Alpha-galactosidase mutant modified based on activity and thermal stability and application of alpha-galactosidase mutant

Abstract

The invention discloses an alpha-galactosidase mutant with high specific activity and thermal stability and application of the alpha-galactosidase mutant. According to the present invention, alpha-galactosidase AgaV derived from Anxybacillus vitaminiphilus is adopted as a template, and amino acid mutation is performed by using a molecular biological technology to obtain mutants with improved catalytic efficiency, including Y340E, N549Q and T550N; the mutant disclosed by the invention has excellent properties of high catalytic efficiency and strong thermal stability, the hydrolysis efficiency of raffinose family oligosaccharides (rof), such as raffinose and stachyose, is improved by 4.0-6.0 times, and the mutant has a wide application prospect in hydrolysis and removal of rfo in soybean products.

Description

Technical Field

[0001] The invention belongs to the technical field of functional gene modification, and specifically relates to a class of alpha-galactosidase mutants and applications thereof. Background Art

[0002] Raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are natural substrates for α-galactosidase and are found in high concentrations in various legume seeds and vegetables. These RFOs are known as anti-nutritional factors and cause flatulence and other gastrointestinal disorders in α-galactosidase-deficient humans and monogastric animals. The hydrolysis and removal of RFOs using α-galactosidase found in soy products is an important area of ​​research, as soymilk, with its rich protein profile and balanced amino acid content, can serve as a substitute for cow's milk for lactose-intolerant patients.

[0003] However, the practical application of α-galactosidase is limited because of its low thermal stability and insufficient hydrolysis activity against RFO. Therefore, it is of great significance to rationally modify α-galactosidase and simultaneously improve its activity and thermal stability for removing RFOs from soybean products. Summary of the invention

[0004] The present invention rationally transforms α-galactosidase from Anoxybacillus vitamininiphilus through computational design of substrate binding pocket, significantly improves its hydrolysis activity, and then increases its stability through reasonable design of conservative amino acid residues away from the active site, thereby obtaining a combination variant N549Q / T550N / Y634F with improved catalytic efficiency and thermal stability, with the catalytic efficiency increased by 6.2 times and the thermal stability increased by 2.0 times.

[0005] The specific technical solutions of the present invention are as follows:

[0006] An alpha-galactosidase mutant has one or more of the 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 present invention performs saturation mutation of the original amino acids at three positions Y340, N549 and T550 in the amino acid sequence of α-galactosidase from Anoxybacillus vitaminiphilus into 19 other amino acids, and the obtained mutants have different degrees of improvement in hydrolysis activity, preferably the 550th threonine is mutated into asparagine. Further on this basis, the 549th amino acid and the 340th amino acid are iteratively saturated mutated. The mutant N549Q / T550N has the highest hydrolysis activity. On the other hand, based on the amino acid sequence shown in SEQ ID NO: 1, the present invention has 27 potential mutants away from the active site, 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, and K617I, among which 124V, M309R, S489A and Y634F significantly improve the thermal stability without losing the hydrolysis activity.

[0008] A preferred embodiment of the present invention is to combine the double mutant N549Q / T550N with single point mutations 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 present invention is to provide a DNA molecule encoding the α-galactosidase mutant of the present invention.

[0011] Another object of the present invention is to provide an expression vector of an α-galactosidase mutant, which expresses the α-galactosidase mutant of the present invention. The expression vector contains a DNA molecule encoding the α-galactosidase mutant of the present invention. The expression vector is a plasmid, a phage, a virus or a host cell.

[0012] The host cell is a prokaryotic cell or a eukaryotic cell, and may be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma, preferably Escherichia coli.

[0013] Another object of the present invention is to provide the use of the above-mentioned α-galactosidase mutant, its DNA molecule or its expression vector in catalyzing the hydrolysis of raffinose family oligosaccharides. The raffinose family oligosaccharides include raffinose, stachyose, verbascose and ajuga.

[0014] A specific example is to use soy milk as a substrate to hydrolyze raffinose and stachyose in the soy milk.

