Method for improving hydrolytic activity of alpha-galactosidase by semi-rational engineering modification of active site of alpha-galactosidase and construction of mutant

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for improving hydrolytic activity of alpha-galactosidase through semi-rational engineering modification of an active site of the alpha-galactosidase and construction of a mutant. According to the invention, based on semi-rational design methods such as sequence alignment, homologous modeling, molecular docking, FoldX energy calculation and the like, the alpha-galactosidase active site from lactobacillus amyloliquefaciens L6 is accurately modified. The mutant enzyme H203P of which the performance is remarkably improved is successfully obtained through directional mutation of active site amino acid residues, the hydrolytic activity of the mutant enzyme H203P reaches 363.95 U / mg and is improved by 2.83 times compared with that of a wild type, the mutant enzyme H203P shows excellent catalytic performance, the enzymatic reaction efficiency is greatly improved, industrial application of alpha-galactosidase is promoted, and the mutant enzyme H203P has a wide application prospect. The method has a wide application prospect and a remarkable economic value in the fields of food processing, feed addition, pharmaceutical and chemical engineering and the like, and is of great significance in promoting the upgrading of related industrial technologies.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a method for semi-rational engineering of the active site of α-galactosidase to improve its hydrolysis activity and the construction of a mutant. Background Art

[0002] α-Galactosidase (α-GLA) is an exoglycosidase that specifically hydrolyzes α-galactosidic bonds at the non-reducing ends of various compounds. It is widely distributed and found in a variety of organisms, including bacteria, fungi, plants, and animals. Its unique catalytic properties have broad applications in food, feed, papermaking, and medicine. In the food and feed industries, it effectively removes flatulence from soybean meal and soy products, significantly alleviating post-consumption flatulence. In the papermaking industry, it hydrolyzes α-galactose from the mannose backbone, enhancing gelation and thus optimizing pulp quality. In medicine, α-GLA hydrolyzes the α-galactosyl group at the end of ceramide trihexosyl groups, enabling the treatment of Fabry disease. It also has potential applications in blood type conversion and other fields. However, the α-GLA used in industrial production suffers from insufficient hydrolytic activity, which has hindered its efficient application in related fields. Currently, the main methods for improving the hydrolytic activity of α-GLA include traditional screening and random mutagenesis. Traditional screening methods primarily rely on selecting α-galactosidase-producing strains with high hydrolytic activity from a large number of microbial resources. However, this method relies on pre-existing enzyme resources in nature, limiting the scope of screening and resulting in low efficiency. It also requires significant human, material, and time resources. Furthermore, strains with ideal hydrolytic activity are scarce in nature, making it difficult to meet the needs of large-scale industrial production. Random mutagenesis involves randomly inducing mutagenesis in the α-galactosidase gene and then screening for mutants with enhanced hydrolytic activity from a large mutant library. While this technique theoretically allows for the generation of a wide range of mutations, the random nature of the mutations results in a complex mutant library and an extremely arduous screening process. Furthermore, random mutagenesis can disrupt the enzyme's original structure and function, reducing the probability of identifying effective mutants and leading to high R&D costs and a long development cycle.

[0003] In summary, existing technologies for increasing the hydrolytic activity of α-galactosidase still suffer from shortcomings such as low efficiency, high cost, and difficulty in accurately obtaining highly active mutants. This makes it difficult to precisely regulate the increase in α-galactosidase activity and fails to meet the urgent demand for highly active enzymes in actual production and applications. Therefore, there is an urgent need to develop a technology that combines high efficiency and precision to construct mutants with higher hydrolytic activity by targeting key sites of α-galactosidase for modification, thereby promoting the development of related industries. Summary of the Invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the present invention takes α-galactosidase (AglB) derived from Lactobacillus amyloliquefaciens L6 as the research object, determines the active sites of the enzyme catalyzing melibiose hydrolysis and substrate binding through homology modeling and molecular docking, then uses semi-rational engineering technology to mutate, replace or introduce relevant amino acid sites, and verifies the key amino acid sites affecting the hydrolysis of AglB, thereby screening AglB mutants with improved hydrolysis activity, providing a theoretical basis for the application of this enzyme in food, papermaking, etc.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides an engineered enzyme with improved hydrolytic activity of α-galactosidase, wherein the amino acid H at position 203 in the amino acid sequence of wild-type α-galactosidase is mutated to P, thereby obtaining an engineered enzyme with improved hydrolytic activity. The amino acid sequence of the wild-type α-galactosidase is shown in SEQ ID NO.2.

[0007] Preferably, the wild-type α-galactosidase is derived from Lactobacillus amylolyticus L6, and the preservation number of the strain is CGMCC NO.9090.

