A β-glucosidase mutant and its application in hydrolyzing malonylgenistin

Abstract

The present invention discloses a β-glucosidase mutant and its application in the hydrolysis of malonyl genistin. The present invention is based on the β-glucosidase from uncultured marine microorganisms, and is designed and modified through homology modeling and molecular docking to obtain a mutant enzyme with improved efficiency and stability in hydrolyzing malonyl genistin. Under the conditions of the same mass of enzyme protein, the ratio of the mutant enzyme to hydrolyze malonyl genistin is 1.5 times higher than that of the starting enzyme; under the conditions of 30-45°C, the half-life of the mutant enzyme is 3.7 times higher than that of the starting enzyme. The mutant has potential application value in the hydrolysis of soybean isoflavones to produce aglycones.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a beta-glucosidase mutant and application thereof in hydrolyzing malonyl genistin. Background Art

[0002] β-Glucosidase (EC 3.2.1.21) belongs to the class of glycoside hydrolases. First discovered in bitter almonds in the 1830s by Liebig and Wohler, it hydrolyzes the β-D-glycosidic bond of terminal, non-reducing alkyl-β-glucosides or aromatic-β-glucosides, releasing glucose and the corresponding ligand. β-Glucosidase is active against a wide range of substrates and plays a vital role in industries such as biofuel production, food processing, and pharmaceuticals.

[0003] Soy isoflavones, a mixture of polyphenols, play an important role in the prevention and treatment of cancer, cardiovascular disease, and osteoporosis. Natural isoflavones exist in 12 monomeric forms: glycosides and aglycones. Aglycones are far more active than glycosides and are readily absorbed by the body. Therefore, converting isoflavone glycosides into aglycones to increase the content of isoflavone aglycones in soy products has attracted considerable interest. Malonyl genistin is a soy isoflavone in which the 6'-hydroxyl group of genistin is substituted with a malonyl group. Malonyl glycosides are the predominant form of soy isoflavones reported in soy flour, accounting for approximately 70% of the total soy isoflavone content. Soybean slurry contains soy isoflavones, of which malonyl genistin accounts for approximately 40%. β-glucosidase can hydrolyze soy isoflavones to aglycones. However, most β-glucosidases are strictly specific for the sugar groups of soy isoflavones and are inactive against malonyl glucosides. Therefore, β-glucosidase capable of hydrolyzing malonyl genistin was obtained through design and modification, and used for hydrolyzing soybean isoflavones in soybean yellow slurry, thereby avoiding waste of resources. Summary of the Invention

[0004] The present invention provides a β-glucosidase mutant and its application in the hydrolysis of malonylgenistin. Based on a semi-rationally engineered mutant of a β-glucosidase derived from an uncultured marine microorganism, the invention designed the mutant through homology modeling and molecular docking. The mutant was recombinantly expressed and purified in Escherichia coli, followed by characterization and screening. The resulting β-glucosidase exhibited improved efficiency and stability in the hydrolysis of malonylgenistin. Using pNPG as a substrate, the mutant's stability was measured to be 3.7-fold higher. In a soybean isoflavone conversion experiment, 8 mg of the mutant enzyme hydrolyzed malonylgenistin in yellow slurry by approximately 88% when added to a 35°C reaction system and hydrolyzed for 30 minutes. The hydrolysis rate of the original enzyme with the same protein mass under the same reaction conditions was only 35%. These results demonstrate the potential application of the mutant in the hydrolysis of soybean isoflavones to produce aglycones.

[0005] The amino acid sequence of the β-glucosidase mutant of the present invention is shown in SEQ ID NO: 1. In the amino acid sequence of the β-glucosidase mutant, the glutamine at position 301 is mutated to alanine.

[0006] The nucleotide sequence of the gene encoding the β-glucosidase mutant is shown in SEQ ID NO: 2.

[0007] The strain expressing the beta-glucosidase mutant contains the mutant plasmid.

[0008] The strain expressing the β-glucosidase mutant of the present invention is classified and named Escherichia coli BL21(DE3) / p ET22b(+)-bgl2A:Q301A, has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with a preservation number of CCTCC NO: M 20241888, a preservation date of September 2, 2024, and a preservation address of Wuhan University, Wuhan, China.