[0015] The advantages of the present invention are as follows:

[0016] The present invention uses α-galactosidase AgaV (nucleotide sequence as shown in SEQ ID NO: 2) from Anoxybacillus vitaminiphilus as a template, uses the Alphafold3 algorithm to predict the three-dimensional structure of AgaV, and then uses AutoDock1.5.7 and default parameters to dock the substrate pNPG to the active site ( Figure 1 ), the active pocket is defined as the The amino acid residues in the region were composed of 20 residues: W119, N334, W336, E337, Y340, F341, D366, D367, R373, W411, E413, R443, K476, N480, C526, G528, G529, N549, T550, and E604. TurNup kcat predicted the hydrolysis activity of virtual saturation mutations, and found that the hydrolysis activities of N334, E337, Y340, F341, R373, E413, N549, and T550 were improved to varying degrees. At the same time, the ΔG values ​​before and after the mutation were predicted by fold X for the residues with improved activity. ΔG<0, which has little effect on the stability of the enzyme. The results showed that the mutation results of Y340, N549 and T550 were more stable. Saturation mutation was performed on Y340, N549 and T550, and the mutants with the highest activity and the least loss of stability were selected for iterative saturation of the remaining two sites. Finally, the combined mutation of N549Q / T550N was obtained, whose activity was increased by 6.0 times, and the residual activity was only 0.7 times that of the wild type. To compensate or further improve the loss of thermostability caused by increased activity, the hotspots affecting the thermostability of AgaV were found by consensus sequence design, and 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 were obtained. Among them, the residual activities of I124V, M309R, S489A and Y634F at 65°C were 2 to 3 times that of wild-type AgaV ( Figure 5A). These mutants retained more than 70% of the catalytic activity of wild-type AgaV, and notably, mutant Y634F showed a 2-fold increase in residual activity with little loss of catalytic activity. N549Q / T550N was combined with the four thermostable mutants, which showed minimal loss of catalytic activity. All four triple mutants showed improvements in catalytic activity and thermostability compared to the wild-type ( Figure 5 B). Compared with the wild-type AgaV, the catalytic activity of the triple mutant N549Q / T550N / Y634F increased by 6 times, and the residual activity increased by 2 times. This indicates that the mutant retains both the enhanced catalytic activity of N549Q / T550N and the thermal stability of Y634F. Therefore, by adopting a computationally assisted double-site mutagenesis strategy, the present invention successfully modified the residues in the active site pocket to improve the catalytic efficiency, while combining these mutations with distal mutations to enhance thermal stability. In the actual application of hydrolyzing soy milk, the efficiency of hydrolyzing rofs by the triple mutant N549Q / T550N / Y634F is much higher than that of the wild type ( Figure 6 ), confirmed the improvement of enzyme performance, effectively reduced the anti-nutritional effects of raffinose family oligosaccharides, and provided a robust and general strategy for simultaneously enhancing enzyme activity and thermal stability, with potential application prospects in industrial biocatalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 Docking of α-galactosidase AgaV with pNPG molecule and surrounding amino acids.

[0019] Figure 2 Amino acids determined by consensus sequence design for α-galactosidase AgaV.

[0020] Figure 3 It is the relative enzyme activity and relative residual activity of the mutants Y340, N549 and T550 described in the present invention.

[0021] Figure 4 It is the relative enzyme activity and relative residual activity of the iterative saturation mutants described in the present invention.

[0022] Figure 5 The relative enzyme activity and relative residual activity (A) of the consensus sequence mutants of the present invention; the relative enzyme activity and relative residual activity (B) of the active center and distal consensus sequence combination mutants of the present invention.

[0023] Figure 6The TLC results of soy milk hydrolyzed by the original enzyme and the mutant of the present invention (A); the concentration of reducing sugar in soy milk hydrolyzed by the original enzyme and the mutant of the present invention (B).

[0024] Figure 7 Liquid phase analysis of soy milk hydrolyzed by the original enzyme and the mutant of the present invention. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described below in conjunction with the accompanying drawings and implementation examples. It should be pointed out that this embodiment is only used to explain the present invention, rather than to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work should belong to the scope of protection of the present invention.

[0026] Example 1 Determination of key amino acids for improving the hydrolytic activity and thermal stability of α-galactosidase AgaV and construction of α-galactosidase AgaV mutants.