[0008] The applicant previously isolated a strain of Lactobacillus amyloliquefaciens L6 (see patent CN104130956B, accession number CGMCC No. 9090) with excellent fermentation performance and probiotic properties. This strain secretes α-galactosidase (AglB), which hydrolyzes melibiose. Cloning and expression of this enzyme revealed low activity in catalyzing melibiose hydrolysis, limiting its potential for application. To address this scientific problem, the present invention identified the enzyme's active sites for melibiose hydrolysis and substrate binding through homology modeling and molecular docking. Using semi-rational engineering techniques, the inventors mutated, replaced, or introduced the relevant amino acid sites, and verified the key amino acid sites that influence AglB's hydrolytic activity. The results revealed that the mutant enzyme, H2O3P, exhibited significantly enhanced hydrolytic activity, reaching 363.95 U / mg, a 2.83-fold increase compared to the wild-type. This will allow the screening of AglB mutants with enhanced hydrolysis activity, providing a theoretical basis for the application of this enzyme in food, papermaking, and other areas.

[0009] Preferably, the nucleotide sequence of the wild-type α-galactosidase is shown as SEQ ID NO.1.

[0010] The second aspect of the present invention further provides a nucleotide sequence encoding the engineered enzyme described in the first aspect, and the nucleotide sequence is shown as SEQ ID NO.11.

[0011] The third aspect of the present invention further provides a recombinant expression vector for expressing the engineered enzyme described in the first aspect, wherein the recombinant expression vector carries the nucleotide sequence encoding the engineered enzyme described in the second aspect.

[0012] The fourth aspect of the present invention also provides a method for constructing the engineered enzyme described in the first aspect, namely, first constructing a recombinant expression vector using the primer pair H203P-F / R of SEQ ID NO.3 and SEQ ID NO.4, then transforming the Escherichia coli competent cell BL21, and then obtaining the engineered enzyme after inducing expression of the enzyme protein and purifying the enzyme protein by affinity chromatography.

[0013] The fifth aspect of the present invention further provides use of the engineered enzyme described in the first aspect in catalyzing the hydrolysis of melibiose.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This study, based on semi-rational design methods such as sequence alignment, homology modeling, molecular docking, and FoldX energy calculation, precisely modified the active site of α-galactosidase from Lactobacillus amyloliquefaciens L6. Through targeted mutagenesis of the amino acid residues in the active site, a significantly improved mutant enzyme, H203P, was successfully obtained. Its hydrolytic activity reached 363.95 U / mg, a 2.83-fold increase compared to the wild-type, demonstrating excellent catalytic performance and significantly enhancing the efficiency of the enzymatic reaction.

[0016] This invention breaks through the limitations of traditional screening and achieves efficient improvement of enzyme activity through semi-rational design, providing a biocatalyst with better performance for the industrial application of α-galactosidase, effectively reducing production costs and improving production efficiency. It has broad application prospects and significant economic value in the fields of food processing, feed additives, pharmaceutical chemicals, etc., and is of great significance for promoting technological upgrading of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 is the sequence alignment result of AglB;

[0018] Figure 2 The homology modeling structure diagram of AglB (a is tetramer, b is single chain);

[0019] Figure 3 Figure 1 is a structural alignment of AglB and 2XN0;

[0020] Figure 4 This is the molecular docking conformation diagram of AglB and melibiose;

[0021] Figure 5 Figure 1 is the binding site map of AglB-melibiose;

[0022] Figure 6 The results of enzyme activity determination of AglB and mutant crude enzyme solutions are shown;

[0023] Figure 7 is the specific activity of AglB and mutants in hydrolyzing pNPG;

[0024] Figure 8 Surface view of the docking pocket of AglB;

[0025] Figure 9 Combined pocket surface comparison diagram (a is WT, b is H203P, c is D370N, d is W415Y, and e is K480R). DETAILED DESCRIPTION

[0026] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0028] In the following examples, the method for isolating AglB enzyme from Lactobacillus amyloliquefaciens L6 refers to the paper: Fei Yongtao. Analysis of bacterial flora and metabolic research of key lactic acid bacteria during acidification of tofu yellow slurry [D]. South China University of Technology, 2018.

[0029] The present invention aims to improve the hydrolytic activity of α-galactosidase by semi-rational engineering of the active site, and to construct a mutant with higher hydrolytic activity. The specific implementation is as follows:

[0030] 1. Experimental methods

[0031] 1.1 Structural Modeling

[0032] SWISS-MODEL is a template-based protein structure prediction server that provides tools for predicting protein three-dimensional structures using homology modeling. It allows users to submit a protein sequence and generate a predicted structural model by aligning it with known protein structures. The server utilizes advanced algorithms to provide users with high-quality structural models and allows for model quality assessment. In this study, homology modeling was performed using SWISS-MODEL using the α-galactosidase from Lactobacillus acidophilus NCFM (PDB ID: 2XN0, sequence similarity 78.28%) from the PDB repository. First, the AglB sequence was uploaded to the SWISS-MODEL platform. Template 2XN0 was manually specified, and an automated sequence alignment was performed. After verifying the alignment of key functional residues (such as the catalytic site), a three-dimensional model was constructed using default parameters. The generated model was saved as a PDB file and used for subsequent substrate binding or mutation site design studies.

