Mutant of beta-glucosidase Cel1A and application in improving biosynthesis of sophorose

Abstract

The application discloses a mutant of beta-glucosidase Cel1A and application of the mutant in improving biosynthesis of sophorose, and belongs to the technical field of enzyme engineering.The application aims to solve the technical problems of low catalytic activity and poor product selectivity of wild-type beta-glucosidase Cel1A in the process of sophorose biosynthesis.A mutant of beta-glucosidase Cel1A is provided, wherein any one or more of the following mutations is made in the sequence of SEQ ID NO.1: T at the 299th position is mutated into G, and K at the 240th position is mutated into H.The developed reverse hydrolysis reaction process with high-concentration glucose as a substrate does not need to add expensive high-energy sugar nucleotide donors, the reaction condition is mild, the process flow is simplified, and the production cost of sophorose is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a mutant of β-glucosidase Cel1A and its application in improving the biosynthesis of sophorose. Background Technology

[0002] Sophorose (β-1,2-glucose disaccharide) is a disaccharide composed of two glucose molecules linked by a β-1,2-glycosidic bond. Studies have shown that sophorose is the strongest inducer for cellulase synthesis in filamentous fungi (such as *Trichoderma reesei* and *Aspergillus*), with an induction efficiency far exceeding that of other inducing compounds such as cellobiose and lactose, reaching tens to thousands of times higher. Adding trace amounts of sophorose to the industrial production of cellulase can significantly improve the yield and quality of cellulase, thus making it of significant value in the economic production of cellulase. However, sophorose currently relies mainly on chemical synthesis or extraction from natural products, resulting in high production costs and low yields. Only reagent-grade products are available on the market, and industrial-scale mass production has not yet been achieved, severely restricting its widespread application in cellulase induction and other fields. Therefore, developing a low-cost, high-efficiency biosynthetic method for sophorose has significant economic implications and application prospects.

[0003] β-glucosidase (EC 3.2.1.21) is a class of glycoside hydrolases widely found in nature and an important component of the cellulase system. This enzyme not only hydrolyzes oligosaccharides such as cellobiose to produce glucose, but also possesses transglycosylation activity, catalyzing the synthesis of sophorose from glycosidic bonds between glucose molecules via reverse hydrolysis or transglycosylation. Among them, β-glucosidase Cel1A (also known as BGLII), derived from *Trichoderma reesei*, belongs to family 1 (GH1) of glycoside hydrolases and is a typical cellobiase. Studies have found that Cel1A has the highest affinity for cellobiose, its transglycosylation reaction primarily synthesizes sophorose, and it exhibits the lowest sensitivity to glucose inhibition. Therefore, Cel1A is considered an ideal candidate enzyme for improving sophorose synthesis activity through enzyme engineering.

[0004] Currently, modifications to β-glucosidase mainly focus on improving its hydrolytic activity, thermal stability, and glucose tolerance, while research on enhancing its transglycosylation activity for sophorose synthesis is still very limited. Existing technologies have few reports on modifying β-glucosidase through directed evolution or rational design to improve its activity in synthesizing specific oligosaccharides, and these modifications generally suffer from low efficiency and limited activity enhancement in mutants. Therefore, there is an urgent need to develop a molecular modification method that can efficiently improve the sophorose synthesis activity of β-glucosidase Cel1A to obtain mutants with significantly enhanced catalytic efficiency, thereby promoting the enzymatic synthesis and industrial application of sophorose. Summary of the Invention

[0005] The purpose of this invention is to overcome the core technical bottlenecks in the biosynthesis of sophorose by wild-type β-glucosidase Cel1A, such as low catalytic activity, poor product selectivity, and high production cost.

[0006] This invention provides a mutant of β-glucosidase Cel1A, with the sequence of SEQ ID NO.1 as the starting sequence, and any one or more of the following mutations: T at position 299 is mutated to G and K at position 240 is mutated to H.

[0007] The present invention provides a gene encoding the above-mentioned mutant.

[0008] This invention provides a reload vector containing the above-mentioned genes.

[0009] Further specifying, the launch vehicle is PET-28a.

[0010] The present invention provides a recombinant microbial cell containing the above-mentioned genes.

[0011] To further specify, the starting cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0012] The present invention provides the application of the above-mentioned mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell in the preparation of a product with increased sophorose yield or increased β-glucosidase activity.

[0013] This invention provides a method for preparing sophora sugar, which involves reacting the above-mentioned mutant with glucose as a substrate.

