Method for enhancing alpha-galactosidase oligosaccharide synthesis capability through site-directed mutagenesis and engineered enzyme

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for enhancing alpha-galactosidase oligosaccharide synthesis capacity through site-specific mutagenesis and an engineered enzyme. The mutant enzyme K480R for enhancing the oligosaccharide synthesis capability of the alpha-galactosidase is obtained by carrying out site-directed mutagenesis on the alpha-galactosidase from lactobacillus amyloliquefaciens L6 by utilizing a site-directed mutagenesis technology, has efficient and accurate catalytic capability, can efficiently convert melibiose into oligosaccharide, has the conversion rate of 34.30%, and can be used for preparing a mutant enzyme K480R for enhancing the oligosaccharide synthesis capability of the alpha-galactosidase. The activity of the alpha-galactosidase is higher than that of alpha-galactosidase derived from bacteria reported in China, side reactions are greatly reduced, and the production efficiency is improved. The synthesized oligosaccharide provides a new effective component for developing novel functional foods and medical products in the food field and the medical health care aspect. Therefore, the mutant enzyme has application potential in multiple industries such as food, medicine, cosmetics and the like, industrial innovation can be promoted, and considerable economic and social benefits can be created.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a method for enhancing the oligosaccharide synthesis ability of α-galactosidase by site-directed mutagenesis and an engineered enzyme. Background Art

[0002] Galactooligosaccharides (GOS) in prebiotics have obvious advantages over other functional oligosaccharides in promoting the proliferation of Bifidobacterium and Lactobacillus and inhibiting harmful intestinal bacteria. However, compared with isomaltooligosaccharides and fructooligosaccharides with a production scale of thousands of tons, the development of the galactooligosaccharide industry is relatively slow, and the lack of high-conversion enzyme preparations is one of the important reasons for the slow development of this industry.

[0003] The applicant previously isolated a strain of Lactobacillus amylolyticus L6 with good fermentation performance and probiotic functional characteristics (specifically see patent CN104130956B, and the preservation number of the strain is CGMCC NO.9090). This bacterium can secrete α-galactosidase (AglB) with oligosaccharide synthesis activity. After cloning and expressing this enzyme, it was found that the conversion rate of its catalyzing melibiose to synthesize oligosaccharides reached 31.56%, which is higher than that of most reported enzymes. However, due to the inherent catalytic characteristics of α-galactosidase AglB, while this enzyme catalyzes the synthesis of galactooligosaccharides from melibiose, it also catalyzes the hydrolysis of melibiose and oligosaccharides, resulting in restrictions on the application of this enzyme in synthesizing galactooligosaccharides. Therefore, the present invention uses homology modeling and molecular docking technologies to find amino acid sites related to oligosaccharide synthesis, hydrolysis, and substrate binding, and uses site-directed mutagenesis technology to mutate, replace, or introduce relevant amino acid sites, and develop key amino acid sites that affect the oligosaccharide synthesis and hydrolysis of AglB, in order to improve the oligosaccharide synthesis activity of AglB and the conversion rate of galactooligosaccharides, provide a theoretical basis for the production and application of this enzyme in galactooligosaccharides, and promote the rapid development of the prebiotic industry. Summary of the Invention

[0004] In order to overcome the above deficiencies of the prior art, the present invention modifies α-galactosidase (AglB) secreted from Lactobacillus amylolyticus L6 by site-directed mutagenesis to improve its oligosaccharide synthesis activity and the conversion rate of galactooligosaccharides. The mutant enzyme K480R obtained by molecular modification has a conversion rate of using melibiose to synthesize oligosaccharides reaching 34.30%, which is higher than the activity of α-galactosidases reported from bacteria in China, and greatly promotes the production and application of AglB enzyme.

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

[0006] The present invention provides an engineered enzyme with enhanced α-galactosidase oligosaccharide synthesis ability. By mutating the 480th amino acid K in the amino acid sequence of wild-type α-galactosidase (AglB) to R (i.e., lysine is mutated to arginine), an engineered enzyme of α-galactosidase based on point mutation is obtained; the amino acid sequence of the wild-type α-galactosidase is shown in SEQ ID NO.1.

[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 present invention also provides a nucleotide sequence encoding the engineered enzyme.

[0009] The present invention also provides a recombinant expression vector expressing the engineered enzyme, and the recombinant expression vector carries a nucleotide sequence encoding the engineered enzyme.

[0010] The present invention also provides the application of the engineered enzyme in catalyzing the synthesis of oligosaccharides from melibiose.