[0033] Gene sequence of AglB (SEQ ID NO.1):

[0034]

[0035] 1.2 Molecular docking

[0036] Molecular docking is a computer simulation method used to predict the interaction patterns and binding affinities between molecules (usually proteins and ligand molecules, such as small molecule drugs). Molecular docking is widely used in drug design, protein function research, enzymology research, and other fields. Its core goal is to simulate the binding process between ligands and receptors, identify possible binding modes, and predict the binding strength between them. In this study, the 3D structural model of melibiose (CID: 92845) was obtained from the PubChem database, and the molecular docking of melibiose and enzyme protein was performed using AutoDock 4.2.6: First, the wild-type model of AglB (PDB format) obtained in step 1.1 was removed of water molecules using AutoDock Tools, and polar hydrogen and Gasteiger charges were added, and saved as a PDBQT receptor file; then the melibiose structure was downloaded from PubChem in SDF format, converted to PDB format using Pymol, and the protonation state and charge distribution were optimized using AutoDock Tools to generate a ligand PDBQT file; then the docking parameters were set in AutoDock (the grid center covered the active pocket), the Lamarkian genetic algorithm was run (population size 150, energy evaluation 2.5 million times), and the conformation with the optimal binding energy was output in PDBQT format; finally, the docking results were loaded using PyMOL, the AglB active pocket was displayed using surface electrostatic potential, the melibiose molecules were superimposed, and the hydrogen bonds were annotated ( angle >120°) and hydrophobic interactions, a high-resolution docking visualization image (PNG format) was generated, and the AglB-melibiose complex structure was converted into PDB format.

[0037] 1.3 FoldX Energy Calculation

[0038] Based on the AglB-melibiose docking complex structure generated in step 1.2, the FoldX program was integrated into the YASARA software environment to perform mutation energy calculations: first, the docked optimized PDB file was imported into YASARA, and the built-in FoldX plug-in was called to perform alanine scanning. Alanine mutants were generated for key residues in the binding pocket, and the BuildModel and Stability commands were run to calculate the Gibbs free energy change (ΔΔG) of each mutant; then, the ScanPosition saturation mutagenesis strategy was executed on the selected residues, and all 20 amino acid substitutions were systematically enumerated. The protein stability parameter (ΔΔG_fold) of each mutant was accurately calculated with the help of the EnergyAnalysis module of the YASARA software.

[0039] 1.4 Sequence Alignment

[0040] Based on the amino acid sequence of AglB, homologous α-galactosidases were compared in the UniProt database using the NCBI BLAST tool. Sequences with a similarity greater than 40% were screened, and α-galactosidases from Lactobacillus hamsteri (WP_056941429.1), Lactobacillus delbrueckii (WP_231546993.1), and Streptococcus oralis (WP_308687821.1) were selected for sequence alignment. The target sequences were imported into the CLUSTALW webpage (https: / / www.genome.jp / tools-bin / clustalw), and the ClustalW algorithm was used for multiple sequence alignment to generate .aln format results. The alignment files were then uploaded to the ESPript 3.0 website for visualization and export as high-resolution PDF images.

[0041] Amino acid sequence of AglB (SEQ ID NO. 2):

[0042] 。

[0043] 1.5

[0044] Based on the results of the aforementioned structural modeling, molecular docking, and sequence alignment, key amino acid residues surrounding the active center pocket were selected as mutation sites. Molecular docking revealed that certain amino acid residues form key hydrogen bonds or hydrophobic interactions with the substrate melibiose. Sequence alignments revealed that these sites are less conserved in homologous enzymes or have room for modification. Therefore, these sites were selected for mutation in the hope of optimizing the active pocket structure, enhancing the enzyme's ability to bind to the substrate, and thereby improving hydrolytic activity. For example, by mutating the amino acids at specific sites to those with different side chain lengths, charges, or polarities, the microenvironment of the active pocket is altered, making it easier for the substrate to enter the active site and undergo hydrolysis.

[0045] 1.6. Gene synthesis and expression vector construction

[0046] (1) In this study, the primers used for PCR amplification and the plasmid pET-32a(+) were synthesized by GeneWeiZhi Biotechnology Co., Ltd. (Suzhou).

