A beta-glucosidase mutant and its use in preparing oligostilbenes

Abstract

The application discloses a beta-glucosidase mutant and application thereof in preparation of oligomeric gentio-oligosaccharides, and belongs to the technical field of enzyme engineering and functional oligosaccharide preparation. A beta-glucosidase gene derived from Thermotoga maritima STB25 is subjected to site-directed mutation, and high-efficiency expression of the beta-glucosidase gene in Bacillus subtilis is realized. The beta-glucosidase prepared by using the mutant is added to a reaction system with 480 g / L glucose as a substrate, and reaction is carried out under the condition of pH 6.0 and 60 DEG C, and the yield of oligomeric gentio-biose can reach 22.12%, which is increased by 66.44% compared with the wild type. The beta-glucosidase mutant has good acid and heat stability and transglycosylation capacity, can catalyze the preparation of gentio-biose by using glucose as a substrate, and has a good application prospect in the field of oligomeric gentio-oligosaccharide production.

Description

Technical Field

[0001] This invention relates to a β-glucosidase mutant and its application in the preparation of oligogentisoses, belonging to the fields of enzyme engineering and functional oligosaccharide preparation technology. Background Technology

[0002] β-glucosidase is a glycoside hydrolase that specifically hydrolyzes the non-reducing terminal β-D-glucosidic bond of a substrate, releasing glucose and the corresponding ligand. Furthermore, β-glucosidase also possesses transglycosylation activity, enabling the synthesis of oligosaccharides with higher degrees of polymerization from monosaccharides or oligosaccharides, thus it is commonly used in the preparation of gentian oligosaccharides. According to current research, the catalytic mechanism of β-glucosidase is a retention-type acid-base catalysis mechanism, involving two steps: glycosylation and deglycosylation. In the first step, glycosylation, nucleophilic catalytic residues nucleophilically attack the anomeric carbon, generating a glycosyl-enzyme covalent intermediate complex. In the second step, deglycosylation, acid-base activated water molecules act as nucleophiles, breaking the glycosidic bond and releasing the product glucose and the corresponding ligand. Simultaneously, β-glucosidase can also transfer glycosyl groups to other non-aqueous acceptor ligands during deglycosylation, thereby catalyzing the transglycosylation reaction.

[0003] Gentian oligosaccharides are a class of oligosaccharides composed of glucose units linked by β-1,6 glycosidic bonds, including gentiobiose and small amounts of gentiotriose and gentiotetraose. Compared to other oligosaccharides, gentian oligosaccharides have a unique, mild bitterness that can have a refreshing and invigorating effect, thus finding wide application in products such as candy, chocolate, ice cream, and coffee. Furthermore, it is also a functional oligosaccharide, capable of balancing the intestinal environment and promoting the proliferation of beneficial intestinal bacteria, making it a high-value-added sugar product with significant development potential. As mentioned above, the enzymatic preparation of gentian oligosaccharides utilizes the transglycosidic activity of β-glucosidase, typically using a high concentration of glucose as a substrate. Compared to traditional extraction methods, it offers advantages such as a milder reaction, lower energy consumption, and easier separation, representing the current main trend in production.

[0004] Due to the limited availability of β-glucosidase, the conversion rate of enzymatic preparation of gentiooligosaccharides remains low, thus hindering industrial-scale production in China. There are two main approaches to improving the yield of gentiooligosaccharides: first, increasing the concentration of the reaction substrate and reducing the water activity of the reaction system to decrease hydrolysis and allow for the accumulation of transglycosylation products; second, enhancing the transglycosylation capacity of the enzyme itself, thereby altering the balance between hydrolysis and transglycosylation to significantly improve the yield. Given the limited solubility of glucose and the difficulty in finding novel β-glucosidases suitable for industrial production, using site-directed mutagenesis to improve the enzymatic properties and product specificity of existing β-glucosidases is of great significance for the industrial-scale preparation of gentiooligosaccharides. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a β-glucosidase mutant and its application in the preparation of oligogentisoses, aiming to solve the technical problem of the lack of β-glucosidase suitable for the industrial production of oligogentisoses.

