Beta-glucosidase mutants with improved stability and uses thereof

Abstract

The application discloses a beta-glucosidase mutant with improved stability and an application thereof. The beta-glucosidase mutant is based on a beta-glucosidase from a marine uncultured microorganism, and is obtained by computer-aided design, clear mutation sites, and heterologous expression in Escherichia coli, and has improved stability. The stability of the mutant enzyme is 4.3 times that of the original enzyme under the condition of 30-45 DEG C. Under the condition of the same enzyme amount, the substrate amount converted by the mutant enzyme is twice that of the original enzyme. The mutant has potential application value in the hydrolysis of isoflavones.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a beta-glucosidase mutant with improved stability and application thereof. 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] Many bioactive compounds in food exist as glycosides. When hydrolyzed by β-glucosidase, these compounds release bioactive aglycones that can be absorbed by humans, providing additional health benefits. Soybeans, the highest-protein food crop, contain isoflavones, primarily in the form of glycosides such as daidzein and genistein, making them an important food supplement due to their health benefits. These inactive glycosides are converted to daidzein and genistein by β-glucosidase, where they exert their active effects. β-glucosidase with good stability has potential applications. Summary of the Invention

[0004] The present invention provides a β-glucosidase mutant with improved stability and its application. Based on a semi-rationally modified mutant of β-glucosidase derived from uncultured marine microorganisms, the present invention obtains a mutant gene through computer-aided design. After inducing expression of a recombinant plasmid containing the mutation in Escherichia coli, a β-glucosidase with improved stability was obtained. Using pNPG as a substrate, the mutant's stability was measured to be 4.3 times greater. In an isoflavone conversion experiment, the addition of 0.75 U of the mutant enzyme to the reaction system hydrolyzed 1.6 mg of isoflavones by over 90%. Under the same reaction conditions, the same amount of enzyme, when added to the starting enzyme, converted half the amount of substrate as the mutant enzyme, while maintaining the same hydrolysis rate. This mutant has potential application value in the hydrolysis of isoflavones.

[0005] The amino acid sequence of the β-glucosidase mutant of the present invention is shown in SEQ ID No: 1, and the serine at position 360 is mutated to lysine.

[0006] The amino acid sequence of the β-glucosidase mutant of the present invention may further include a combination of nonsense mutations or synonymous mutations in the sequence.

[0007] The nucleotide sequence of the gene encoding the β-glucosidase mutant of the present invention is shown in SEQ ID No: 2.

[0008] The mutant plasmid of the present invention contains the gene encoding the β-glucosidase mutant as described in SEQ ID No: 2.

[0009] The strain expressing the beta-glucosidase mutant contains the mutant plasmid.

[0010] The engineered strain expressing the β-glucosidase mutant of the present invention is classified and named Escherichia coli BL21 (DE3) / pET22b (+) bgl3A: S360K, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20232148, the preservation time is November 8, 2023, and the preservation address is: Wuhan University, Wuhan, China.

[0011] The method for constructing an engineered strain expressing a β-glucosidase mutant of the present invention comprises the following steps:

[0012] First, using the structure of β-glucosidase from Paenibacillus polymyxa as a template, Swiss-Model was used to perform homology modeling of the β-glucosidase Bgl3A. Using a computer-aided design strategy, three energy calculation functions were selected for calculation, and pairwise intersection was used to identify mutation sites for positive screening. Coevolutionary analysis and sequence conservation analysis were then used for negative screening to identify target amino acids for mutation.

[0013] Based on the gene sequence of β-glucosidase Bgl3A, mutant primers were designed and synthesized. Using the recombinant plasmid containing the β-glucosidase Bgl3A gene as a template and the above-mentioned synthetic mutant primers as primers, site-directed mutagenesis was performed based on the overlap extension PCR method to obtain a mutant gene of β-glucosidase with improved stability.

[0014] 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.

[0015] The expression plasmid vectors described in the above construction method include pCold, pET15, pET22 or pET28, etc.

[0016] The host bacteria in the above construction method include E. coli BL21 (DE3), E. coli DH5α, E. coli JM109 or E. coli Rosetta, etc.

[0017] The beta-glucosidase mutant of the present invention can be obtained by fermenting the engineered strain.

[0018] The application of the β-glucosidase mutant of the present invention is to hydrolyze isoflavones. When pNPG is used as a substrate, the stability of the mutant is measured to be 4.3 times that of the original enzyme.

[0019] Furthermore, the hydrolysis temperature is 30-35° C., and the system pH value is 7.0-7.5.

[0020] In an isoflavone hydrolysis experiment, adding 0.75 U of the mutant enzyme to the reaction system hydrolyzed 1.6 mg of isoflavones by over 90%. While maintaining the same hydrolysis rate, the same amount of enzyme, added under the same reaction conditions, converted half the amount of substrate as the mutant enzyme. This mutant has potential application in isoflavone hydrolysis.

[0021] The present invention measured and compared the optimal pH, temperature, and stability of the mutant protein and the original enzyme protein. The mutant showed improved stability. At 30-35°C and pH 7.0-7.5, the half-life of the mutant was 4.3 times that of the original enzyme Bgl3A. The mutation did not cause changes in the optimal temperature and pH. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 、 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 S360K-S, and S360K-X, respectively; Figure 2 The lanes in the middle are the PCR amplification products using DNA marker, S360K-S and S360K-X as templates.

[0023] Figure 3 Figure 3 is the SDS-PAGE profile of the purified mutant protein and the starting enzyme Bgl3A: 1 is the supernatant of Bgl3A fragmentation, 2 is the fragmentation precipitate, 3 is the pure Bgl3A enzyme, 4 is the supernatant of S360K fragmentation, 5 is the fragmentation precipitate of S360K, 6 is the pure S360K enzyme, and M is a protein marker.