[0047] Table 1 PCR amplification primers

[0048]

[0049] (2) The expression vector was constructed as follows:

[0050] 1) Prepare the reaction system according to Table 2, and perform primer-bridge amplification of the target gene. The reaction procedure is: 98°C pre-denaturation for 5 minutes, 98°C denaturation for 10 seconds, 55°C annealing for 15 seconds, 72°C extension for 40 seconds, 30 cycles of amplification, and a final extension at 72°C for 5 minutes. Detect the PCR product by electrophoresis, then purify and recover the PCR product using a PCR purification kit, and perform DNA sequencing on the PCR product.

[0051] Table 2 PCR reaction system

[0052]

[0053] Note: The template AglB DNA was obtained by constructing a recombinant plasmid based on the AglB gene sequence and then following the DNA extraction process.

[0054] 2) Perform double enzyme digestion according to the reaction system in Table 3 and incubate at 37°C for 4 hours. After digestion, verify complete linearization by agarose gel electrophoresis and purify the linearized vector using a gel recovery kit.

[0055] Table 3 Double enzyme digestion reaction system

[0056]

[0057] 3) Ligate the target gene fragment and the pET-32a(+) digestion product after double enzyme digestion and linearization using T4 DNA ligase according to Table 4 (16°C overnight). After T4 DNA ligase ligates the target gene fragment and the vector, a recombinant plasmid containing the target gene fragment is obtained.

[0058] Table 4 T4 DNA ligase ligation reaction system

[0059]

[0060] 1.7 Preparation and transformation of E. coli competent cells BL21

[0061] (1) Remove the frozen glycerol tube of E. coli BL21 (DE3) from the refrigerator and place it on ice. Before the glycerol tube thaws, quickly remove a small amount of the bacterial mass from the top and add it to two 10 mL bottles of LB medium. Incubate the LB medium at 37°C overnight.

[0062] (2) Select a well-growing bacterial solution as a seed, inoculate 0.5 mL into 100 mL of LB medium, add 1 mL of 2M MgCl2 solution, and make two bottles in parallel. One bottle is used as a control for measuring OD. 600 and the other bottle is used to prepare competent cells.

[0063] (3) Cultivate with vigorous shaking at 37°C for 2-5 hours (37°C, 180-220 rpm). At the same time, pre-cool the flask containing CaCl2-MgCl2 solution (CaCl2 20mM, MgCl2 80mM) on ice. During the cultivation process, when turbidity is observed, remove the control bottle and quickly remove 1 mL in the clean bench to measure the OD value. When the OD value grows to 0.35-0.4, ice-bath the other bottle of culture and centrifuge it (4°C, 5000 rpm×10 min). Discard the supernatant and remove as much liquid as possible.

[0064] (4) Resuspend the E. coli pellet in 50 mL of CaCl2-MgCl2 solution and place the resuspended centrifuge tube on ice for 10 min. Centrifuge again (4°C, 5000 rpm for 10 min), discard the supernatant, and invert the centrifuge tube for 1 min (place one end of the tube on the clean bench and the other end on the centrifuge tube cap).

[0065] (5) Resuspend the cell pellet in 2 mL of ice-cold calcium chloride glycerol solution (15% glycerol, 50 mM CaCl2). Aliquot the calcium chloride solution into 1.5 mL centrifuge tubes pre-cooled at -20°C, 200 μL per tube, and store directly in a -80°C refrigerator.

[0066] 1.8. Plasmid transformation

[0067] Add 20 μL of sterile water to the bottom of the tube to dissolve the plasmid and let it stand at room temperature for 1 minute. Take 200 μL of competent cells and 5 μL of plasmid, mix them, and let them stand on ice for 30 minutes. Place them in a 42°C water bath and let them stand for heat shock for 90 seconds. Take out the centrifuge tube and ice bathe for 2 minutes, then add 800 μL of nutrient broth preheated at 37°C to the mixture. After incubation for 1 hour, centrifuge to remove part of the culture medium, spread the resuspended bacteria on nutrient agar containing 50 μg / mL ampicillin, and culture at 37°C overnight. At the same time, use empty plasmid pET-32a(+) as a positive control and sterile water as a negative control. Other operation steps are the same as above. Pick a single colony on the resistance plate and transfer it to the corresponding resistance LB liquid culture medium and culture overnight.

[0068] 1.9. Target protein expression and purification

[0069] (1) Induced expression of enzyme protein: The preserved recombinant bacteria were inoculated into a nutrient broth containing 50 μg / mL ampicillin and cultured overnight on a shaking platform. The activated seed liquid was inoculated into a 500 mL conical flask containing 200 mL of resistance nutrient broth at a ratio of 2%, mixed thoroughly, and cultured on a shaking platform at 37°C and 180 rpm. During the culture process, the OD value of the bacterial solution was detected at 600 nm using a spectrophotometer. When the OD value of the bacterial solution reached about 0.5, the growth of E. coli was in the logarithmic growth phase. Then, 5 mL of IPTG inducer was added to make its final concentration in the culture medium reach 1 mM. The bacterial solution was cultured on a shaking platform at 30°C and 180 rpm for 4 h to induce the expression of the target protein gene in E. coli BL21.