[0027] 1. The α-galactosidase AgaV selected in the present invention can hydrolyze raffinose family oligosaccharides (ROF) to generate galactose and sucrose. Studies have shown that due to the poor thermal stability of α-galactosidase and insufficient hydrolysis activity on ROS, the practical application of α-galactosidase in removing ROS from soybean products is limited. The present invention adopts an integrated strategy to simultaneously improve the hydrolysis activity and thermal stability of α-galactosidase AgaV, namely, the modification of amino acids in the active pocket region to improve the hydrolysis activity and the modification of amino acids distal to the active pocket to improve the thermal stability. Figure 1 A strategy for modifying the surface amino acids of α-galactosidase AgaV. Figure 2The substrate channel and surrounding amino acids of α-galactosidase AgaV were predicted by the Alphafold3 algorithm to determine the amino acids in the active center. Then, the hydrolysis activity of virtual saturation mutations was predicted using TurNup kcat. It was determined that the hydrolysis activities of the eight amino acid sites, N334, E337, Y340, F341, R373, E413, N549, and T550, were all improved to varying degrees. In order to avoid the influence of the activity-enhancing enzyme structure, the ΔG values ​​before and after the mutation were predicted for these eight amino acid residues using fold X. ΔG < 0 had little effect on the stability of the enzyme. It was determined that the mutations of Y340, N549, and T550 had better stability, and these three sites were subjected to iterative saturation mutations. The above mutants were constructed as follows, and the primers used are shown in the table below: The codons corresponding to different amino acids of 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 for different amino acids in 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 α-galactosidase AgaV gene sequence as a template, referring to the Vazyme biological product and operation manual, using the mutation primer pair, the whole plasmid amplified the site-directed mutation sequence. The PCR product was treated with Dpn I. After the template digestion, it was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and spread on LB agar plates containing 100μg / ml kanamycin sulfate, and inverted and cultured at 37℃ overnight. The mutation results were verified by sequence determination by Anhui General Biological Company. The construction of the mutant used conventional PCR technology, and the α-galactosidase AgaV expression vector was used as a template for full plasmid amplification to introduce mutations. The mutants obtained were successfully constructed by sequencing verification.

[0032] The PCR reaction system is as follows:

[0033]

[0034] The PCR program was set as follows:

[0035]

[0036] Repeat steps 2-5 30 times.

[0037] After the full plasmid amplification is completed, 2 μL of PCR product is taken for nucleic acid electrophoresis verification. After the verification is completed, DpnI digestion enzyme is used to degrade the initial template.

[0038] The digestive system is as follows:

[0039] PCR product 1 μL

[0040] DpnI enzyme 1 μL

[0041] 10×QuickCut Buffer 2μL

[0042] The digestion procedure was as follows:

[0043] 37℃, 30min.

[0044] After digestion, the PCR product was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and spread on LB agar plates containing 100 μg / ml kanamycin sulfate and cultured at 37°C for 14-16 hours. The mutation results were verified by sequence determination (completed by Anhui General Biological Company) to obtain the corresponding mutants.

[0045] 2. Fermentation expression of the above recombinant α-galactosidase mutant in Escherichia 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 culture medium containing 100 μg / mL kanamycin sulfate, and cultured overnight at 37°C, 180 rpm to prepare seed liquid. (2) The seed liquid was inoculated into 30 mL of fresh LB liquid culture medium at a 2% inoculum, and cultured at 37°C, 180 rpm until OD600 was 0.6-1.0, then taken out and cooled in an ice water bath for 5 min, and the inducer IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) was added, and the expression was induced at 20°C, 180 rpm for 20 h.

[0048] (3) Take the fermentation broth induced by expression, centrifuge at 12000 rpm for 20 min, discard the supernatant, and then add 50 mM Na 2 HPO 4 -KH 2 PO 4The cells were resuspended in buffer (pH 7.5) and washed, centrifuged at 12000 rpm for 20 min, the supernatant was discarded, the cells were resuspended in buffer, and then ultrasonically disrupted. The disrupted liquid was centrifuged at 12000 rpm for 20 min, and the supernatant was taken to obtain the crude enzyme solution of each mutant of α-galactosidase AgaV.

[0049] 3. Determination of α-galactosidase activity of the above mutants

[0050] The present invention uses pNPG as a substrate to determine the enzyme activity of each mutant of α-galactosidase AgaV. 2 PO 4 -Na 2 HPO 4 Prepare 10mM pNPG solution in buffer (50mM, pH7.5) as substrate solution. Add 240μL substrate solution and 10μL appropriately diluted enzyme solution to the ELISA plate, and react in an ELISA reader at 35℃ for 10min. Measure the absorbance at 410nm. Use 10μL inactivated enzyme solution with the same dilution factor as control.

[0051] Definition of enzyme activity unit: the amount of enzyme required to catalyze the hydrolysis of pNPG to produce 1 μmol pNP per minute at 35°C and pH 7.5.

[0052] The method for preparing the pNP standard curve is as follows:

[0053] Accurately weigh 0.139 g of pNP and use KH 2 PO 4 -Na 2 HPO 4 Buffer (50mM, pH7.5) was dissolved and the volumetric flask was made up to 100mL. 2 PO 4 -Na 2 HPO 4 Buffer is used to dilute the prepared mother solution to prepare pNP solutions of different concentrations. 10 μL of buffer is added to the ELISA plate, and then 240 μL of pNP standard solutions of different concentrations are added in sequence. The absorbance value at a wavelength of 410 nm is measured with an ELISA instrument. The concentration of the standard substance is used as the horizontal axis (X) and the absorbance value is used as the vertical axis (Y). A standard curve is drawn, and the equation is: Y = 5.51X + 0.077, R 2 =0.9980.