[0011] The present invention uses site-directed mutagenesis technology to perform molecular modification (site-directed mutagenesis) on α-galactosidase from Lactobacillus amylolyticus L6. The conversion rate of the mutant enzyme K480R for synthesizing oligosaccharides from melibiose reaches 34.30%. Among the α-galactosidases reported in China, its conversion rate is relatively high, higher than the conversion rate of α-galactosidase from Bacteroides fragilis, which is 32.4% (Gong Wei. Screening of transglycosylating α-galactosidase and enzymatic synthesis of Globotriose [D]. Shandong University, 2016.).

[0012] Preferably, the oligosaccharide is oligosaccharide trisaccharide.

[0013] The present invention also provides a method for synthesizing oligosaccharides from melibiose, specifically: using McIlvaine with pH = 6.0 as a buffer, adding 0.02 - 0.05 mg / mL of the engineered enzyme described in claim 1 or 2, and a reaction substrate of melibiose with a final concentration of 35 - 45%, w / v. After the reaction, oligosaccharides are obtained.

[0014] Preferably, the reaction time is 48h - 60h.

[0015] Preferably, the reaction temperature is 25 - 45 °C.

[0016] Preferably, the pH of the reaction system is 5 - 6.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The present invention uses site-directed mutagenesis technology to perform molecular modification (site-directed mutagenesis) on α-galactosidase derived from Lactobacillus amylolyticus L6, and obtains a mutant enzyme K480R with enhanced ability to synthesize oligosaccharides from α-galactosidase. This mutant enzyme has efficient and precise catalytic ability, can efficiently convert melibiose into oligosaccharides, and the conversion rate can reach 34.30%, which is higher than the activity of α-galactosidase derived from bacteria reported in China. It greatly reduces side reactions and improves production efficiency. At the same time, the synthesized oligosaccharides have novel structures and special biological activities. In the food field, they are expected to be used as prebiotics to specifically promote the growth and reproduction of beneficial intestinal flora and regulate the balance of intestinal microecology; in the aspect of medicine and health care, they have potential effects such as antioxidant and immunomodulatory effects, providing new active ingredients for the development of new functional foods and pharmaceutical products. It can be seen that this mutant enzyme has application potential in many industries such as food, medicine, and cosmetics, can promote industrial innovation, and create considerable economic and social benefits. Description of the Drawings

[0019] Figure 1 It is a homologous modeling structure diagram of AglB;

[0020] Figure 2 It is a docking conformation diagram of AglB and melibiose molecule;

[0021] Figure 3 It is a binding site diagram of AglB-melibiose;

[0022] Figure 4 It is the determination result of the enzyme activity of AglB and the crude enzyme solution of the mutant;

[0023] Figure 5 It is a liquid phase analysis diagram of the synthesis of oligosaccharides from melibiose catalyzed by K480R (a is at 0 h of the reaction, b is at 48 h);

[0024] Figure 6 It is the reaction time curve of the synthesis of oligosaccharides by AglB and the mutant in the synthesis of oligosaccharides;

[0025] Figure 7 It is the LC-MS spectrum of the synthesis of oligosaccharides from melibiose catalyzed by the mutant K480R (a is the HPLC spectrum, b, c, d, e, f are the mass spectra of peaks 1, 2, 3, 4, 5). Detailed Embodiments

[0026] The following further describes the detailed embodiments of the present invention. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are all commercially available through conventional channels unless otherwise specified.

[0028] In the following examples, the method for isolating the AglB enzyme from Lactobacillus amylolyticus L6 was referred to the paper: Fei Yongtao. Analysis of the microbial community during the acidification of soybean milk wastewater and the metabolic study of its key lactic acid bacteria [D]. South China University of Technology, 2018.

[0029] The amino acid sequence of the original enzyme AglB (SEQ ID NO.1) is shown as follows:

[0030] MNHELITFDQEQKVFHLHNDKISYLLAIEDGGTLSHLYFGKRVKNYHGQLRYPRRDRGFSGNLPGSLDRTFSRDSILKEYSTAGEMDYHTPAAIVRHPDGANALFLTYQSYRIEDGKPDLKGLPHAWVKNKNEAQTLIIRLEDKKSQLDFDLYYTIYRDRPVVTRSVQVLNNGQETVFLEKAASMQIDFADRNFEVITLPGAHANERHVEREKIGQGIHVYSSIRGTSSHQMNPFLALVDPETTENNGDAYGFSLVYSGNHKFEVERDQFNQIHLNIGINDFNFKWQLAPKAEFQTPEVLMVYSAHGLNQMSQTFHRLIHDRIMRSKFKDQLRPIVVNNWEATYFDFDEDKLRPIVDKAKDLGIEMFVLDDGWFGHRDDDNSSLGDWYVDHKKFPKGLGHFADYVHHQGLKFGLWFEPEMISYDSDLYRQHPDYLMHVPGRKPSPSRNQYLLDLGRKEVRDNIYEQMIKILDGGKIDYIK (mutation point to be introduced) WDMNRHLSDIYEADLPADRQGEAYHRYVLSYYDLLDRLVTRYPNILFEGCSGGGGRFD AGQAYYTPQIWASDNSDAIARLVIQYGTSLVYPQSMMTSHVSVSPNEQNGRITPFDTRGAVAMWGDLGYELDLTKLSAADSRKVAKQVSKYKKIRQITQYGKFYRLKSPMTGNQCAWMTVSPDQSEAVVTVVDIMAYAQPYCTKTKLMGLDPSKRYEDVATQEIFGGDELMNLGFYDPFANGDFQAKMYHFKAVD.