[0006] The first technical solution provided by this invention is a β-glucosidase mutant with enhanced ability to produce oligogentisic acid, wherein the β-glucosidase mutant is obtained by performing any of the following mutations on the β-glucosidase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The glycine at position 343 is mutated to histidine, leucine or valine; (2) The isoleucine at position 455 is mutated to alanine; (3) Alanine at position 468 is mutated to glycine.

[0007] Specifically, the amino acid sequence of the β-glucosidase parent is shown below: .

[0008] In some embodiments, the nucleotide sequence encoding the β-glucosidase mutant gene is shown in SEQ ID NO. 2. Specifically, the nucleotide sequence is as follows:

[0009] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0010] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0011] In some embodiments, the recombinant vector is expressed using plasmids such as pP43NMK, pHT01, pHT43, and pHCMC05, with pP43NMK being the preferred expression vector.

[0012] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0013] In some embodiments, the recombinant cells use Bacillus subtilis or Escherichia coli as expression hosts.

[0014] The fifth technical solution provided by this invention is a method for improving the ability of β-glucosidase to produce oligogentisoses, wherein the method involves performing any one of the following mutations on the β-glucosidase parent cell with the amino acid sequence shown in SEQ ID NO.1: (1) The glycine at position 343 is mutated to histidine, leucine or valine; (2) The isoleucine at position 455 is mutated to alanine; (3) Alanine at position 468 is mutated to glycine.

[0015] The sixth technical solution provided by the present invention is a method for producing oligogentisic acid, wherein the method comprises adding the β-glucosidase mutant described in the first technical solution to a reaction system with glucose as a substrate for conversion.

[0016] In some embodiments, the reaction conditions of the reaction system are 40-80°C and pH 3.0-6.0 for 12-60 h.

[0017] In some embodiments, the concentration of the substrate is at least 480 g / L.

[0018] In some embodiments, the amount of β-glucosidase added to the reaction system is at least 50 U / g.

[0019] The seventh technical solution provided by the present invention is the application of the β-glucosidase mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution in the preparation of gentiosaccharides or products containing gentiosaccharides.

[0020] Beneficial effects (1) This invention, through the analysis of the source Thermotoga maritima Mutating the β-glucosidase of STB25 alters the enzyme's key active site, transforming the β-glucosidase, which was originally inefficient in acting on glucose substrates, into a mutant with enhanced ability to produce oligogentisose. This expands its application range in the field of oligosaccharide preparation, and it has shown good results in the production process of preparing oligogentisose using glucose-related products as substrates.

[0021] (2) The β-glucosidase mutant provided by the present invention is derived from thermophilic bacteria. Compared with the existing β-glucosidase which is difficult to use in high-temperature industrial environments due to insufficient heat resistance, the β-glucosidase mutant provided by the present invention has good thermal stability and can maintain its activity at 80°C without loss. Therefore, it can meet the requirements of different reaction temperatures in the production of oligogentisose and provide a solution for reference in high-temperature industrial processes.

[0022] (3) The β-glucosidase mutant provided by the present invention can prepare oligo-gentian sugar under the condition of glucose as substrate. The conversion rate of oligo-gentian sugar in the product obtained by G343H catalysis is 1.42 times higher than that of wild type, reaching 22.12%. The high conversion rate provides a new option for industrial production of oligo-gentian sugar and has good application prospects in the field of starch functional sugar preparation.

[0023] In summary, this invention, through the analysis of substances derived from... Thermotoga maritima Site-directed mutagenesis of the STB25 β-glucosidase gene yielded β-glucosidase mutants G343L, G343V, G343H, I455A, A468G, G343T, and G343D, which exhibit enhanced gentiooligosaccharide production. Building upon the existing reaction with glucose to produce gentiooligosaccharides, these mutants further improved the yield of gentiooligosaccharides. Specifically, G343L, G343V, G343H, I455A, and A468G showed yield increases of 1.37, 1.20, 1.42, 1.10, and 1.09 times compared to the wild-type β-glucosidase, respectively, demonstrating superior performance in industrial production. G343T and G343D, on the other hand, showed yield decreases of 0.35 and 0.61 times compared to the wild-type β-glucosidase, respectively. Attached Figure Description