[0054] 4. Thermal stability test of the above mutants

[0055] The cells were incubated at 65°C for 2.5 hours, and samples were collected every 0.5 hours. The enzyme activities and residual activities of the crude enzyme solutions of the mutants and the original enzyme of α-galactosidase AgaV were determined using pNPG as a substrate. The relative enzyme activities and relative residual activities of the mutants are shown in Table 1. Figure 3 and Figure 4 As shown, the results showed that the mutation results of the three amino acid sites were that site 550 was better than site 549, and site 340 was the worst. The hydrolysis activity of T550N in the single mutant was increased by 4.0 times compared with the wild type, and the relative residual activity remained unchanged. Using T550N as a template, after iterative saturation mutation of N549 and Y340, the results showed that the double mutant N549Q / T550N had a 6.0-fold increase in hydrolysis activity and a 0.7-fold residual activity. While the hydrolysis activity was improved, the thermal stability was slightly reduced. The results of N549Q / T550N and Y340 after iteration were not as good as N549Q / T550N.

[0056] Example 2

[0057] By consensus sequence alignment, 27 potential distal amino acid residues were identified as mutants that could improve the thermal stability of AgaV, 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 mutants were constructed as follows. The primers used are shown in the table below:

[0059]

[0060]

[0061] The mutants were constructed according to the method of Example 1 and the residual activity was detected. The results are as follows Figure 5 As shown in A, the results showed that the residual activity of I124V, M309R, S489A and Y634F at 65°C was 2.0-3.0 times higher than that of the wild type, and the relative hydrolysis activity remained 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 four single point mutations of I124V, M309R, S489A and Y634F to construct mutants, and the enzyme activity and residual activity were tested. The results are as follows: Figure 5As shown in B, the results showed that the hydrolysis 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 mutant N549Q / T550N / Y634F and wild type in hydrolyzing rofs

[0065] Methods: Soybeans were ground into fine powder and then defatted with n-hexane at a ratio of 1:1 (w / v). After removing the oil phase, the defatted soybean powder was resuspended in distilled water at a concentration of 1:10 (w / v) and boiled in a water bath for 5 min. Insoluble matter was removed by centrifugation at 8000 rpm for 10 min. The resulting supernatant constituted soymilk, which was incubated with 4.0 U / mL de-α-galactosidase AgaV and its mutant N549Q / T550N / Y634F at 65 °C for different times and then boiled for 5 min 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 hydrolyzate on a silica gel plate (10 cm × 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 and sulfuric acid (4:1), and then heated at 110°C for 10 min to detect sugar spots. The hydrolyzate was appropriately diluted and filtered through a microporous filter membrane, and the reaction was analyzed by liquid chromatography. The liquid chromatography used a Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm), an injection volume of 20 μL, and a mobile phase of 5 mM H 2 SO 4 , flow rate 0.4ml·min-1; column temperature: 50℃, detection by differential refractometer. Galactose elution time: 13.8min, sucrose elution time: 19.1min, raffinose elution time: 23.3min, stachyose elution time: 24.2min.

[0066] Results: The test results are as follows: Figure 6 and Figure 7 As shown, the results showed that the triple mutant had completely hydrolyzed all the ROS in soy milk into sucrose and galactose within 20 minutes, while the wild type still had most of the ROS remaining after 30 minutes. Therefore, the present invention successfully modified α-galactosidase to have high hydrolysis activity and thermal stability, and can efficiently hydrolyze ROS in soybean products, which has important industrial application value.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An α-galactosidase mutant, characterized in that The mutant has one or more of the mutations Y340, N549, T550, 124V, M309R, S489A or Y634F in the amino acid sequence shown in SEQ ID NO:

1.

2. The α-galactosidase mutant according to claim 1, characterized in that The mutants are T550N and N549Q mutations.

3. The α-galactosidase mutant according to claim 2, characterized in that The mutant also has one or more of 124V, M309R, S489A and Y634F mutations.

4. A DNA molecule, characterized in that The DNA molecule encodes the α-galactosidase mutant according to any one of claims 1-3.

5. An expression vector for an α-galactosidase mutant, characterized in that Expressing the α-galactosidase mutant according to claim 4.

6. The expression vector according to claim 4, characterized in that Express the DNA molecule of claim 4.

7. The expression vector according to claim 6, characterized in that The expression vector is a plasmid, a phage, a virus or a host cell.

8. The expression vector according to claim 7, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

9. The expression vector according to claim 8, characterized in that The host cell is selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

10. Use of the α-galactosidase mutant according to any one of claims 1 to 3, the DNA molecule according to claim 4, or the expression vector of the α-galactosidase mutant according to any one of claims 5 to 9 in catalyzing the hydrolysis of raffinose family oligosaccharides.

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