[0031] The gene sequence of the original enzyme AglB (SEQ ID NO.2) is shown as follows:

[0032]

[0033] Example: A method for enhancing the ability of α-galactosidase to synthesize oligosaccharides by site-directed mutagenesis

[0034] 1. Experimental method

[0035] 1.1. Structure modeling

[0036] SWISS-MODEL is a template-based protein structure prediction server that provides tools for predicting the three-dimensional structure of proteins by 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 uses advanced algorithms to provide users with high-quality structural models and can evaluate the quality of the models. Using the α-galactosidase of Lactobacillus acidophilus NCFM in the PDB library (PDB ID: 2XN0, sequence similarity 78.28%) as a template, homology modeling was performed through SWISS-MODEL: First, the AglB sequence was uploaded to the SWISS-MODEL platform, the template 2XN0 was manually specified and an automatic sequence alignment was performed. After verifying the alignment of key functional residues (such as catalytic sites), a three-dimensional model was constructed using the default parameters; after generating the model, it was saved as a PDB file and used for subsequent substrate binding or mutation site design studies.

[0037] 1.2. Molecular docking

[0038] Molecular docking is a computer simulation method used to predict the interaction modes and binding affinities between molecules (usually proteins and ligand molecules, such as small molecule drugs). Molecular docking is widely used in fields such as drug design, protein function research, and enzymology research. Its core goal is to find possible binding modes and predict the binding strength between them by simulating the binding process of the ligand and the receptor. Based on the PubChem database, the 3D structure model of melibiose (CID: 92845) was obtained, and AutoDock4.2.6 was used for the molecular docking of melibiose and the enzyme protein: First, the wild-type model of AglB (in PDB format) was processed by AutoDockTools to remove water molecules, add polar hydrogens, and Gasteiger charges, and saved as a PDBQT receptor file; after downloading the melibiose structure in SDF format from PubChem, it was converted to PDB format by AutoDock Tools, and the protonation state and charge distribution were optimized to generate a ligand PDBQT file; docking parameters were set in AutoDock (the grid center covers the active pocket), and the Lamarkian genetic algorithm was run (population number 150, energy evaluation 2.5 million times) to output the conformation with the optimal binding energy; finally, the docking results were loaded by PyMOL, the active pocket of AglB was displayed with surface electrostatic potential, the melibiose molecule was superimposed and hydrogen bonds were labeled ( Angles > 120°) and hydrophobic interactions to generate a high-resolution docking visualization image.

[0039] 1.3. FoldX Energy Calculation

[0040] Based on the AglB-melibiose docking complex structure, FoldX was integrated in YASARA for mutant energy calculation: First, the optimized PDB file after docking was imported into YASARA, and the built-in FoldX plugin was called to perform alanine scanning. Alanine mutants were generated for the 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; Subsequently, saturation mutagenesis of the selected residues was performed by ScanPosition, enumerating all 20 amino acid substitutions, and the protein stability (ΔΔG_fold) was calculated through the EnergyAnalysis module.

[0041] 1.4. Sequence Alignment

[0042] Based on the amino acid sequence of AglB, homologous α-galactosidases were aligned in the UniProt database using the NCBI BLAST tool, and sequences with similarity > 40% were screened. The α-galactosidases of Lactobacillus hamsteri, Lactobacillus delbrueckii, and Streptococcus oralis were selected for sequence alignment; The target sequences were imported into the CLUSTALW web page (https: / / www.genome.jp / tools-bin / clustalw), and the ClustalW algorithm was used for multiple sequence alignment to generate the.aln format result; Subsequently, the alignment file was uploaded to the ESPript 3.0 website to draw a visualization map and export a high-resolution PDF format image.