[0024] Figure 1 SDS for crude enzyme solutions of wild type and mutant PAGE electrophoresis analysis, where M: protein molecular weight standard, lane 1: B. subtilis WB600 / pP43NMK / tm-bgl Extracellular crude enzyme, lane 2: B. subtilis WB600 / pP43NMK / tm-bgl / G343L extracellular crude enzyme, lane 3: B. subtilis WB600 / pP43NMK / tm-bgl / G343V extracellular crude enzyme, lane 4: B. subtilis WB600 / pP43NMK / tm-bgl / G343H extracellular crude enzyme, lane 5: B. subtilis WB600 / pP43NMK / tm-bgl / I455A extracellular crude enzyme, lane 6: B. subtilis WB600 / pP43NMK / tm-bgl / A468G extracellular crude enzyme, lane 7: B. subtilis WB600 / pP43NMK / tm-bgl / G343T extracellular crude enzyme, lane 8: B. subtilis WB600 / pP43NMK / tm-bgl / G343D extracellular crude enzyme.

[0025] Figure 2 The yield of oligogentiobiose from the secondary mother liquor of crystalline glucose is determined by the action of wild-type β-glucosidase and its mutants. Detailed Implementation

[0026] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0027] Test method: (1) Method for determining the enzyme activity of β-glucosidase mutants (refer to the following literature: Zhu Qihui, Gao Zexin, He Laping, et al. Screening and identification of β-glucosidase strains producing hydrolyzed ginkgo flavonoid glycosides. China Brewing. 2017;36(9):5.) A 1% (w / v) salicin solution was prepared using a 50 mM, pH 4.8 acetate-sodium acetate solution as the substrate. 900 μL of the solution was placed in a centrifuge tube and preheated at 60 °C for 3 min. Then, 100 μL of enzyme solution diluted to an appropriate concentration was added, and the mixture was stirred thoroughly and reacted for 10 min. Finally, 1 mL of DNS reagent was added, and the mixture was boiled in a water bath for 5 min. The absorbance was measured at 540 nm. The enzyme solution inactivated at high temperature served as a blank control.

[0028] Enzyme activity definition: Under the above conditions, the amount of enzyme required to hydrolyze salicin to produce 1 μmol of reducing sugar (calculated as glucose) per minute is defined as 1 unit of enzyme activity (U).

[0029] (2) Analytical methods for reaction products The sample, diluted to an appropriate ratio, was centrifuged. The supernatant was then filtered through a 0.22 μm aqueous filter membrane. The carbohydrate components of the sample were analyzed using a high-performance anion exchange chromatography system equipped with a pulsed amperometric detector. The analytical conditions were: Thermo Fisher Scientific ICC-5000, CarboPac PA 200 column, gradient elution with 100 mM NaOH and 500 mM NaAc solutions, flow rate 0.5 mL / min, column temperature 35℃, injection volume 10 μL, and external standard method for quantification. The yield of gentiobiose oligosaccharides in the product was calculated as follows: The yield of gentiobiose oligosaccharides = (total mass of gentiobiose oligosaccharides / dry weight of substrate) × 100%.

[0030] Raw materials used in the examples: SDS-PAGE gel preparation kit, protein molecular weight standard, DNA loading buffer, DNA molecular weight standard, Dpn I enzyme, etc., were purchased from Beyotime Biotechnology Research Institute; plasmid mini-extraction kit, supercompetent cell preparation kit, ampicillin, kanamycin, etc., were purchased from Sangon Biotech (Shanghai) Co., Ltd.; yeast extract and tryptone were purchased from Oxoid Ltd., UK; sodium chloride, agar powder, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, and glycerol were purchased from Sinopharm Shanghai Laboratory; secondary mother liquor of crystalline glucose was purchased from Heilongjiang Jinxiang Biochemical Co., Ltd.; STAR Primer GXL kit was purchased from TaKaRa.

[0031] LB liquid medium: 0.5% (w / v) yeast extract, 1% (w / v) tryptone, 1% (w / v) sodium chloride, pH 7.0.