[0014] Further specifying the reaction conditions, the reaction temperature is 50°C and the reaction time is 24 hours.

[0015] Further specified, the glucose concentration is 2.2 M, the concentration of the mutant according to claim 1 is 4 μg / mL, the reaction buffer is 50 mM sodium acetate, and the pH is 4.8.

[0016] Beneficial effects: Significantly improved catalytic performance: Through computer-aided semi-rational design, the key mutation sites of T299G and K240H were successfully obtained. Among them, the optimal double mutant Cel1A-T299G-K240H showed that the sophorose synthesis activity was increased by about 4.36 times compared with the wild type, and the catalytic efficiency (kcat / Km) was increased by about 3.76 times. At the same time, the formation of the byproduct gentiobiose was significantly inhibited, achieving dual optimization of sophorose synthesis activity and selectivity.

[0017] Production costs are effectively reduced: The developed reverse hydrolysis reaction process using high-concentration glucose as a substrate has mild reaction conditions (pH 4.8, 37-50℃), which simplifies the process and significantly reduces the production cost of sophora japonica syrup.

[0018] Breakthrough improvement in production efficiency: Through the synergy of the above-mentioned enzyme modification and process optimization, the sophora sugar biosynthesis system finally constructed achieved a significant increase in sophora sugar production under optimal conditions, demonstrating strong potential for industrial application. Attached Figure Description

[0019] Figure 1 The results of the prediction of saturation mutations in the whole protein of β-glucosidase Cel1A using the UniKP method; Figure 2 To analyze the binding energy of target proteins with different ligands using molecular dynamics methods; A: MM / PBSA results of Cel1A binding to sophorose; B: MM / PBSA results of Cel1A binding to glucose.

[0020] Figure 3 The relative activities of Cel1A wild-type and mutant in the synthesis of sophorose are: A: Relative activity of single mutant in the synthesis of sophorose; B: Relative activity of K240 site saturated mutant in the synthesis of sophorose; C: Relative activity of T299 site saturated mutant in the synthesis of sophorose; D: Relative activity of combined mutant in the synthesis of sophorose.

[0021] Figure 4 The protein purification status of Cel1A wild-type and mutant (A) and the activity of the purified enzyme in synthesizing sophorose (B).

[0022] Figure 5 The effect of different pH values ​​on the enzyme activity of Cel1A mutant.

[0023] Figure 6 The effect of different temperatures on the enzyme activity of Cel1A mutant.

[0024] Figure 7 The effect of substrate concentration on the reaction rate of Cel1A is shown. Detailed Implementation

[0025] Example 1: Screening of potential key sites and verification of K240 site saturation mutations based on the machine learning UniKP method In this embodiment, the deep learning model UniKP was used to predict the catalytic efficiency (kcat) of single-point mutations in the full sequence of Trichoderma reesei β-glucosidase Cel1A (amino acid sequence shown in SEQ ID NO.1), screen out potential key sites, and verify their impact on sophorose synthesis activity through saturation mutation experiments.

[0026] 1. UniKP Prediction Process Prepare the amino acid sequence of Cel1A (FASTA format) and the SMILES string of the substrate glucose. Using the UniKP code (https: / / github.com / ziyewang / UniKP), select the "kcat prediction" mode, input the protein sequence and substrate SMILES, and perform a full-sequence single-point mutation scan. The model outputs the predicted kcat fold change relative to the wild type for each mutant. Set the screening threshold to a predicted kcat fold increase ≥ 1.5. The results are as follows. Figure 1 and Figure 3 Eleven candidate mutation sites were identified in the A group, with the following predicted fold increases in kcat: K240A (2.3-fold), A13K (2.1-fold), W160A (2.0-fold), F114R (1.9-fold), L275I (1.8-fold), S79K (1.7-fold), A58T (1.6-fold), A238N (1.6-fold), P264R (1.5-fold), D227P (1.5-fold), and S79R (1.5-fold). K240A showed the highest predicted fold increase at 2.3-fold. Therefore, site K240 was selected for subsequent saturation mutation experiments.