[0043] 1.5. Gene Synthesis and Construction of Expression Vectors

[0044] In this study, primers for PCR amplification were synthesized by GenScript Biotech Corporation (Suzhou), and the mutant sequences were ligated with pET-32a(+) to obtain recombinant vectors.

[0045] Table 1 PCR Amplification Primers

[0046]

[0047]

[0048] 1.6. Preparation and Transformation of Escherichia coli Competent Cells BL21

[0049] (1) Take out the glycerol tube of the E. coli BL21(DE3) strain stored frozen from the refrigerator and place it on ice. Before the glycerol tube thaws, quickly pick up a little of the bacterial mass at the top and add it to two 10-mL LB media bottles. Incubate the LB media overnight at 37°C.

[0050] (2) Select a well-growing bacterial solution as the seed, inoculate 0.5 mL into 100 mL of LB media, add 1 mL of 2M MgCl2 solution, and prepare two bottles in parallel. One bottle is used as a control specifically for measuring OD 600 , and the other bottle is used for preparing competent cells.

[0051] (3) After culturing vigorously at 37°C for 2 - 5 h (37°C, 180 - 220 rpm), place the Erlenmeyer flask containing the CaCl2-MgCl2 solution on ice for pre-cooling. During this period, the control bottle can be taken out after seeing turbidity, and 1 mL is quickly taken out in the laminar flow hood to measure the OD value. When it grows to 0.35 - 0.4, ice-bath the other bottle of culture and then centrifuge (4°C, 5000 rpm × 10 min), discard the supernatant, and try to remove the liquid as clean as possible.

[0052] (4) Resuspend the E. coli precipitate with 50 mL of CaCl2-MgCl2 solution, and place the resuspended centrifuge tube on ice for 10 min. Centrifuge (4°C, 5000 rpm × 10 min), discard the supernatant, and invert the centrifuge tube for 1 min (one end of the tube mouth is placed on the laminar flow hood, and the other end is placed on the centrifuge tube cap).

[0053] (5) Resuspend the cell precipitate with 2 mL of pre-cooled calcium chloride glycerol solution, dispense the calcium chloride solution into 1.5-mL centrifuge tubes pre-cooled at -20°C, 200 μL per tube, and directly store them in the -80°C refrigerator.

[0054] 1.6. Transformation of plasmid

[0055] Add 20 μL of sterile water to the bottom of the recombinant vector dry powder to dissolve the plasmid, and let it stand at room temperature for 1 min. Take 200 μL of competent cells and 5 μL of plasmid, mix them well, and place them on ice for 30 min. Put them into a 42°C water bath and let them stand for heat shock for 90 s. Take out the centrifuge tube and ice-bath it for 2 min, then add 800 μL of nutrient broth pre-warmed at 37°C to the mixture, culture for 1 h, then centrifuge to remove part of the medium, and spread the resuspended bacterial solution on nutrient agar containing 50 μg / mL ampicillin, and culture overnight at 37°C. At the same time, use the empty plasmid pET-32a(+) as the positive control and sterile water as the negative control, and the other operation steps are the same as above. Pick a single colony on the resistant plate into the corresponding resistant LB liquid medium and culture overnight.

[0056] 1.7. Expression and purification of the target protein

[0057] Induced expression of enzyme protein: The preserved recombinant bacteria were inoculated into nutrient broth containing 50 μg / mL ampicillin and cultured overnight on a shaker. The activated seed liquid was inoculated into a 500 mL conical flask containing 200 mL of resistant nutrient broth at a ratio of 2%, and after thorough mixing, it was cultured with shaking at 37 °C and 180 rpm. During the culture process, the OD value of the bacterial solution was measured at 600 nm using a spectrophotometer. When the OD value of the bacterial solution reached about 0.5, the growth of Escherichia coli was in the logarithmic growth phase, and then 5 mL of IPTG inducer was added to make its final concentration in the medium reach 1 mM. Then the bacterial solution was cultured with shaking at 30 °C and 180 rpm for 4 h to induce the expression of the target protein gene in Escherichia coli BL21.

[0058] The induced Escherichia coli bacterial solution was centrifuged and collected to extract the crude enzyme solution. The operation steps were as follows: The induced expression bacterial solution was aliquoted into disposable, pre-cooled 50 mL centrifuge tubes and centrifuged at 4 °C and 7000 rpm for 5 min. The supernatant was discarded, and the bacterial cells were collected. Then 25 mL of sterile distilled water was added to the centrifuge tube, pipetted and washed, and centrifuged under the same conditions as above. The supernatant was discarded. The bacterial cells obtained from every 100 mL of Escherichia coli bacterial solution were resuspended in 10 mL of pre-cooled McIlvaine buffer (pH = 6), and then the Escherichia coli cells were ultrasonically disrupted under ice bath conditions. The disruption parameters were 55 w, 2 s / 2 s, 10 min. After ultrasonic disruption, the 50 mL centrifuge tube was taken out and centrifuged at 4 °C and 9000 rpm for 10 min. Then the supernatant obtained by centrifugation was transferred to a clean centrifuge tube, and this solution was the crude enzyme solution, which was stored at 4 °C in the refrigerator for standby.