[0032] LB solid medium: 1.5% (w / v) agar is added to LB liquid medium.

[0033] TB liquid medium: 2.4% (w / v) yeast extract, 1.2% (w / v) tryptone, 17 mM KH2PO4, 72 mM K2HPO4·3H2O, 0.5% (w / v) glycerol, pH 7.0.

[0034] E. coli E. coli JM109 and Bacillus subtilis B. subtilis All WB600 strains are commercial strains.

[0035] Plasmids pP43NMK, pHT01, pHT43, and pHCMC05 are all commercial plasmids.

[0036] The secondary mother liquor of crystalline glucose contains 60%~80% glucose, 5%~10% maltose, 1%~5% isomaltose, 0.1%~5% maltotriose, 0.5%~5% isomalttriose, 1%~5% panose, and 1%~5% maltotetraose by mass fraction.

[0037] Example 1: Preparation of β-glucosidase mutant (1) Based on publicly available data from NCBI Thermotoga maritima The protein sequence of STB25 β-glucosidase was used as a template (SEQ ID NO.1) to construct the vector pP43NMK. The restriction sites NcoI and XhoI were used, with GC bases added at the NcoI restriction site to prevent frameshift. No stop codon was added at the end of the target gene to obtain the recombinant expression vector pP43NMK / tm-bgl.

[0038] (2) Following the steps above, replace plasmid pP43NMK with pHT01, pHT43, and pHCMC05 to obtain recombinant expression vectors pHT01 / tm-bgl, pHT43 / tm-bgl, and pHCMC05 / tm-bgl.

[0039] (3) The recombinant expression vector obtained in the above steps was transformed into Bacillus subtilis WB600, plated on LB plates containing antibiotics, and transformants were picked for sequencing and colony PCR verification to extract the recombinant plasmid containing the β-glucosidase gene. The recombinant plasmid was then transformed into Bacillus subtilis WB600. B. subtilis In WB600, genetically engineered bacteria were obtained. B. subtilis WB600 / pP43NMK / tm-bgl, B. subtilis WB600 / pHT01 / tm-bgl, B. subtilis WB600 / pHT43 / tm-bgl, B. subtilis WB600 / pHCMC05 / tm-bgl .

[0040] (4) Based on the amino acid sequence of the β-glucosidase coding gene shown in SEQ ID NO.1, primers for introducing mutations G343L, G343V, G343H, G343T, G343D, I455A, and A468G were designed and synthesized. Site-directed mutagenesis was performed on the β-glucosidase gene, and the DNA coding sequence was determined. The following mutations were identified: Gly codon at position 343 changed to Leu codon; Gly codon at position 343 changed to Val codon; Gly codon at position 343 changed to His codon; Gly codon at position 343 changed to Thr codon; Gly codon at position 343 changed to Asp codon; Ile codon at position 455 changed to Ala codon; and Ala codon at position 468 changed to Gly codon. The mutant gene was placed in an expression vector and introduced into Bacillus subtilis for expression to obtain a single-mutant β-glucosidase. Using rapid PCR technology, single mutants G343L, G343V, G343H, G343T, G343D, I455A, and A468G were constructed using the recombinant plasmid pP43NMK-tm-bgl as a template. Complementary primer chains required for the experiment were designed (see Table 1). The primers were synthesized by Genewiz Biotechnology Co., Ltd., and site-directed mutagenesis was performed according to the instructions of the TaKaRa STAR Primer GXL kit. To reduce the possibility of PCR errors, the upstream and downstream primers were designed to be complementary only in the middle, with the ends offset.

[0041] Table 1. Introduction of β-glucosidase mutation sites

[0042] Note: 1 The underlined bases correspond to the mutated amino acids.

[0043] This mutation was performed using a two-step PCR method. The PCR reaction system was configured according to the conditions specified in the STAR Primer kit instructions: 5×PrimeSTAR Buffer (Mg 2+ 10 μL of the primer (plus), 1 μL of template DNA, 1 μL each of the forward and reverse primers (10 μM), 1 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), 4 μL of dNTPs (2.5 mM each), and finally 32 μL of ultrapure water.