[0009] The method for constructing a strain expressing a β-glucosidase mutant of the present invention comprises the following steps:

[0010] First, using the structure of Agrobacterium tumefaciens β-glucosidase SghA (PDB code: 6rjm) as a template, homology modeling of the β-glucosidase Bgl2A structure was performed using Swiss-Model. The receptor macromolecule (enzyme protein) was preprocessed using Autodock software through steps such as hydrogenation, charge calculation, and atom type addition, and then exported to the pdbqt format for future use. The ligand small molecule malonylgenistin was downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) and preprocessed using Autodock software through steps such as charge adjustment and selection of ligand-twistable bonds, and then exported to the pdbqt format for future use. Molecular docking calculations were performed using Autodock vina, and the docking results were analyzed using Pymol. Docking has 19 kinds of conformations, according to the glucose molecule on the malonyl genistin molecule in substrate channel towards, docking result is divided into two classes, the one is the glucose molecule on the malonyl genistin molecule towards substrate channel inside, the one is the glucose molecule on the malonyl genistin molecule towards the outside of substrate channel.By the distance analysis of binding energy and the glycosidic bond of malonyl genistin from protein catalytic residues (E170, E352), it is preliminarily speculated that glycosidase is when hydrolyzing malonyl genistin, and glucose molecule is towards substrate channel inside.Therefore glucose molecule is analyzed towards six kinds of conformations inside substrate channel, and the amino acid with malonyl group hydrogen bond interaction is selected.Wherein comprising Bgl2A:Q301, it is mutated into the amino acid that side chain is less i.e. alanine, purpose makes the substrate channel of protein become larger, can accommodate larger malonylation glucose group, and then improves the activity of glycosidase to malonyl substrates.

[0011] According to the gene sequence of β-glucosidase Bgl2A, mutation primers were designed and synthesized (Table 1). The recombinant plasmid containing the β-glucosidase Bgl2A gene was used as a template and the above-mentioned synthetic mutation primers were used as primers to perform site-directed mutagenesis based on the overlap extension PCR method ( Figure 1 and Figure 2 ).

[0012] The mutant gene is ligated with the pEASY-T3 plasmid and transformed into Escherichia coli Trans1-T1 competent cells. Positive clones are selected and the mutant gene is subjected to DNA sequencing. A clone with the correct sequence is selected and a plasmid is extracted to obtain a pEASY-T3 recombinant plasmid containing the mutant β-glucosidase gene. The pEASY-T3 recombinant plasmid and the expression plasmid vector are double-digested with Nde I and Xho I, and then the digested mutant gene and the expression plasmid vector are ligated with T4 DNA ligase to obtain a ligation product. The ligation product is then transformed into a host bacterium, and positive clones are screened to obtain an engineered strain containing the mutant gene of the present invention.

[0013] The expression plasmid vectors described in the above construction method include pCold, pET15, pET22 or pET28, etc.

[0014] The host bacteria in the above construction method include E. coli BL21 (DE3), E. coli DH5α, E. coli JM109 or E. coli Rosetta, etc.

[0015] The β-glucosidase mutant of the present invention can be obtained by fermentation, expression and purification of the engineered strain ( Figure 3 ).

[0016] The invention relates to an application of the beta-glucosidase mutant in the hydrolysis of malonyl genistin.

[0017] The present invention measured and compared the optimal pH, optimal temperature, and stability of the mutant protein and the original starting enzyme protein. The stability of the mutant was improved. At 30-35°C and pH 7.0-7.5, the half-life of the starting enzyme was 1.5 hours, and the half-life of the mutant Bgl2A:Q301A was 7 hours. The stability of the mutant was increased by 3.7 times, and the mutation did not cause changes in the optimal temperature and optimal pH. Figure 4 ).

[0018] In the soy isoflavone hydrolysis experiment, adding 8 mg of the mutant enzyme to the reaction system at 35°C and hydrolyzing for 30 minutes can hydrolyze malonyl genistin in soybean yellow slurry to about 88%, which is 1.5 times higher than the hydrolysis rate of the original enzyme with the same protein amount under the same reaction conditions (35%). Figure 5 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 、 Figure 2 The electrophoretic pattern of the PCR amplification product of the present invention is: Figure 1 The lanes in the middle are DNA marker, PCR amplified fragments Q301A-S, and Q301A-X, respectively; Figure 2 The lanes in the middle are the PCR amplification products using DNA marker, Q301A-S and Q301A-X as templates.