[0059] Purification of enzyme protein by affinity chromatography: In order to accurately characterize the enzymatic properties, a relatively pure enzyme solution was required. In this study, affinity chromatography was used for purification. First, the binding buffer and elution buffer were prepared. Among them, the binding buffer was prepared as follows: 19 mL of 200 mM NaH2PO4 solution and 81 mL of 200 mM Na2HPO4 solution were mixed. At the same time, 29.22 g of NaCl and 1.36 g of imidazole were weighed, first dissolved in 700 mL of distilled water, the pH was adjusted to 7.4, and then the volume was fixed to 1 L. The elution buffer was prepared as follows: 19 mL of 200 mM NaH2PO4 solution and 81 mL of 200 mM Na2HPO4 solution were mixed. At the same time, 29.22 g of NaCl and 34 g of imidazole were weighed, the pH was adjusted to 7.4 and the volume was fixed to 1 L.

[0060] After filtering the crude enzyme solution through a 0.22 μm filter membrane, it was purified using a HisTrap FF chromatography column: A syringe was used to aspirate 5 mL of distilled water and flush the chromatography column at a rate of 1 mL / min. Then, a new syringe was used to aspirate 5 mL of binding buffer and flush and equilibrate the chromatography column at the above rate. A new syringe was used to aspirate the filtered crude enzyme solution and load the sample at the above rate. A new syringe was used to aspirate the binding buffer to wash the impurities from the loaded chromatography column, and the washed samples were collected in 1.5 mL centrifuge tubes, 1 mL per tube. Then, the absorbance was measured at 280 nm using an enzyme-linked immunosorbent assay (ELISA) reader. When the absorbance value remained unchanged, the elution buffer was started to be added using a syringe. The eluted samples were collected in separate tubes, 1 mL per tube, and their enzyme activities were measured according to Method 1.8, and the active filtrates were collected and stored.

[0061] The purified enzyme solution was concentrated using an ultrafiltration tube, the concentration of the enzyme was measured using a BCA protein concentration assay kit, and the enzyme solution was diluted to the same concentration, i.e., 0.1 mg / mL.

[0062] 1.8. Research on the activity of oligosaccharide synthesis

[0063] The reaction system (4 mL) for measuring the oligosaccharide content was as follows: Melibiose at a final concentration of 40% (w / v) was used as the reaction substrate, the addition amount of the 0.1 mg / mL enzyme solution was 0.3 mL, McIlvaine was used as the buffer (pH = 6.0), the mixture was placed in a 50 mL centrifuge tube and transferred to a shaker for reaction. The reaction temperature was 37 °C, the rotation speed was 150 rpm, and samples of 350 μL were taken at time points of 0 h, 1 h, 12 h, 24 h, 36 h, 48 h, and 60 h, then inactivated at 95 °C for 10 min and stored in a -80 °C refrigerator for subsequent measurement.

[0064] The sugar components in the samples were measured using high-performance liquid chromatography (HPLC), and the pretreatment of the samples was referred to GB5009.8-2023. 350 μL of each type of sample was respectively aspirated into a 1.5 mL centrifuge tube, then 100 μL of 0.25 M potassium ferrocyanide and 100 μL of 1 M zinc acetate solution were added and shaken well, and then made up to 1 mL with distilled water. After standing for 30 min, the samples were centrifuged at 12000 rpm for 5 min, and the supernatant was collected. After filtering the supernatant through a 0.45 μm filter membrane, it was put into an injection vial for liquid phase determination. The GL Sciences Inertsil NH2 column used for HPLC determination had a specification of 4.6 mm × 250 mm, 5 μm, a differential refractive index detector was used, the flow rate was 1.0 mL / min, the mobile phase was 68% acetonitrile - 32% water, the analysis time was 20 min, the injection volume was 20 μL, and the column temperature was 35 °C.

[0065] 1.9. Structural identification of the synthesis product

[0066] The product structure was determined by liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF / MS) and Fourier transform infrared spectroscopy.

