme structure on activity improvement, fold X was used to predict the AG value of the 8 amino acid residues before and after mutation, AG < 0, which had less effect on the stability of the enzyme. It was determined that the mutation stability of Y340, N549, and T550 was better, and iterative saturation mutation was performed on these 3 sites. The above mutants were constructed, and the construction method was as follows, and the primers used are shown in the table below: The codons of different amino acids corresponding to ndt are as follows: TGT (Cys), GAT (Asp), TTT (Phe), GGC (Gly), CAT (His), ATT (Ile), AAA (Lys), AAT (Asn), CGT (Arg), TCA (Ser), GTT (Val), TAT (Tyr).
[0028] The codons of different amino acids corresponding to vhg are as follows: GCA (Ala), GAA (Glu), AAA (Lys), CTG (Leu), ATG (Met), CCG (Pro), CAG (Gln), ACA (Thr), GTT (Val)
[0029]
[0030]
[0031] Using the pET-28a(+) plasmid with the alpha-galactosidase AgaV gene sequence as the template, referring to the Vazyme biological product and operation manual, the whole plasmid was amplified with mutation primer pairs. The PCR product was treated with Dpn I for enzyme digestion, and after the template digestion was completed, it was transformed into E. coli competent cells E. coli BL21(DE3) by heat shock method, and coated on LB agar plate containing 100 μg / ml kanamycin sulfate, and incubated at 37°C overnight. The mutation result was verified by sequence determination completed by Anhui General Biological Company. The mutants were constructed using conventional PCR technology, and the alpha-galactosidase AgaV expression vector was used as the template for whole plasmid amplification to introduce mutations, and each mutant was verified by sequencing to be successfully constructed.
[0032] The PCR reaction system is as follows:
[0033]
[0034] The PCR program is set as follows:
[0035]
[0036] 2-5 step cycle 30 times.
[0037] After the completion of the whole plasmid amplification, 2 μL of PCR product was taken for nucleic acid electrophoresis verification. After verification, DpnI digestion enzyme was used to degrade the initial template.
[0038] The digestion system is as follows:
[0039] PCR product 1 μL
[0040] DpnI enzyme 1 μL
[0041] 10x QuickCut Buffer 2 μL
[0042] The digestion procedure is as follows:
[0043] 37°C, 30 min.
[0044] After the digestion was completed, the PCR product was transformed into E. coli competent cells E. coli BL21 (DE3) by heat shock method, and was coated on a LB agar plate containing 100 μg / ml kanamycin sulfate, and was cultured at 37°C for 14-16 h. The mutation results were verified by sequence determination (completed by Anhui General Biotechnology Co., Ltd.), and the corresponding mutant was obtained.
[0045] 2. Fermentation expression of the above-mentioned recombinant α-galactosidase mutant in E. coli:
[0046] The specific expression method is as follows:
[0047] (1) The mutant constructed in Example 1 and the original enzyme α-galactosidase AgaV were inoculated into 30 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate, respectively, and were cultured at 37°C, 180 rpm overnight to prepare seed liquid. (2) The seed liquid was inoculated into fresh 30 mL of LB liquid medium at a 2% inoculation amount, and was cultured at 37°C, 180 rpm until the OD600 was 0.6-1.0. Then, it was taken out, cooled in an ice water bath for 5 min, and IPTG (isopropyl-β-D thiogalactoside) was added (final concentration 0.1 mmol / L) to induce expression at 20°C, 180 rpm for 20 h.
[0048] (3) Take the induced expression of the fermentation broth, 12000 rpm centrifugal 20 min, discard the supernatant, and then use 50 mM Na2HPO4-KH2PO4 (pH 7.5) buffer resuspended the bacteria, 12000 rpm centrifugal 20 min, discard the supernatant, resuspend with buffer, and then ultrasonic disruption. The broken liquid is centrifuged at 12000 rpm for 20 min, and the supernatant is taken to obtain the above-mentioned α-galactosidase AgaV each mutant crude enzyme solution.
[0049] 3. The above-mentioned mutant is determined for α-galactosidase enzyme activity
[0050] The present application determines the enzyme activity of each mutant of α-galactosidase AgaV by using pNPG as a substrate. A 10 mM pNPG solution is prepared by using KH2PO4-Na2HPO4 buffer (50 mM, pH 7.5) as a substrate solution. In the enzyme-labeled plate, 240 μL of the substrate solution and 10 μL of the appropriately diluted enzyme solution are added, and the reaction is carried out in the enzyme marker instrument at a reaction temperature of 35°C for 10 min. The absorbance value is measured at 410 nm. The control uses 10 μL of the inactivated enzyme solution with the same dilution factor.
[0051] Definition of enzyme activity unit: the amount of enzyme required to catalyze the hydrolysis of pNPG to generate 1 μmol of pNP per minute at 35°C and pH 7.5.
[0052] The preparation method of the pNP standard curve is as follows:
[0053] Accurately weigh 0.139 g of pNP, dissolve it in KH2PO4-Na2HPO4 buffer (50 mM, pH 7.5), and dilute the prepared mother liquor with KH2PO4-Na2HPO4 buffer to prepare pNP solutions with different concentrations. In the enzyme-labeled plate, 10 μL of the buffer is added, and then 240 μL of the pNP standard solution with different concentrations is added in turn. The absorbance value at 410 nm is measured by the enzyme marker instrument. The concentration of the standard substance is taken as the abscissa (X), and the absorbance value is taken as the ordinate (Y). The standard curve is drawn, and the equation is Y=5.51X+0.077, R 2 = 0.9980.