[0027] 2. Design of saturation mutant primers at the K240 site For the K240 site, degenerate primers covering all 19 amino acid substitutions were designed (using the NNK degenerate codon, N=A / T / C / G, K=G / T). The forward primer sequence is: 5′-CCTGATCGAGAAGNNKAAGGCCCTGGAG-3′, SEQ ID NO.1; the reverse primer sequence is: 5′-CTCCAGGGCCTTNNKCTTCTCGATCAGG-3′, SEQ ID NO.2. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and were of HPLC purity. Reverse PCR amplification was performed using the pET-28a-Cel1A plasmid (PET-28a vector linking the Cel1A gene) as a template, employing Phanta Flash Master Mix. The PCR system (50 μL) consisted of: 1 μL template plasmid (50 ng / μL), 25 μL 2× high-fidelity premix, 2 μL each of the forward and reverse degenerate primers (10 μM), and sterile water to a final volume of 50 μL. PCR program: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 3 minutes, 30 cycles; final extension at 72℃ for 5 minutes. The product was digested with DpnI (1 μL, 20 U / μL) at 37℃ for 1 hour, then transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. Ninety-six single clones were randomly selected and seeded into 96-well plates, incubated overnight at 37℃, and plasmids were extracted and sequenced (using T7 primers). Sequencing results showed that 19 different amino acid substitutions were obtained at the K240 site (all mutations except for the stop codon), including K240A, K240H, K240R, K240N, K240S, K240T, K240D, K240E, K240Q, K240G, K240V, K240L, K240I, K240M, K240F, K240Y, K240W, K240P, and K240C. Plasmids containing the above mutants were constructed, and the mutants were ligated to PET-28a.

[0028] 3. Saturation mutant expression and activity screening Nineteen K240 mutant plasmids, confirmed by sequencing, were transformed into E. coli BL21(DE3) expression strains for small-scale induction expression in 96-well plates (0.5 mM IPTG, 25°C, 24 hours). Bacterial cells were collected, and 200 μL of lysis buffer (20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.5) was added to each well. After sonication and centrifugation, the supernatant was collected. High-throughput purification was performed using Ni-NTA magnetic beads (GE Healthcare). 20 μL of magnetic bead suspension was added to each well, incubated at room temperature for 30 minutes, washed, and eluted to obtain purified protein. Protein concentration was determined using the Bradford method and adjusted to 0.1 mg / mL. Results are as follows: Figure 4 As shown.

[0029] The catalytic reaction system (200 μL) consisted of 50 mM sodium acetate buffer (pH 4.8), 2.2 M glucose, and 20 μL purified enzyme solution. The reaction was carried out at 50℃ for 24 hours. After the reaction, 20 μL of 1 M NaOH was added to terminate the reaction. The yield of sophorose was determined by HPLC. Chromatographic conditions: Shodex Asahipak NH2P-50 4E column (4.6 mm × 250 mm), mobile phase acetonitrile:water = 75:25 (v / v), flow rate 1.0 mL / min, column temperature 30℃, injection volume 10 μL, evaporative light scattering detector (ELSD), drift tube temperature 80℃, and nitrogen flow rate 1.5 L / min. The retention time of the sophorose standard (Sigma-Aldrich, purity ≥98%) was approximately 12.5 minutes. The relative activities of each mutant were calculated with the activity of wild-type Cel1A as 100%.

[0030] 4. Results The results of the K240 site saturation mutation are as follows: Figure 3 B in the diagram shows that the K240H mutant has the highest relative activity, at 251% of the wild type; K240A has a relative activity of 189%; K240R has 187%; K240N has 163%; K240S has 142%; and the activities of the remaining mutants are all below 130% or below the wild type. It is noteworthy that the optimal mutant predicted by UniKP, K240A (predicted up 2.3-fold), only showed an activity of 189% in the actual experiment, not the highest; while K240H (predicted up 2.1-fold) showed the highest actual activity, reaching 251%. This indicates a certain deviation between the UniKP prediction and the experimental results, but the K240 site is indeed a key site, and K240H is the optimal single-point mutation. Therefore, K240H is determined to be the optimal mutation for the K240 site.

[0031] Example 2: Screening of potential key sites and verification of T299 site saturation mutation based on molecular dynamics methods This embodiment uses molecular docking, molecular dynamics simulation, and MM / PBSA binding free energy decomposition method to identify key residues that contribute significantly to the binding of sophorose products, and verifies the saturation mutation of the screened T299 site.