[0067] For liquid chromatography - mass spectrometry (LC-MS), the chromatographic column used was an Agilent NH2 column with a specification of 4.6 mm × 250 mm, 5 μm. The injection volume was 2 μL, the column temperature was 30 °C, the flow rate was 1.0 mL / min, the mobile phase was 75% acetonitrile - 25% water, the scanning mode was MS, the detection was carried out in the negative ion mode, the fragmentation voltage was 120 V, the electrospray ESI source was used, and the scanning range was 100 - 1000.

[0068] Fourier transform infrared spectroscopy was carried out using a centrifugal ultrafiltration tube with a 50 kDa MWCO. Centrifugation was performed at 5000 × g at room temperature for 10 min to retain the protein in the filter and collect the filtrate in the centrifuge tube. The mixed sample solution was placed in a dialysis bag (300 Da), and the dialysis bag was placed in an appropriate volume of deionized water for dialysis for two days. Then the sample solution was freeze-dried. About 2 mg of the sample was weighed, thoroughly mixed with 200 mg of KBr powder, ground with an agate mortar, and pressed into a light-transmitting thin slice using a tablet press. Finally, it was placed in an infrared spectrometer for infrared scanning in the range of 4000 - 400 cm -1 for infrared scanning.

[0069] 2. Experimental Results

[0070] 2.1. Structure Simulation and Molecular Docking of AglB

[0071] Using SWISS-MODEL, with the α-galactosidase of Lactobacillus plantarum (ID: 2XN0, 78.28% similarity) as a template, homology modeling of AglB was carried out as Figure 1 shown, and a 3D structural model of AglB was obtained. The enzyme was confirmed to be a homotetramer, and its four subunits (chains A - D) were arranged in a ring around the central symmetry axis, with C4 symmetry. The stability of the tetramer was maintained by a hydrophobic core and an inter-subunit hydrogen bond network. Among them, the proportion of α-helix was 20.8%, the proportion of β-sheet was 28.6%, and the proportion of random coil was 50.6%.

[0072] In the α-galactosidase 2XN0 from NCFM, which is highly similar to AglB, residues Trp340, Asp370, Asp371, Trp415, Lys480, Asp482, Cys530, and Asp552 form a conserved semi-pocket that is the active center of the enzyme. Among them, Trp340 is highly conserved. The salt bridge of Cys530 can form multiple hydrogen bonds with surrounding residues. Asp552 is a key acid-base catalyst, and this pocket is selected as the active center of AglB.

[0073] The structural model of AglB was processed using PyMol. Three subunits in the tetramer were deleted, and only one subunit was retained as the protein receptor for molecular docking. The Grid Box was set near the active pocket. Molecular docking was performed using this receptor molecule and the melibiose molecule through Autodock. The conformational diagram of the molecular docking is as Figure 2 shown. As can be seen from Figure 2 , the amino acid residues that play a key role in the binding of AglB and melibiose are Hip203, Trp340, Tyr344, Phe345, Asp370, Asp371, Asp380, Trp415, Arg447, Lys480, Asp482, Asn484, Cys530, Gly532, Gly533, Trp549, Asp552, Glh608 (Hip203 is a protonated state of histidine His, and Glh608 is a protonated state of glutamate Glu). In addition, there are numerous hydroxyl groups in the melibiose structure, which have hydrogen bond interactions with the amino acid residues Asp370, Trp415, Lys480, and Asp552 of α-galactosidase.

[0074] The mutation strategy is to mutate the amino acid residues around the active center pocket into short-chain amino acids, which enlarges the binding pocket and increases the volume of the active pocket, enabling melibiose to more easily enter and contact the active site. In the binding pocket of α-galactosidase, the sites of W340, C530, and D552 are highly conserved. Mutations at these sites may disrupt the functionality of the protein and have a significant negative impact on the enzyme structure, leading to enzyme inactivation. Therefore, D370, D371, W415, K480, and D482 are selected as the mutation sites. When selecting the amino acid residues for substitution, those with similar properties to the original site amino acids are usually chosen to better maintain the stability of the protein and improve the predictability of the mutation. Among them, D370 and D371 can be conservatively mutated to asparagine (Asn, N), which has a similar chemical property. N 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; W415 is mutated to tyrosine (Tyr, Y) or phenylalanine (Phe, F). Tyrosine and phenylalanine are aromatic amino acids like tryptophan. Mutating W to Y introduces a hydroxyl group, and the side chains of Y and F are smaller than that of W; K480 can be mutated to arginine (Arg, R). Both K and R are positively charged basic amino acids, so they are very similar in chemical properties in many aspects. The side chain of R contains multiple amino groups, making it stronger than K in forming ionic bonds, hydrogen bonds, and interactions with other molecules, or it can be mutated to short-chain glycine (Gly, G). D482 can be mutated to N. In the docking results ( Figure 3 ), it can be seen that H203 is located at the entrance of the substrate binding pocket. When melibiose binds to the receptor protein, the imidazole group on the histidine side chain may interact with melibiose to maintain the functional state of the protein. Therefore, H203 is selected as the mutation site. H203 can be mutated to short-chain proline (Pro, P), and P is rigid, making the pocket larger while making the protein more stable. In summary, a total of eight mutants, namely H203P, D370N, D371N, W415Y, W415F, K480R, K480G, and D482, are obtained.