[0070] The induced E. coli bacterial solution was centrifuged and collected to extract the crude enzyme solution. The steps were as follows: the induced expression bacterial solution was divided into disposable, pre-cooled 50mL centrifuge tubes, centrifuged at 4°C, 7000rpm for 5 minutes, discarded the supernatant, and the bacterial cells were collected. Then, 25mL of sterile distilled water was added to the centrifuge tubes, washed by pipetting, and centrifuged under the same conditions as above, and the supernatant was discarded. The bacterial cells obtained from each 100mL of E. coli bacterial solution were resuspended in 10mL of pre-cooled McIlvaine buffer (pH=6), and then the E. coli cells were ultrasonically disrupted in an ice bath at 55W for 2s / 2s for 10 minutes. After ultrasonic disruption, the 50mL centrifuge tubes were removed and centrifuged at 4°C, 9000rpm for 10 minutes. The supernatant obtained by centrifugation was then transferred to a clean centrifuge tube. This solution was the crude enzyme solution and was placed in a 4°C refrigerator for storage.

[0071] (2) Purification of enzyme protein by affinity chromatography: In order to accurately characterize the enzymatic properties, a relatively pure enzyme solution needs to be obtained. This study uses affinity chromatography for purification. First, prepare the binding buffer and the binding buffer. The binding buffer is: mix 19 mL of 200 mM NaH2PO4 solution and 81 mL of 200 mM Na2HPO4 solution, add 29.22 g of NaCl and 1.36 g of imidazole, first dissolve them in 700 mL of distilled water, adjust the pH to 7.4, and then make the volume to 1 L. The elution buffer is: mix 19 mL of 200 mM NaH2PO4 solution and 81 mL of 200 mM Na2HPO4 solution, add 29.22 g of NaCl and 34 g of imidazole, adjust the pH value (7.5) and make the volume to 1 L.

[0072] Filter the crude enzyme solution with a 0.22μm filter membrane and purify it with a HisTrap FF chromatography column. Use a syringe to draw 5mL of distilled water at a rate of 1mL / min to rinse the chromatography column, and then use a new syringe to draw 5mL of binding buffer to rinse the equilibrated chromatography column at the above rate. Use a new syringe to draw the filtered crude enzyme solution and load the sample at the above rate. Use a new syringe to draw binding buffer to wash the chromatography column after loading. Collect the washed sample in a 1.5mL centrifuge tube, 1mL per tube, and then measure the absorbance at 280nm with a microplate reader. When the absorbance value remains unchanged, start adding elution buffer with a syringe. Collect the eluted sample in a separate tube, 1mL per tube, and measure its enzyme activity according to the method in 1.10. When the reaction system turns yellow, it proves that the enzyme solution has hydrolytic activity. Collect and preserve the active filtrate.

[0073] The purified enzyme solution was concentrated using an ultrafiltration tube, the enzyme concentration was determined using a BCA protein concentration assay kit, and the enzyme solution was diluted to the same concentration of 0.1 mg / mL.

[0074] 1.10. Hydrolytic activity assay

[0075] The enzyme activity was determined using the p-nitrophenol method (Carrera-Silva, Eugenio Antonio, et al. "Athermostable α-galactosidase from Lactobacillus fermentum CRL722: genetic characterization and main properties." Current microbiology 53(2006):374-378.). This method uses p-nitrophenyl-α-D-galactopyranoside (pNPG) as a specific substrate, which is decomposed by enzyme catalysis to produce yellow p-nitrophenol. The absorbance of the reaction solution is measured at 405 nm to calculate the enzyme activity. At the same time, the method for determining the standard curve using the pNPG method is as follows: prepare p-nitrophenol solution with 0.1 M McIlvaine buffer to a concentration of 1 mM, then add different volumes of p-nitrophenol (pNP) and 0.1 M McIlvaine buffer according to Table 5, and then add 1500 μL of 1 M Na2CO3 solution to make the total reaction system volume 2 mL, measure the absorbance value, draw a standard curve, and calculate the enzyme activity based on the standard curve.

[0076] Table 5 p-Nitrophenol standard curve

[0077]

[0078] The enzyme activity assay method is as follows: 50 μL of 40 mM pNPG substrate and 140 μL of McIlvaine buffer are added to a 200 μL enzyme reaction system into a 1.5 mL centrifuge tube. 10 μL of enzyme solution is then added. The reaction is incubated in a 37°C water bath for 10 minutes, followed by the addition of 200 μL of Na₂CO₃ solution to terminate the reaction. The reaction solution is then added to a 96-well plate, and the absorbance at 405 nm is measured using a microplate reader to determine the amount of p-nitrophenol released, thereby calculating the enzyme activity. Enzyme activity units (U / mL) are defined as the amount of enzyme required to release 1 μmol of pNP from pNPG per minute in the reaction system.