[0020] Figure 3 Figure 3 is the SDS-PAGE profile of the purified mutant protein and the starting enzyme Bgl2A: 1 is the supernatant of Bgl2A fragmentation, 2 is the fragmentation precipitate, 3 is the pure Bgl2A enzyme, 4 is the supernatant of Q301A fragmentation, 5 is the fragmentation precipitate of Q301A, 6 is the pure Q301A enzyme, and M is a protein marker.

[0021] Figure 4In the figure, a is the optimum temperature measurement result, b is the optimum pH measurement result, and c is the stability measurement result.

[0022] Figure 5 The hydrolysis rates of malonylgenistin by the starting enzyme and mutants are shown in Figure 5. DETAILED DESCRIPTION

[0023] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0024] (1) Construction of an expression strain containing the β-glucosidase mutant gene of the present invention

[0025] 1. Selection of β-glucosidase gene mutation sites

[0026] Based on sequence alignment, Bgl2A is most similar to the β-glucosidase SghA (PDB code: 6rjm) from Agrobacterium tumefaciens A6, with an amino acid sequence identity of 44%. Using the SghA structure as a template, homology modeling of the β-glucosidase Bgl2A was performed using Swiss-Model (http: / / swissmodel.expasy.org / ).

[0027] The receptor macromolecule (enzyme protein) was preprocessed using Autodock software through steps such as hydrogenation, charge calculation, and atom type addition, and then exported to pdbqt format for future use.

[0028] The ligand was downloaded from the NCBI website (https: / / pubchem.ncbi.nlm.nih.gov / ). The ligand was pre-processed using Autodock software by adjusting the charge and selecting ligand-twistable bonds. The ligand was then exported to the pdbqt format for future use. Molecular docking calculations were performed using Autodockvina, and the results were analyzed using Pymol. Nineteen conformations were identified, and the docking results were categorized into two groups based on the orientation of the glucose molecule on the malonyl genistin molecule within the substrate channel: one in which the glucose molecule faces the interior of the substrate channel and one in which the glucose molecule faces the exterior of the substrate channel. Analysis of binding energies and the distance between the glycosidic bond of malonyl genistin and the catalytic residues of the protein led to the hypothesis that the glucose molecule faces the interior of the substrate channel during glycosidase hydrolysis of malonyl genistin. Therefore, six conformations in which the glucose molecule faces the interior of the substrate channel were analyzed, and amino acids that hydrogen bond with the malonyl group were selected. It includes Bgl2A:Q301, which is mutated into alanine, an amino acid with a smaller side chain, in order to enlarge the substrate channel of the protein so that it can accommodate malonyl genistin with a larger side chain, thereby increasing the activity of glycosidase towards malonyl substrates.

[0029] 2. Design of mutation primers and PCR amplification of mutant genes

[0030] Based on the gene sequence of β-glucosidase Bgl2A: SEQ ID NO: 2 (its amino acid sequence is SEQ ID NO: 1), and the selected mutation site 301Q, the following four site-directed mutagenesis primers were designed (Table 1).

[0031] Table 1 Primer sequences

[0032]

[0033] 3. Construction of expression vector

[0034] The PCR amplification product obtained in step 2 was ligated with the pEASY-T3 plasmid to establish the following enzyme digestion system: 10 ng pEASY-T3 vector, 70 ng PCR amplification product, and ligation was carried out at 25°C for 15 min. The ligation product was heat-shock transformed into Escherichia coli Trans1-T1 competent cells, and the obtained transformants were sequenced to verify whether they were mutated; clones with correct sequences were selected to extract plasmids to obtain pEASY-T3 recombinant plasmids containing the mutant gene of β-glucosidase of the present invention; the obtained pEASY-T3 recombinant plasmids and pET-22b(+) vector were double-digested with Nde I and Xho I, and then the mutant gene after enzyme digestion was ligated with the expression plasmid vector using T4 DNA ligase to establish the following enzyme digestion system: 25 ng pET-22b(+) vector, 50 ng mutant gene enzyme fragment, 2 μL 10×ligation buffer, 1 μL T4 DNA ligase, added water to 20 μL, and ligated at 22° C. for 1 hour to obtain a ligation product; the ligation product was transformed into a host bacterium, and the obtained transformants were sequenced to verify whether they were mutated, and the transformants with correct sequences were selected to obtain the engineered strain Escherichia coli BL21(DE3) / p ET-22b(+)-Bgl2A:Q301A containing the mutant gene of the present invention.