[0075] 2.2, Expression and purification of AglB mutants

[0076] The constructed plasmid is transformed into Escherichia coli BL21(DE3) competent cells. IPTG is used to induce the expression of the target protein, and Escherichia coli BL21 without the plasmid is used as a blank control group. Then, the crude enzyme solution is extracted through ultrasonic disruption and freeze centrifugation. Under the conditions of 37 °C and pH 6, the crude enzyme solution is preliminarily screened for the enzyme activity of the mutants. The results are as Figure 4As shown, the reaction solutions of wild type, H203P, W415Y and K480R turned yellow, while the reaction solutions of the blank control group and D370N were colorless. This indicates that wild type, H203P, W415Y and K480R have hydrolysis activity and can decompose pNPG into yellow pNP, while the blank control group and D370N do not have enzyme activity.

[0077] 2.3. Study on the oligosaccharide synthesis activity of AglB mutants

[0078] Previous studies have reported that α-galactosidases from various sources not only have hydrolysis activity but also have oligosaccharide synthesis function at high substrate concentrations. Using melibiose as the substrate, α-galactosidase can decompose it into a glucose molecule and an intermediate with galactose, and transfer the galactosyl group to glucose to form a new glycosidic bond, generating a new oligosaccharide or oligosaccharide synthesis product. Using equal amounts of wild type enzyme and mutants (0.1 mg / mL), the oligosaccharide synthesis activity of the mutants was explored at 37 °C, pH 6, and melibiose concentration of 40% (w / v). Figure 5 Figure 8 is the liquid chromatogram of the K480R reaction system at 0 h and 48 h. It can be clearly seen from it the increase and decrease of each component. The peaks of glucose and galactose are at 5.9 min. The two peaks (glucose at 5.9 min and galactose at 6.1 min) are too close, and because many galactose residues are transferred into melibiose, the galactose peak is very small, resulting in inability to separate them; the peak of melibiose is at 8.2 min; the peak of oligosaccharide is at 11.3 min. By comparing 0 h and 12 h, it can be seen the increase of glucose-galactose components and oligosaccharide components and the decrease of melibiose component. Therefore, by comparing the peak areas of oligosaccharides, the oligosaccharide synthesis activity of the mutants can be analyzed.

[0079] Figure 6The effects of wild-type AglB and mutants on the synthesis activity of oligosaccharides were shown. The peak area of oligosaccharides of K480R reached the highest value at 48 h and then stabilized at 60 h. The peak area of oligosaccharides of W415Y showed an upward trend within 60 h. The oligosaccharide synthesis reaction of H203P reached the highest value at 24 h but started to decline after reaching the highest value. The wild-type oligosaccharide area reached the highest value at 36 h and then showed a stable and slightly decreasing trend. Compared with the wild type, the oligosaccharide synthesis activity of K480R was significantly improved. Combining with the hydrolysis activity of this mutant, it indicated that after mutating lysine at position 480 to arginine, the activity of the enzyme hydrolyzing the oligosaccharide synthesis product was reduced, thus improving the oligosaccharide synthesis activity of the enzyme. The decrease in the oligosaccharide peak area in the H203P reaction system was because the hydrolysis activity of H203P was increased, and the oligosaccharides produced by the oligosaccharide synthesis reaction were hydrolyzed again. According to the calculation of the consumption of melibiose ((total consumption of melibiose - hydrolysis amount of melibiose) / initial amount of melibiose = conversion rate of the melibiose oligosaccharide synthesis reaction), it was obtained that at 48 h, the conversion rate of the maximum oligosaccharide synthesis activity of K480R was 34.30%, and that of the wild type was 11.70%, an increase of 22.60%. The functional focus of the enzyme was shifted from hydrolysis to oligosaccharide synthesis, realizing the inversion of catalytic preference.