[0024] Figure 4 In the figure, a is the optimum temperature measurement result, b is the optimum pH measurement result, and c is the stability measurement result.

[0025] Figure 5 a is the optimization of the starting enzyme hydrolysis substrate concentration, b is the optimization of the S360K hydrolysis substrate concentration, c is the optimization of the starting enzyme hydrolysis enzyme amount, d is the optimization of the S360K hydrolysis enzyme amount, e is the optimization of the starting enzyme hydrolysis pH, f is the optimization of the S360K hydrolysis pH, g is the optimization of the starting enzyme hydrolysis temperature, h is the optimization of the S360K hydrolysis temperature, i is the optimization of the starting enzyme hydrolysis time, and j is the optimization of the S360K hydrolysis time. DETAILED DESCRIPTION

[0026] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0027] (1) Construction of an expression strain containing the β-glucosidase mutant gene of the present invention

[0028] 1. Selection of β-glucosidase gene mutation sites

[0029] Based on sequence alignment, Bgl3A is most similar to the β-glucosidase Bg1B (PDB code: 2O9R) from Paenibacillus polymyxa, with an amino acid sequence identity of 43%. Using the structure of Bg1B as a template, homology modeling of the β-glucosidase Bgl3A was performed using Swiss-Model (http: / / swissmodel.expasy.org / ).

[0030] The serine at position 360 was mutated to lysine based on the calculation of the energy function.

[0031] 2. Design of mutation primers and PCR amplification of mutant genes

[0032] Based on the gene sequence of β-glucosidase Bgl3A: SEQ ID No: 2 (its amino acid sequence is SEQ ID No: 1), and the selected mutation site 360S, the following four site-directed mutagenesis primers were designed (Table 1).

[0033] Table 1 Primer sequences

[0034]

[0035] 3. Construction of expression vector

[0036] The PCR amplification product obtained in step 2 was ligated with pEASY-T3 plasmid (TaKaRa) 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 (TaKaRa), 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 ET22b(+)bgl3A:S360K containing the mutant gene of the present invention.

[0037] The engineered strain expressing the β-glucosidase mutant of the present invention is classified and named Escherichia coli BL21 (DE3) / pET22b (+) bgl3A: S360K, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20232148, the preservation time is November 8, 2023, and the preservation address is: Wuhan University, Wuhan, China.

[0038] (2) Expression and protein purification of genetically engineered bacteria containing the β-glucosidase mutation of the present invention

[0039] The engineered strain Escherichia coli BL21(DE3) / p ET22b(+)bgl3A:S360K obtained in (I) was inoculated into 400 mL LB liquid medium containing ampicillin and cultured at 37°C and 200 rpm until OD 600The cell suspension was cultured in LB medium as a blank control. IPTG was added to a final concentration of 0.2 mM for induction and cultured at 16°C and 120 rpm for 16 hours. The cells were harvested by centrifugation at 8000 g at 4°C, three times the volume of the culture suspension in Binding Buffer was added, 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 the elution volume was 3 column volumes. The resulting protein was tested for purity by SDS-PAGE.

[0040] Using pNPG as substrate, the mutant has an optimum pH of 6.5, and the enzyme can show more than 70% of its activity within the pH range of 6.0-7.5. The mutant has catalytic activity within the range of 30-55 degrees Celsius, with an optimum temperature of 45 degrees Celsius.

[0041] (III) Detection of the specific activity of the β-glucosidase mutants of the present invention

[0042] 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 adding 500 μL of 1 M Na₂CO₃. Three replicates 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.

[0043] (IV) Detection of the stability of the β-glucosidase of the present invention

[0044] Bgl3A 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%.

[0045] The measurement results showed that the half-life of the mutant under these conditions was 130 minutes, which was 4.3 times that of the starting enzyme.

[0046] (V) Application of the β-glucosidase mutant of the present invention in isoflavone hydrolysis

[0047] 40% isoflavone powder was extracted with ethanol at 70°C for 1 hour at a 1:20 solid-liquid ratio, followed by centrifugation at 10,000 rpm / min for 10 minutes to obtain an isoflavone extract solution. The reaction system consisted of 500 μL of isoflavone extract (final ethanol concentration: 22.5%), 100 μL of isoflavone extract solution (final ethanol concentration: 22.5%), an appropriate amount of β-glucosidase, and 50 mmol of citric acid-phosphate buffer (pH 6.5) to make up to 500 μL. The mixture was thoroughly mixed and reacted at 40°C for 10 minutes. After the reaction, 4.5 mL of 90% ethanol was added, and the mixture was filtered and analyzed by HPLC.

[0048] After the isoflavone conversion conditions were optimized, adding 0.75U of the mutant enzyme to the reaction system could hydrolyze 1.6mg of isoflavones by more than 90%. While ensuring the same hydrolysis rate, when the same amount of starting enzyme was added under the same reaction conditions, the amount of substrate converted was half that of the mutant enzyme. This mutant has potential application value in the hydrolysis of glycoside isoflavones.

Claims

1. A β-glucosidase mutant with improved stability, characterized in that Its amino acid sequence is shown in SEQ ID No:

2.

2. The gene encoding the β-glucosidase mutant according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID No:

1.

3. An engineered strain expressing the β-glucosidase mutant of claim 1, characterized in that: The strain is classified and named Escherichia coli BL21(DE3) / pET22b(+)bgl3A:S360K, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20232148, the preservation time is November 8, 2023, and the preservation address is: Wuhan University, Wuhan, China.

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

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

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