[0079] 2. Experimental results

[0080] 2.1. AglB mutation site selection

[0081] (1) Amino acid sequence comparison analysis

[0082] Multiple sequence alignment can be used to analyze conserved regions, variable regions, and possible functional or structural features of proteins. In the BLAST comparison, sequences with a similarity greater than 40% were screened, and α-galactosidases with high length and sequence similarity to AglB from different sources were found. α-galactosidases from Lactobacillus hamsteri, Lactobacillus delbrueckii, and Streptococcus oralis were selected for sequence alignment. Figure 1 As shown, the amino acid residues marked completely in red are highly conserved, such as FVLDDGWFG from F367 to G375 and KFGLWPEMIS from K411 to S422. Sites with red letters indicate relative conservation, while sites with black letters indicate non-conservation. Amino acids that occur frequently at specific positions in similar sequences have a stronger impact on protein catalytic activity than amino acids that occur less frequently at that position. Among them, His203 is non-conserved. At position 203, only the amino acid from oral Streptococcus is tryptophan; all other sources are histidine. The imidazole ring of histidine can be protonated and deprotonated at physiological pH and is often involved in acid-base catalysis and metal ion coordination. Tryptophan, on the other hand, lacks this function, potentially leading to the failure of key catalytic reactions. Furthermore, tryptophan is relatively large, and its indole ring may introduce steric hindrances into the active site, affecting substrate binding and proper positioning.

[0083] (2) AglB structure simulation

[0084] The homology modeling of AglB was performed using SWISS-MODEL with the α-galactosidase of Lactobacillus plantarum (ID: 2XN0, 78.28% similarity) as a template. Figure 2 ) to obtain a 3D structural model of AglB. The enzyme is a homotetramer with four subunits (chains AD) arranged in a ring around a central axis of symmetry, exhibiting C4 symmetry. The tetramer is stabilized by a hydrophobic core and a network of intersubunit hydrogen bonds. The α-helix ratio is 20.8%, the β-sheet ratio is 28.6%, and the random coil ratio is 50.6%.

[0085] (3) Molecular docking

[0086] The structural model of AglB was processed using PyMol, and the three subunits in the tetramer were deleted, leaving only one subunit. The single chain of AglB was used to align the structure with 2XN0 ( Figure 3 ), RMSD is far below This indicates that the structures of the two proteins are highly similar, so the active centers of the two enzymes are in the same region. The single chain of AglB is used as the protein receptor for molecular docking, and the GridBox is set near the active pocket. The receptor molecule and the melibiose molecule are used for molecular docking via Autodock. The molecular docking conformation diagram is shown in the figure below. Figure 4 As shown. Figure 5 From the binding site map, we can see that the amino acid residues that play a key role in the binding of AglB and melibiose are His203, Trp340, Tyr344, Phe345, Asp370, Asp371, Asp380, Trp415, Arg447, Lys480, Asp482, Asn484, Cys530, Gly532, Gly533, Trp549, Asp552, and Glu608, among which the amino acid residues Asp370, Trp415, Lys480, and Asp552 have hydrogen bonds with melibiose. At the same time, Trp340, Asp370, Asp371, Trp415, Lys480, Asp482, Cys530 and Asp552 are also key amino acid residues in the active center of 2XN0. However, Trp340 is highly conserved, the salt bridge of Cys530 can form multiple hydrogen bonds with the surrounding, and Asp552 is a key acid-base catalyst. Therefore, Asp370, Asp371, Trp415, Lys480 and Asp482 were selected for subsequent research. In addition, His203 is located at the entrance of the substrate binding pocket ( Figure 8 ). When melibiose binds to the receptor protein, the imidazole group on the histidine side chain may interact with melibiose to maintain protein functionality. Combined with the previous sequence alignment results, H203 is not conserved, so His203 was selected as the mutation site. Therefore, further analysis was conducted on six sites: His203, Asp370, Asp371, Trp415, Lys480, and Asp482.