[0032] 1. Molecular docking and molecular dynamics simulation Protein preparation: Using the 3D structure of Cel1A predicted by AlphaFold2 (pLDDT>90), water molecules were removed, polar hydrogen was added, and Gasteiger charge was calculated in AutoDock Tools 1.5.7, and the result was saved as a .pdbqt file. Ligand preparation: The 3D structures of sophorose (CID 439573) and glucose (CID 5793) were downloaded from PubChem, and energy minimization was performed using OpenBabel 3.1.1 (MMFF94 force field, 500 steps). The center of the docking box was set as the active pocket (midpoint of catalytic residues Glu172 and Glu367), with dimensions of 60 Å × 60 Å × 60 Å. The Lamarckian genetic algorithm was used to run 200 docking iterations, and the conformation with the lowest binding free energy was selected.

[0033] Place the Cel1A-sophorose and Cel1A-glucose complexes (the names of the structural models used in molecular dynamics) into TIP3P water boxes respectively, and add Na. + / Cl - Neutralization was performed using GROMACS 2023 for a 200 ns molecular dynamics simulation (AMBER99SB-ILDN force field, 300 K, 1 bar). 200 frames were uniformly extracted from the last 50 ns trajectory, and residue-level binding free energy decomposition was performed using the gmx_MMPBSA tool. The difference in binding free energy contribution of each residue to sophorose and glucose was calculated (ΔΔG = ΔGsophose - ΔGglucose). Screening criteria: ΔΔG ≤ -1.5 kcal / mol, i.e., residues whose contribution to sophorose binding is significantly greater than their contribution to glucose binding.

[0034] The results are as follows Figure 2 As shown, one key residue was identified: T299 (ΔΔG = -1.92 kcal / mol). This residue contributes -2.34 kcal / mol to the binding free energy of sophorose, but only -0.42 kcal / mol to glucose, a difference of -1.92 kcal / mol, indicating that T299 is the key site leading to product inhibition. Therefore, the T299 site was selected for saturation mutagenesis experiments.

[0035] 2. Primer design for saturation mutation at the T299 site For the T299 site, degenerate primers covering all 19 amino acid substitutions were designed (using NNK degenerate codons). The forward primer sequence is: 5′-CGACTGGTACGGCGGCGACTACATCGTCAAC-3′, SEQ ID NO.4 (where the mutation site is located in the middle of the primer, and the corresponding codon is replaced by NNK); the reverse primer sequence is: 5′-GTTGACGATGTAGTCGCCGCCGTACCAGTCG-3′, SEQ ID NO.5. Primer synthesis, PCR amplification, DpnI digestion, transformation, and sequencing were performed using the same methods as in Example 1. Sequencing results showed that 18 different amino acid substitutions were obtained at the T299 site (all mutations except the stop codon, except for T299C, which was not obtained).

[0036] 3. Saturation mutant expression and activity screening The expression, purification, and activity assay methods were the same as in Example 1. The sophorose synthesis activity of 18 T299 mutants and wild-type Cel1A was simultaneously measured.

[0037] 4. Results The results of the T299 site saturation mutation are as follows: Figure 3 The C-values ​​in the data show that the T299G mutant has the highest relative activity, at 329% of the wild type; T299A has a relative activity of 215%; T299S has 198%; T299V has 156%; and the activities of the remaining mutants are all below 140% or below the wild type. Therefore, T299G is determined to be the optimal mutation for the T299 site.

[0038] 5. Construction of combinatorial mutants The optimal single-point mutation K240H was combined with T299G to construct the double mutant T299G-K240H. Using pET-28a-Cel1A-T299G plasmid (PET-28a linked to the coding gene of Cel1A-T299G, the sequence of the coding gene of Cel1A-T299G is shown in SEQ ID NO.2) as a template, reverse PCR was performed using K240H site-directed mutagenesis primers (forward: 5′-CCTGATCGAGAAGCACGCCAAGGCCCTGGAG-3′, SEQ ID NO.6; reverse: 5′-CTCCAGGGCCTTGGCGTGCTTCTCGATCAGG-3′, SEQ ID NO.7). After DpnI digestion, transformation, and sequencing verification, the double mutant plasmid pET-28a-Cel1A-T299G-K240H (PET-28a linked to the coding gene of Cel1A-T299G-K240H, the sequence of the coding gene of Cel1A-T299G-K240H is shown in SEQ ID NO.3) was obtained. After expression and purification, the activity of sophorose synthesis was measured. Results are as follows: Figure 3 The D-values ​​in the data show that the relative activity of the double mutant T299G-K240H reached 436% of that of the wild type, which is significantly better than either single-point mutant (329% for T299G and 251% for K240H), indicating that the two mutations have a synergistic effect.