[0054] 4. The above-mentioned mutant is determined for thermal stability
[0055] Incubate at 65°C for 2.5 hours, collect samples every 0.5 hour, and determine the enzyme activity and residual activity of the crude enzyme solution of each mutant of α-galactosidase AgaV and the original enzyme by using pNPG as a substrate. The relative enzyme activity and the relative residual activity of each mutant are as follows: Figure 3 and Figure 4As shown in Table A, the results showed that the mutation of three amino acid sites was better at site 550 than at site 549, and the worst was at site 340. The hydrolytic activity of the single mutant T550N was 4.0 times higher than that of the wild type, and the residual activity remained unchanged. Using T550N as a template, iterative saturation mutation was performed on N549 and Y340. The results showed that the hydrolytic activity of the double mutant N549Q / T550N was increased by 6.0 times, and the residual activity was 0.7 times. The thermal stability was slightly reduced while the hydrolytic activity was increased, and the results of N549Q / T550N iterative Y340 were all worse than N549Q / T550N.
[0056] Example 2
[0057] The 27 potential distal amino acid residues that can improve the thermal stability of AgaV mutants were determined by the method of consensus sequence alignment, including M394L, S132T, N305G, S489A, N309R, S143G, Y634F, F93L, C728V, Q613E, I124V, H342D, Y242D, S396D, Q236R, V212P, F678R, N695T, D7E, V719Q, A63E, F186R, R453E, H138E, K617I, V140E and M45L.
[0058] The above mutants were constructed by the following construction method, and the primers used are shown in the following table:
[0059]
[0060]
[0061] The mutants were constructed by referring to the method of Example 1, and the residual activity was detected, and the results are shown in Table A Figure 5 As shown in Table A, the results showed that I124V, M309R, S489A and Y634F increased the residual activity at 65℃ by 2.0-3.0 times compared with the wild type and the relative hydrolytic activity was basically unchanged.
[0062] Example 3 Construction of triple mutants
[0063] Referring to the method of Example 1, the double mutant N549Q / T550N was combined with I124V, M309R, S489A and Y634F four single point mutations to construct mutants, and the enzyme activity and residual activity were detected, and the results are shown in Table B Figure 5 As shown in Table B, the results showed that the hydrolytic activity of the triple mutant N549Q / T550N / Y634F was increased by 6.0 times and the residual activity was increased by 2.0 times.
[0064] Example 4 Application of α-galactosidase mutants N549Q / T550N / Y634F and wild type to hydrolyze rofs
[0065] Method: Soybean was ground into fine powder, then defatted with n-hexane at a ratio of 1:1 (w / v). After removing the oil phase, defatted soybean powder was resuspended in distilled water at a concentration of 1:10 (w / v) and boiled in a water bath for 5 minutes. Insoluble substances were removed by centrifugation at 8000 rpm for 10 minutes. The resulting supernatant constituted soy milk, which was incubated with 4.0 U / mL of deα-galactosidase AgaV and its mutants N549Q / T550N / Y634F at 65°C for different times, and then boiled for 5 minutes to stop the enzyme reaction. The treated mixture was centrifuged to separate the supernatant, which was then analyzed using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). TLC analysis was performed by spotting 2.0 μL of the hydrolysate on a silica gel plate (10 cm x 10 cm) in a mixed solvent system of n-butanol / acetic acid / water (3:2:1, v / v / v). The plate was sprayed with a mixture of ethanol:sulfuric acid (4:1) and then heated at 110°C for 10 min to detect sugar spots; the hydrolysate was diluted as appropriate and filtered with a microporous membrane, and liquid-phase detection was used to analyze the reaction; liquid-phase detection used a Bio-Rad Aminex HPX-87H chromatographic column (300 mm x 7.8 mm), with a sample size of 20 μL, a mobile phase of 5 mM H2SO4, a flow rate of 0.4 ml·min-1; the column temperature was 50°C, and a differential refractometer detector was used for detection. The peak time for galactose was 13.8 min, the peak time for sucrose was 19.1 min, the peak time for raffinose was 23.3 min, and the peak time for stachyose was 24.2 min.
[0066] Results: The detection results are shown in Figure 6 and Figure 7 The results show that the three mutants have completely hydrolyzed all of the rofs in the soy milk to sucrose and galactose in 20 min, while the wild type still has a large portion of rofs remaining in 30 min, thus the present application successfully modifies the α-galactosidase to have high hydrolysis activity and thermal stability, enabling it to efficiently hydrolyze rofs in soybean products, and having important industrial application value.
[0067] The above description is merely preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. An α-galactosidase mutant, characterized in that... The mutant is based on the amino acid sequence shown in SEQ ID NO:1, with simultaneous mutations of N549Q, T550N, and Y634F.
2. A DNA molecule, characterized in that, The DNA molecule encodes the α-galactosidase mutant of claim 1.
3. An expression vector for an α-galactosidase mutant, characterized in that... Express the α-galactosidase mutant of claim 1.
4. The expression vector according to claim 3, characterized in that... Express the DNA molecule as described in claim 2.
5. The expression vector according to claim 4, characterized in that... The expression vector is a plasmid, a virus, or a host cell.
6. The expression vector according to claim 5, characterized in that... The host cell is a prokaryotic cell or a eukaryotic cell.
7. The expression vector according to claim 5, characterized in that... The host cells are selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma.
8. The application of the α-galactosidase mutant according to claim 1, the DNA molecule according to claim 2, or the expression vector of the α-galactosidase mutant according to any one of claims 3-7 in the catalytic hydrolysis of raffinose family oligosaccharides.