[0035] The strain expressing the β-glucosidase mutant of the present invention is classified and named Escherichia coli BL21(DE3) / p ET22b(+)-bgl2A:Q301A, has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with a preservation number of CCTCC NO: M 20241888, a preservation date of September 2, 2024, and a preservation address of Wuhan University, Wuhan, China.

[0036] (2) Expression and protein purification of genetically engineered bacteria containing the β-glucosidase mutation of the present invention

[0037] (1) The genetically engineered strain Escherichia coli BL21(DE3) / pET22b(+)-bgl2A:Q301A was inoculated into 400 mL of LB liquid medium containing ampicillin and cultured at 37°C and 200 rpm until the OD 600The enzyme was cultured in LB medium as a blank control. IPTG was added to a final concentration of 0.2 mM for induction and cultured for 16 hours at 16°C and 120 rpm. The cells were harvested by centrifugation at 8000 g at 4°C, three times the volume of the culture medium was added to Binding Buffer, and the cells were disrupted by sonication at 350 W for 30 minutes on ice. The supernatant was collected by centrifugation at 12000 g to obtain a crude enzyme solution. The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the eluent was 60 mM, and elution was performed over three column volumes. The resulting protein was tested for purity by SDS-PAGE.

[0038] (III) Detection of the specific activity of β-glucosidase mutants

[0039] The reaction system consisted of 500 μL of pNPG in a pH 6.5 citrate-Na₂HPO₄ buffer solution, added to a final concentration of 5 mM. The solution was preheated at 45°C for 5 minutes before the addition of enzyme solution. After 5 minutes of reaction, the reaction was terminated by the addition of 500 μL of 1 M Na₂CO₃. Three parallel experiments were performed in the experimental group, while the control group used buffer instead of enzyme solution. The control group was used as the zero value, and absorbance at 405 nm was measured. Enzyme activity (U) was defined as the amount of enzyme required to produce 1 μmol of pNP per minute.

[0040] (IV) Detection of β-glucosidase stability

[0041] Bgl2A and the mutant were heat-treated at 30-35°C and pH 7.0-7.5. Samples were taken every half hour or hour. The initial enzyme activity was taken as 100%. The residual enzyme activity after a certain period of heat treatment was calculated using the following formula: residual enzyme activity = (enzyme activity before heat treatment - lost enzyme activity) / heat-treated enzyme activity × 100%.

[0042] (V) Application of β-glucosidase mutants in the hydrolysis of soybean isoflavones

[0043] The yellow slurry water was treated using a previously established laboratory process. The volume of the yellow slurry water was first concentrated to 1 / 5. Anhydrous ethanol, 3 / 4 of the concentrate volume, was then added. After thorough mixing at room temperature, the concentrate was centrifuged at 8000 rpm for 20 minutes to produce an isoflavone extraction solution. Because high final ethanol concentrations can easily inactivate β-glucosidase, the initial process opted to dilute the ethanol concentration to approximately 20% (v / v) by adding an equal volume of water. The pH was adjusted with NaOH, and β-glucosidase was then added to hydrolyze the isoflavones. 10 mL of the yellow slurry water was incubated in a 35°C waterbath for five minutes. 8 mg of enzyme was added and the reaction was allowed to proceed for 30 minutes. A 1 mL sample was then taken and immediately terminated with 1 mL of 70% ethanol. A control without enzyme was used. The terminated reaction solution was aspirated with a 1 mL syringe and filtered through 0.22 μm organic and aqueous filters. The consumed components of the glycoside substrate were analyzed by HPLC.

Claims

1. A β-glucosidase mutant, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A gene encoding the β-glucosidase mutant according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO:

2.

3. A strain expressing the β-glucosidase mutant according to claim 1, characterized in that: The strain is classified and named Escherichia coli BL21(DE3) / p ET22b(+)-bgl2A:Q301A, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20241888, the preservation date is September 2, 2024, and the preservation address is: Wuhan University, Wuhan, China.

4. Use of the β-glucosidase mutant according to claim 1 in the hydrolysis of malonyl genistin.

5. The use according to claim 4, characterized in that: The temperature of the hydrolysis system is 30-35° C., and the pH value is 7.0-7.5.

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