[0044] The conditions for the first PCR amplification were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 4 min 30 s, 35 cycles of amplification, and 68℃ incubation for 10 min.

[0045] The conditions for the second PCR amplification were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 9 min, 35 cycles of amplification, and 68℃ incubation for 10 min.

[0046] Example 2: Construction of genetically engineered bacteria The PCR products obtained in Example 1 were treated with Dpn I at 37°C for more than 2 hours, and then the treated PCR products were transformed into Escherichia coli. E. coli JM109 was plated onto LB agar medium containing 100 μg / mL ampicillin and incubated overnight at 37°C for 12 h. Single colonies were selected and inoculated into LB liquid medium containing 100 μg / mL ampicillin, and incubated overnight at 37°C and 200 r / min. Plasmids were then extracted, identified, and sequenced according to the plasmid extraction kit instructions. The plasmid containing the correctly sequenced β-glucosidase mutant gene and the recombinant plasmid pP43NMK-tm-bgl were transformed into the expression host. B. subtilis In WB600 competent cells, genetically engineered bacteria were finally obtained. B. subtilis WB600 / pP43NMK / tm-bgl / G343L、 B. subtilis WB600 / pP43NMK / tm-bgl / G343V、 B. subtilis WB600 / pP43NMK / tm-bgl / G343H、 B. subtilis WB600 / pP43NMK / tm-bgl / G343T、 B. subtilis WB600 / pP43NMK / tm-bgl / G343D、 B. subtilis WB600 / pP43NMK / tm-bgl / I455A、 B. subtilis WB600 / pP43NMK / tm-bgl / A468G、 B. subtilis WB600 / pP43NMK / tm-bgl .

[0047] Example 3: Expression of β-glucosidase mutant (1) The single colony prepared in Example 2 B. subtilis WB600 / pP43NMK / tm-bgl / G343L、 B. subtilis WB600 / pP43NMK / tm-bgl / G343V、B. subtilis WB600 / pP43NMK / tm-bgl / G343H、 B. subtilis WB600 / pP43NMK / tm-bgl / G343T、 B. subtilis WB600 / pP43NMK / tm-bgl / G343D、 B. subtilis WB600 / pP43NMK / tm-bgl / I455A、 B. subtilis WB600 / pP43NMK / tm-bgl / A468G was inoculated into 50 mL of LB liquid medium containing 20 μg / mL kanamycin and cultured in shake flasks at 37℃ and 200 rpm for 8–12 h to prepare seed culture.

[0048] (2) The seed liquid prepared above was transferred to 50 mL of TB liquid culture medium containing 20 μg / mL kanamycin at an inoculation rate of 2%~4% (v / v), and then placed in a shake flask at 25~30℃ and 200 rpm for 48~96 h to prepare fermentation broth.

[0049] (3) Centrifuge the fermentation broth at 4℃ and 10000 rpm for 20 min, and collect the supernatant as the crude enzyme solution of β-glucosidase mutant.

[0050] Example 4: Expression and Enzyme Activity Determination of Wild-Type and Mutant β-Glucosidase Take the recombinant strain obtained in Example 2 out of the refrigerator. B. subtilis WB600 / pP43NMK / tm-bgl / G343L、 B. subtilis WB600 / pP43NMK / tm-bgl / G343V、 B. subtilis WB600 / pP43NMK / tm-bgl / G343H、 B. subtilis WB600 / pP43NMK / tm-bgl / G343T、 B. subtilis WB600 / pP43NMK / tm-bgl / G343D、 B. subtilis WB600 / pP43NMK / tm-bgl / I455A and B. subtilis WB600 / pP43NMK / tm-bgl / A468G、 B. subtilis WB600 / pP43NMK / tm-bgl100 μL of bacterial culture was drawn from each glycerol tube and inoculated into 50 mL LB liquid medium containing 20 μg / mL kanamycin. The culture was incubated at 37°C with shaking for 10–12 h to obtain the culture medium. 2 mL of this culture medium was then transferred to 50 mL TB medium containing 20 μg / mL kanamycin and incubated at 30°C for 48 h. After centrifugation at 10,000 rpm for 20 min at 4°C, the supernatant was collected as the crude enzyme solution containing wild-type and mutant G343L, G343V, G343H, G343T, G343D, I455A, and A468G. The crude enzyme solution was analyzed by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown, the results indicate that both wild-type and mutant β-glucosidase were expressed, and, from Figure 1 The SDS-PAGE electrophoresis results of crude enzyme solutions from wild-type and mutant strains showed that the expression level of mutants was correspondingly increased.