[0080] 2.4. Identification of oligosaccharide structure:

[0081] To deeply explore the molecular weight and structural characteristics of oligosaccharides, in this study, liquid chromatography-mass spectrometry and Fourier transform infrared spectroscopy were used to analyze the oligosaccharides synthesized in the K480R reaction system at 48 h. The results are as Figure 7 shown in a. Five peaks appeared in the HPLC chromatogram, and mass spectrometry detection was performed on the five peaks. By performing mass spectrometry detection on peak 1 ( Figure 7 b), a monosaccharide M+113 (CF-3CO2 anion) with m / z of 293 was detected, and the relative intensity was 36.64%. It was speculated to be the peak of glucose and galactose. By performing mass spectrometry detection on peaks 2 and 3 ( Figure 7 c and d), a disaccharide M+113 (CF-3CO2 anion) with m / z of 455 was detected in both, and the relative intensities were 22.88% and 267.83% respectively, indicating that it was the peak of melibiose. By performing mass spectrometry detection on peaks 4 and 5 ( Figure 7 e), an oligosaccharide trisaccharide M+113 (CF-3CO2 anion) with m / z of 617 was detected in both, and the relative intensities were 31.12% and 152.68% respectively ( Figure 7 f). From the above mass spectrometry results analysis, it can be seen that K480R synthesized two kinds of oligosaccharide trisaccharides using melibiose.

[0082] The amino acid sequence of K480R is shown as follows:

[0083] MNHELITFDQEQKVFHLHNDKISYLLAIEDGGTLSHLYFGKRVKNYHGQLRYPRRDRGFSGNLPGSLDRTFSRDSILKEYSTAGEMDYHTPAAIVRHPDGANALFLTYQSYRIEDGKPDLKGLPHAWVKNKNEAQTLIIRLEDKKSQLDFDLYYTIYRDRPVVTRSVQVLNNGQETVFLEKAASMQIDFADRNFEVITLPGAHANERHVEREKIGQGIHVYSSIRGTSSHQMNPFLALVDPETTENNGDAYGFSLVYSGNHKFEVERDQFNQIHLNIGINDFNFKWQLAPKAEFQTPEVLMVYSAHGLNQMSQTFHRLIHDRIMRSKFKDQLRPIVVNNWEATYFDFDEDKLRPIVDKAKDLGIEMFVLDDGWFGHRDDDNSSLGDWYVDHKKFPKGLGHFADYVHHQGLKFGLWFEPEMISYDSDLYRQHPDYLMHVPGRKPSPSRNQYLLDLGRKEVRDNIYEQMIKILDGGKIDYIR(Mutation point)WDMNRHLSDIYEADLPADRQGEAYHRYVLSYYDLLDRLVTRYPNILFEGCSGGGGRFD AGQAYYTPQIWASDNSDAIARLVIQYGTSLVYPQSMMTSHVSVSPNEQNGRITPFDTRGAVAMWGDLGYELDLTKLSAADSRKVAKQVSKYKKIRQITQYGKFYRLKSPMTGNQCAWMTVSPDQSEAVVTVVDIMAYAQPYCTKTKLMGLDPSKRYEDVATQEIFGGDELMNLGFYDPFANGDFQAKMYHFKAVD。

[0084] The gene sequence of mutant enzyme K480R is as follows:

[0085]

[0086] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. An engineered enzyme for enhancing the oligosaccharide synthesis ability of α-galactosidase, characterized in that: The 480th amino acid K in the amino acid sequence of wild-type α-galactosidase is mutated to R, thereby obtaining an engineered α-galactosidase enzyme based on point mutation; the amino acid sequence of the wild-type α-galactosidase is shown in SEQ ID NO.

1.

2. The engineered enzyme for enhancing the oligosaccharide synthesis ability 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.

3. A nucleotide sequence encoding the engineered enzyme according to claim 1 or 2.

4. A recombinant expression vector expressing the engineered enzyme according to claim 1 or 2, characterized in that: The recombinant expression vector carries a nucleotide sequence encoding the engineered enzyme according to claim 1 or 2.

5. Use of the engineered enzyme according to claim 1 or 2 in catalyzing the synthesis of oligosaccharides from melibiose.

6. The use according to claim 5, characterized in that: The oligosaccharide is oligotrisaccharide.

7. A method for synthesizing oligosaccharides using melibiose, characterized in that: McIlvaine at pH=6.0 is used as a buffer, 0.02-0.05 mg / mL of the engineered enzyme according to claim 1 or 2 and a melibiose reaction substrate at a final concentration of 35-45%, w / v are added, and oligosaccharides are obtained after reaction.

8. A method for synthesizing oligosaccharides using melibiose according to claim 7, characterized in that: The reaction time is 48h-60h.

9. The method for synthesizing oligosaccharides using melibiose according to claim 7, characterized in that: The reaction temperature is 25-45°C.

10. The method for synthesizing oligosaccharides using melibiose according to claim 7, characterized in that: The pH of the reaction system is 5-6.

Images