[0087] The amino acid residues in the active center pocket are directly involved in the substrate binding and catalytic process. By changing the size, shape or polarity of the pocket, the substrate binding and product release can be optimized. The mutation strategy of this study is to mutate the amino acid residues around the active center pocket into short-chain amino acids, making the binding pocket larger and increasing the volume of the active pocket, so that melibiose can more easily enter and contact the active site. When selecting amino acid residues to replace, amino acids with similar properties to the original site are selected to better maintain the stability of the protein and improve the predictability of the mutation. Among them, Asp370 and Asp371 can be conservatively mutated into aspartic acid (Asn, N), which has more similar chemical properties. Aspartic acid has an additional amide group that can form more hydrogen bonds with other amino acids or molecules. This may help stabilize the secondary and tertiary structures of the protein, or enhance the binding of the protein to ligands and substrates; Trp415 can mutate to tyrosine (Tyr, Y) or phenylalanine (Phe, F). Tyrosine, phenylalanine and tryptophan are all aromatic amino acids. The mutation of tryptophan to tyrosine introduces a hydroxyl group, and the side chains of tyrosine and phenylalanine are smaller than those of tryptophan; Lys480 can mutate to arginine (Arg, R) or a short chain of glycine (Gly, G). Lysine and arginine are both positively charged basic amino acids, so their chemical properties are very similar in many aspects. The side chain of arginine contains multiple amino groups, making it stronger than lysine in forming ionic bonds, hydrogen bonds and interacting with other molecules; Asp482 can mutate to aspartic acid; His203 can mutate to a short chain of proline (Pro, P). Proline is rigid, making the pocket larger and making the protein more stable. After analyzing the above mutation strategy, eight mutants were finally obtained, including H203P, D370N, D371N, W415Y, W415F, K480R, K480G and D482N.

[0088] (4) Free energy calculation results

[0089] Free energy calculations are primarily used to assess and predict protein stability and the free energy changes associated with mutations. By calculating the free energy changes of a protein under different states, they can aid in studying the effects of mutations on its structure, biological activity, and stability. FoldX software is currently commonly used for energy calculations. Alanine scanning is a commonly used mutagenesis technique primarily used to study protein structure and function. This method systematically replaces amino acid residues in a protein with alanine residues to observe the effects of these mutations on protein function, structure, or stability. In this study, alanine virtual scanning was performed on His203, Asp370, Asp371, Trp415, Lys480, and Asp482 of AglB. The results are shown in Table 6. Higher energy values indicate greater protein structure instability, with W415 being the most destabilizing, followed by D370. This suggests that mutations at these two sites may significantly alter the protein's structure. The free energy changes at other sites are all below 2 kcal / mol, suggesting relatively minor effects on enzyme structure. Therefore, it is inferred that mutations in W415 and D370 may cause significant changes in the enzymatic activity of AglB.

[0090] Table 6 Alanine scanning results

[0091]

[0092] The Position Scan function performs mutation scans on the amino acid at a specified position, mutating it one by one to all other 19 naturally occurring amino acids and then calculating the free energy change (ΔΔG) for each mutant. This helps assess which mutations increase or decrease protein stability. The scan results for H203P, D370N, D371N, W415Y, W415F, K480R, K480G, and D482N are shown in Table 7. Compared with D370N and D371N, D370N has a smaller free energy change. Compared with W415F, W415Y has a smaller free energy change. Among K480G, K480R, and D482N, K480R has a smaller free energy change. The free energy after H203P mutation is -1.14 Kcal / mol, indicating that this mutation has little effect on the protein. Finally, after summary and analysis, H203P, D370N, W415Y, and K480R were selected for site-directed mutagenesis.

[0093] Table 7 Position scanning results

[0094]

[0095] 2.2 Expression and purification of AglB mutants

[0096] The constructed plasmid was transformed into E. coli BL21 (DE3) competent cells, and the expression of the target protein was induced by IPTG. E. coli BL21 without plasmid was used as blank control group, and then the crude enzyme solution was extracted by ultrasonic disruption and refrigerated centrifugation. Under the conditions of 37℃ and pH 6, the crude enzyme solution was preliminarily screened for the activity of mutants using the 1.10 method. The results are as follows Figure 6 As shown in the figure, the reaction solutions of the wild-type, H203P, W415Y, and K480R enzymes turned yellow, while those of the blank control and D370N enzymes remained colorless. This indicates that the wild-type, H203P, W415Y, and K480R enzymes possess hydrolytic activity, breaking down pNPG into yellow pNP, while the blank control and D370N enzymes lacked enzymatic activity.