[0051] The enzyme activities of the above crude enzyme solutions catalyzing the hydrolysis reaction were measured, and the results are shown in Table 2. It can be seen that the enzyme activities of G343L, G343V, G343H, and A468G were all increased compared with the wild type. Among them, the enzyme activity of mutant G343H was increased by 220.9% compared with the wild type, while the enzyme activities of mutants G343T and G343D were decreased.

[0052] Table 2. Enzyme activities of different β-glucosidases in crude enzyme solution

[0053] Example 5: Preparation of gentiooligosaccharides by reacting β-glucosidase with the secondary mother liquor of crystalline glucose. In this embodiment, β-glucosidase prepared from wild type and its mutants was used to prepare oligogentisoses using secondary mother liquor of crystalline glucose as substrate. The specific steps are as follows.

[0054] (1) Adjust the pH of the secondary mother liquor of crystalline glucose with a glucose content of about 480 g / L to 6.0.

[0055] (2) The substrate solution was preheated in a water bath shaker at 60°C for 10 min. Then, β-glucosidase produced by wild type (amino acid sequence as shown in SEQ ID NO.1) and its mutants G343L, G343V, G343H, G343T, G343D, I455A, and A468G was added at an enzyme dosage of 50 U / g glucose. The reaction was carried out in a water bath shaker at 60°C for 24 h. Finally, the reaction was terminated by transferring the solution to boiling water for 30 min to inactivate the enzyme.

[0056] (3) After enzyme inactivation treatment, the reaction solution was centrifuged at 10,000 rpm for 10 min. The supernatant was diluted to an appropriate ratio and then passed through a 0.22 μm aqueous membrane to remove impurities. The sugar composition and content in the reaction product were analyzed by ion chromatography. The results are shown in Table 3 and Figure 2 As shown, using the secondary mother liquor of crystalline glucose as a substrate, the β-glucosidase prepared by the mutant significantly improved the yield of oligogentiobiose compared to the wild type. Among them, G343H showed the best effect, reaching 22.12%, which is 66.44% higher than the wild type.

[0057] Table 3. Yield of gentiobiose from secondary mother liquor of crystalline glucose treated by wild-type β-glucosidase and its mutants.

[0058] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A β-glucosidase mutant, characterized in that, The β-glucosidase mutant was obtained by performing any of the following mutations on the β-glucosidase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The glycine at position 343 is mutated to histidine or valine; (2) The isoleucine at position 455 is mutated to alanine.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vectors are expressed using plasmids pP43NMK, pHT01, pHT43, or pHCMC05.

5. Recombinant cells expressing the mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of claim 3 or 4.

6. A method for improving the ability of β-glucosidase to produce oligogentiobiose, characterized in that, The method involves performing one of the following mutations on the β-glucosidase parent cell containing the amino acid sequence shown in SEQ ID NO.1: (1) The glycine at position 343 is mutated to histidine or valine; (2) The isoleucine at position 455 is mutated to alanine.

7. A method for producing gentiobiose oligosaccharides, characterized in that, The method is as follows: the β-glucosidase mutant of claim 1 is added to a reaction system with glucose as a substrate for transformation.

8. The method according to claim 7, characterized in that, The reaction conditions of the reaction system are 40~80℃ and pH 3.0~6.0 for 12~60 h.

9. The method according to claim 7, characterized in that, The concentration of the substrate is at least 480 g / L; the amount of β-glucosidase added to the reaction system is at least 50 U / g.

10. The use of the β-glucosidase mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3 or 4, or the recombinant cell of claim 5, or the method of claim 6, or the method of any one of claims 7 to 9 in the preparation of gentiobiose oligosaccharide or products containing gentiobiose oligosaccharide.

Images