[0039] Example 3: Enzymatic characterization and reaction condition optimization of the optimal mutant T299G-K240H In this embodiment, the optimal pH, optimal temperature, kinetic parameters and reaction conditions of the best double mutant T299G-K240H were optimized and compared with the wild type Cel1A.

[0040] First, the correctly sequenced mutant plasmid was transformed into... E. coli BL21(DE3) expression strain was induced to express the protein (25℃, 0.5 mM IPTG, 24 h). Bacterial cells were collected and resuspended in Binding Buffer (20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.5) for sonication. The supernatant was filtered through a 0.22 μm filter and loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Binding Buffer. Impurities were washed with 10–15 column volumes of Washing Buffer (20 mM sodium phosphate, 300 mM NaCl, 80 mM imidazole, pH 7.5). The target protein was eluted with Elution Buffer (20 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 7.5), and the elution peak was collected. The eluent was centrifuged in an ultrafiltration centrifuge tube with a 10 kDa molecular weight cutoff at 4°C and 6000×g, and then replaced and concentrated with 50 mM citrate-sodium citrate buffer (pH 4.8) to remove imidazole. The concentration of the purified protein was determined, and the protein was stored at -80°C or used immediately for activity assays.

[0041] 1. Determination of optimal pH Prepare a series of buffer solutions for pH 3.0–7.0: 50 mM citrate-sodium citrate buffer for pH 3.0–5.0, 50 mM sodium acetate buffer for pH 5.0–6.0, and 50 mM sodium phosphate buffer for pH 6.0–7.0. Reaction system (1 mL): corresponding buffer, 2.2 M glucose, 4 μg / mL purified enzyme (wild-type Cel1A or double mutant T299G-K240H), react at 50℃ for 30 min. Sophorose yield was determined by HPLC. The highest activity was considered 100%.

[0042] The results are as follows Figure 5Wild-type Cel1A has an optimal pH of 4.8 and maintains more than 80% activity in the pH range of 4.0–5.5; T299G-K240H also has an optimal pH of 4.8, but maintains more than 85% activity in the pH range of 4.0–6.0, showing a wider pH adaptability.

[0043] 2. Determination of optimal temperature In 50 mM sodium acetate buffer (pH 4.8), with a fixed glucose concentration of 2.2 M and an enzyme concentration of 4 μg / mL, the reaction was carried out at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 30 minutes, respectively. HPLC analysis was performed.

[0044] The results are as follows Figure 6 The optimal temperature for wild-type Cel1A is 50℃, and its activity drops to 72% at 55℃. The optimal temperature for the double mutant T299G-K240H is also 50℃, but it still maintains 89% activity at 55℃ and 68% at 60℃, showing significantly better heat resistance than the wild type.

[0045] 3. Measurement of kinetic parameters Prepare 0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, and 4.0 M glucose solutions. Add 4 μg / mL of purified enzyme at pH 4.8 and 50℃, and react for 15 minutes (initial rate). Perform Michaelis-Menten fitting using GraphPad Prism 9.0.

[0046] The results are as follows Figure 7 The wild-type Cel1A has Km = 2.41 ± 0.09 M and kcat = 0.085 ± 0.003 s. - ¹, kcat / Km = 0.0353 M - ¹·s - ¹; The double mutant T299G-K240H has Km = 1.83 ± 0.07 M and kcat = 0.243 ± 0.008 s. - ¹, kcat / Km = 0.1328 M - ¹·s - ¹. The catalytic efficiency of the mutant was increased to 3.76 times that of the wild type.

[0047] 4. Verification of optimal reaction conditions Based on the above results, the optimal conditions for sophorose biosynthesis were determined to be: substrate glucose concentration of 2.2 M, enzyme concentration (double mutant T299G-K240H) of 4 μg / mL, reaction buffer of 50 mM sodium acetate (pH 4.8), reaction temperature of 50℃, and reaction time of 24 hours. Three parallel experiments were conducted under these conditions, and the average sophorose yield was 1.55 ± 0.04 g / L, 4.56 times that of the wild type (0.34 g / L), with a product purity of 96%, higher than the wild type's 61%. The content of the mutant byproduct gentiobiose was extremely low, at 0.12 ± 0.02 g / L, lower than the wild type's 3.12 ± 0.87 g / L. Specifically, the average sophorose yield of the single mutant T299G was 1.92 ± 0.11 g / L; and the average sophorose yield of the single mutant K240H was 2.17 ± 0.57 g / L.