[0097] 2.3 Hydrolytic activity of AglB mutants

[0098] The purified enzyme was concentrated using a centrifugal ultrafiltration tube, and its protein concentration was determined using a BCA protein quantification kit and diluted to 0.1 mg / mL for subsequent experiments. Figure 7The hydrolysis activities of wild-type and mutant AglB using pNPG as a substrate at 37°C and pH 6 are shown. Only the hydrolysis activity of H203P increased by 2.83-fold, reaching a specific activity of 362.95 U / mg. The hydrolysis activities of W415Y and K480R decreased, losing 62.73% and 90.37% of their hydrolysis activities, respectively. The crude enzyme D370N was found to have lost its activity when assayed. Moreover, the specific activity of H203P is higher than that of α-galactosidase from most sources, such as α-galactosidase from Thermoclostridium stercorarium subsp. thermolacticum, with a specific activity of 9 U / mg, YZ1 from Aspergillus oryzae, with a specific activity of 76.9 U / mg, AglB from Rhizomucor miehei, with a specific activity of 198 U / mg (Katrolia, Priti, et al. "Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides." Bioresourcetechnology 110(2012):578-586.), and AglB from Gibberella, with a specific activity of 48.33 U / mg (Cao YaNan, CaoYaNan, et al. "A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp.F75:gene cloning, expression, and enzymatic characterization." (2009): 875-884.). A possible reason for this is that when the histidine at position 203 is mutated to proline, a cyclic side chain is introduced near the active pocket. This rigid side chain may limit the flexibility of the enzyme, improving its stability and thus promoting substrate-enzyme binding. In W415Y, the tryptophan side chain contains a large, hydrophobic aromatic ring, which promotes catalysis through hydrophobic interactions with the substrate. Although tyrosine is also an aromatic amino acid, the -OH group on its side chain is more polar than the -NH group on tryptophan, which may reduce the hydrophobicity of the active pocket and, in turn, affect the binding of the melibiose substrate to the enzyme.In K480R, both lysine and arginine contain -NH2 side chains, but the lysine side chain is relatively short and flexible, while the arginine side chain contains a larger guanidine group (-C(=NH)NH2) with a nitrogen atom. This larger guanidine group takes up more space, potentially causing steric crowding within the active pocket, hindering substrate access to the active site or correct positioning, thereby reducing the catalytic efficiency of the enzyme. Furthermore, the loss of hydrolytic activity in D370N may be due to the following: D370 is a highly conserved site located in the active center, potentially a key active site. After the mutation, the -NH2 side chain replaces the carboxyl group, altering the acid-base properties of the active site and preventing substrate binding.

[0099] 2.4 Mutant structure modeling and analysis

[0100] Homology modeling was performed on the mutants H203P, D370N, W415Y and K480R, and the surface structure was compared with that of the wild type to observe the changes in the pocket before and after the mutation. Figure 9 As shown in the figure, the effect of structural changes on the activity of α-galactosidase was analyzed. Comparing the wild type and H203P, it was found that the structures of the two were significantly different. Since the 203 site is located at the entrance of the binding pocket, the histidine is mutated to proline, and the amino acid side chain becomes smaller, which Figure 9 The pocket in b becomes significantly more spacious, which may reduce the physical barrier for the substrate to enter the active site, promote the rapid binding of the substrate pNPG or melibiose, and may also make it easier for the product to be released from the enlarged pocket, thereby improving the hydrolysis activity. Figure 9 a) and D370N( Figure 9 c) W415Y( Figure 9 d) structure, it was found that D370N and W415Y were located in the inner part of the binding pocket, and the mutation had little effect on the enzyme structure, and there was no obvious change in the binding pocket. Figure 9 a) and K480R( Figure 9 Comparison of the structures of e) reveals a significantly shallower binding pocket. This is due to the large size of the guanidine group on the arginine side chain, which likely occupies the entrance or critical region of the substrate-binding pocket. This shallower pocket may restrict the ability of water molecules to enter the active center, severely impacting the hydrolysis reaction.

[0101] Gene sequence of mutant H203P (SEQ ID NO.11):

[0102]

[0103] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. An engineered enzyme for improving the hydrolytic activity of α-galactosidase, characterized in that: An engineered enzyme with enhanced hydrolysis activity is obtained by mutating the amino acid H at position 203 in the amino acid sequence of wild-type α-galactosidase to P. The amino acid sequence of the wild-type α-galactosidase is shown in SEQ ID NO.

2.

2. The engineered enzyme for improving the hydrolysis activity of α-galactosidase according to claim 1, characterized in that: The nucleotide sequence of the wild-type α-galactosidase is shown in SEQ ID NO.

1.

3. The engineered enzyme for improving the hydrolysis activity of α-galactosidase according to claim 1, characterized in that: The wild-type α-galactosidase is derived from Lactobacillus amyloliquefaciens L6, and the preservation number of the strain is CGMCC NO.9090.

4. A nucleotide sequence encoding the engineered enzyme according to any one of claims 1 to 3, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

11.

5. A recombinant expression vector expressing the engineered enzyme according to any one of claims 1 to 3, characterized in that: The recombinant expression vector carries the nucleotide sequence encoding the engineered enzyme according to claim 4.

6. The method for constructing the engineered enzyme according to any one of claims 1 to 3, characterized in that: First, a recombinant expression vector was constructed using the primer pair H203P-F / R of SEQ ID NO.3 and SEQ ID NO.4, and then the competent Escherichia coli cells BL21 were transformed. The engineered enzyme was then obtained after enzyme protein induction expression and enzyme protein affinity chromatography purification.

7. Use of the engineered enzyme according to any one of claims 1 to 3 in catalyzing the hydrolysis of melibiose.

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