[0048] Table 1: Kinetic parameters of wild type, T299G, K240H, and T299G-K240H

[0049] SEQ ID NO.1: MLPKDFQWGFATAAYQIEGAVDQDGRGPSIWDTFCAQPGKIADGGSSGVTACDSYNRTAEDIALLKSLGAKSYRFSISWSRIIPEGGRGDAVNQAGIDHYVKFVDD LLDAGITPFITLFHWDLPEGLHQRYGGLLNRTEFPLDFENYARVMFRALPKVRNWITFNEPLCSAIPGYGSGTFAPGRQSTSEPWTVGHNILVAHGRAVKAYRDD FKPASGDGQIGIVLNGDFTYPWDAADPADKEAAERRLEFFTAWFADPIYLGDYPASMRKQLGDRLPTFTPEERALVHGSNDFYGMNHYTSNYIRHRSSPASADDT VGNVDVLFTNKQGNCIGPETQSPWLRPCAAGFRDFLVWISKRYGYPPIYVTENGTSIKGESDLPKEKILEDDFRVKYYNEYIRAMVTAVELDGVNVKGYFAWSLM DNFEWADGYVTRFGVTYVDYENGQKRFPKKSAKSLKPLFDELIAAA; SEQ ID NO.2: MLPKDFQWGFATAAYQIEGAVDQDGRGPSIWDTFCAQPGKIADGSSGVTACDSYNRTAEDIALLKSLGAKSYRFSISWSRIIPEGGRGDAVNQAGIDHYVKFVDDLLDAGITPFITLFHWDLPEGLHQRYGGLLNRTEFPLDFENYARVMFRALPKVRNWITFNEPLCSAIPGYGSGTFAPGRQSTSEPWTVGHNILVAHGRAVKAYRDDFKPASGDGQIGIVLNGDFTYPWDAADPADKEAAERRLEFFTAWFADPIYLGDYPASMRKQLGDRLPTFTPEERALVHGSNDFYGMNHYGSNYIRHRSSPASADDTVGNVDVLFTNKQGNCIGPETQSPWLRPCAAGFRDFLVWISKRYGYPPIYVTENGTSIKGESDLPKEKILEDDFRVKYYNEYIRAMVTAVELDGVNVKGYFAWSLM DNFEWADGYVTRFGVTYVDYENGQKRFPKKSAKSLKPLFDELIAAA; SEQ ID NO.3: MLPKDFQWGFATAAYQIEGAVDQDGRGPSIWDTFCAQPGKIADGSSGVTACDSYNRTAEDIALLKSLGAKSYRFSISWSRIIPEGGRGDAVNQAGIDHYVKFVDDLLDAGITPFITLFHWDLPEGLHQRYGGLLNRTEFPLDFENYARVMFRALPKVRNWITFNEPLCSAIPGYGSGTFAPGRQSTSEPWTVGHNILVAHGRAVKAYRDDFKPASGDGQIGIVLNGDFTYPWDAADPADHEAAERRLEFFTAWFADPIYLGDYPASMRKQLGDRLPTFTPEERALVHGSNDFYGMNHYGSNYIRHRSSPASADDTVGNVDVLFTNKQGNCIGPETQSPWLRPCAAGFRDFLVWISKRYGYPPIYVTENGTSIKGESDLPKEKILEDDFRVKYYNEYIRAMVTAVELDGVNVKGYFAWSLM DNFEWADGYVTRFGVTYVDYENGQKRFPKKSAKSLKPLFDELIAAA。

[0050] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A mutant of β-glucosidase Cel1A, characterized in that, Starting with the sequence of SEQ ID NO.1, any one or more of the following site mutations are made: T mutation at position 299 is changed to G, and K mutation at position 240 is changed to H.

2. The gene encoding the mutant of claim 1.

3. A reload vector containing the gene described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The launch vehicle is PET-28a.

5. A recombinant microbial cell containing the gene described in claim 2.

6. The recombinant microbial cell according to claim 5, characterized in that, The starting cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

7. The use of the mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the preparation of sophora sugar, increasing sophora sugar yield, or increasing β-glucosidase activity.

8. A method for preparing sophora japonica syrup, characterized in that, The mutant described in claim 1 was obtained by reacting glucose as a substrate.

9. The method according to claim 8, characterized in that, The reaction conditions were a reaction temperature of 50℃ and a reaction time of 24 hours.

10. The method according to claim 8, characterized in that, The glucose concentration was 2.2 M, the concentration of the mutant according to claim 1 was 4 μg / mL, and the reaction buffer was 50 mM sodium acetate at pH 4.8.

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