Beta-glucosidase mutant and application thereof in preparation of gentiooligosaccharide

Abstract

The invention discloses a beta-glucosidase mutant and application of the beta-glucosidase mutant in preparation of gentiooligosaccharide, and belongs to the technical field of enzyme engineering and functional oligosaccharide preparation. According to the invention, site-directed mutagenesis is carried out on a beta-glucosidase gene derived from Thermotoga maristima STB25, and high-efficiency expression of the beta-glucosidase gene in bacillus subtilis is realized. Beta-glucosidase prepared from the mutant is added into a reaction system with 480 g / L glucose as a substrate, reaction is carried out under the conditions that the pH is 6.0 and the temperature is 60 DEG C, and the yield of gentiobiose oligomer can reach 22.12% and is increased by 66.44% compared with that of a wild type. The beta-glucosidase mutant provided by the invention has good acid stability, thermal stability and glucoside conversion capability, can generate gentiobiose through catalysis by taking glucose as a substrate, and has a relatively good application prospect in the field of gentiooligosaccharide production.

Description

Technical Field

[0001] The present invention relates to a β-glucosidase mutant and its application in the preparation of gentiobiose oligosaccharides, belonging to the technical fields of enzyme engineering and the preparation of functional oligosaccharides. Background Art

[0002] β-glucosidase is a glycoside hydrolase that can specifically hydrolyze the β-D-glucosidic bond at the non-reducing end of the substrate, releasing glucose and the corresponding ligand. Moreover, β-glucosidase also has transglycosylation activity and can synthesize oligosaccharides with higher degrees of polymerization using monosaccharides or oligosaccharides. Therefore, it is often used in the preparation of gentiobiose oligosaccharides. According to the current research progress, the catalytic mechanism of β-glucosidase is a retention-type acid-base catalytic mechanism, including two steps: glycosylation and deglycosylation. In the first step of glycosylation, the nucleophilic catalytic residue makes a nucleophilic attack on the anomeric carbon, thereby generating a glycosyl-enzyme covalent intermediate complex. The second step is the deglycosylation process, in which the water molecule activated by acid-base catalysis acts as a nucleophilic group to break the glycosidic bond, releasing the product glucose and the corresponding ligand. At the same time, β-glucosidase can also transfer the glycosyl group to other non-water molecule receptor ligands during the deglycosylation process, thereby catalyzing the occurrence of the transglycosylation reaction.

[0003] Gentiobiose oligosaccharides are a class of oligosaccharides composed of glucose units linked by β-1,6 glycosidic bonds, including gentiobiose and a small amount of gentiotriose and gentiotetraose. Compared with other oligosaccharides, gentiobiose oligosaccharides have a unique mild bitterness and can play a role in refreshing and invigorating the mind. Therefore, they are widely used in products such as candies, chocolates, ice creams, and coffees. In addition, it is also a functional oligosaccharide that can play physiological effects such as balancing the intestinal environment and promoting the proliferation of intestinal probiotics. It is a high-value-added sugar product with great development prospects. As mentioned above, the enzymatic preparation of gentiobiose oligosaccharides precisely utilizes the transglycosylation activity of β-glucosidase, usually using high-concentration glucose as the substrate, which has advantages such as mild reaction, low energy consumption, and easy separation compared with the traditional extraction method. It is the main trend in current production.

[0004] Due to the limited sources of β-glucosidase, the conversion rate of enzymatic preparation of gentiobiose remains at a relatively low level at present, so industrial production has not been realized in China. There are mainly two ways to increase the yield of gentiobiose. One is to increase the concentration of the reaction substrate and reduce the water activity of the reaction system, thereby reducing the occurrence of hydrolysis reaction and enabling the accumulation of transglycosylation products. The second way is to improve the transglycosylation ability of the enzyme itself. Changing the balance between hydrolysis and transglycosylation can significantly increase the yield of gentiobiose. Given the certain upper limit of glucose solubility and the great difficulty in finding a new β-glucosidase suitable for industrial production, therefore, using site-directed mutagenesis to improve the enzymatic properties and product specificity of the existing β-glucosidase is of great significance for realizing the industrial preparation of gentiobiose. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned prior art, the present invention provides a β-glucosidase mutant and its application in the preparation of gentiobiose, aiming to solve the technical problem of the lack of β-glucosidase adaptable to the industrial production of gentiobiose.

[0006] The first technical solution provided by the present invention is a β-glucosidase mutant with improved gentiobiose-producing ability, and the β-glucosidase mutant is obtained by any one of the following mutations of the β-glucosidase parent with the amino acid sequence shown in SEQ ID NO.1:

[0007] (1) Glycine at position 343 is mutated to histidine, leucine or valine;

[0008] (2) Isoleucine at position 455 is mutated to alanine;

[0009] (3) Alanine at position 468 is mutated to glycine.

[0010] Specifically, the amino acid sequence of the β-glucosidase parent is as follows:

[0011] merideilsqltteekvklvvgvglpglfgnphsrvagaagethpvprlgipafvladgpaglrinptrendentyyttafpveimlastwnrdlleevgkamgeevreygvdvllapamnihrnplcgrnfeyysedpvlsgemasafvkgvqsqgvgacikhfvannqetnrmvvdtivseralreiylkgfeiavkkarpwtvmsaynklngkycsqnewllkkvlreewgfdgfvmsdwyagdnpveqlkagndmimpgkayqvnterrdeieeimealkegklseevldecvrnilkvlvnapsfkgyrysnkpdleshaevayeagaegvvllenngvlpfdenthvavfgtgqietikggtgsgdthprytisilegikernmkfdeelastyeeyikkmreteeykprtdswgtvikpklpenflsekeikkaakkndvavvvisrisgegydrkpvkgdfylsddeleliktvskefhdqgkkvvvllnigspievaswrdlvdgillvwqagqemgrivadvlvgkinpsgklpttfpkdysdvpswtfpgepkdnpqrvvyeediyvgyryydtfgvepayefgyglsytkfeykdlkiaidgetlrvsytitntgdragkevsqvyikapkgkidkpfqelkafhktkllnpgeseeisleiplrdlasfdgkewvvesgeyevrvgassrdirlrdiflvegekrfkp。

[0012] In some embodiments, the nucleotide sequence encoding the β-glucosidase mutant gene is as shown in SEQ ID NO.2. Specifically, the nucleotide sequence is as follows:

[0013]

[0014] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0015] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0016] In some embodiments, the recombinant vector uses plasmid pP43NMK, pHT01, pHT43, pHCMC05, etc. as expression vectors, and preferably uses pP43NMK as the expression vector.

[0017] 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.

[0018] In some embodiments, the recombinant cell uses Bacillus subtilis or Escherichia coli as an expression host.

[0019] The fifth technical solution provided by the present invention is a method for improving the ability of β-glucosidase to produce oligogentiobiose. The method is to perform any one of the following mutations on the β-glucosidase parent of the amino acid sequence shown in SEQ ID NO.1:

[0020] (1) Mutating glycine at position 343 to histidine, leucine or valine;

[0021] (2) Mutating isoleucine at position 455 to alanine;

[0022] (3) Mutating alanine at position 468 to glycine.

[0023] The sixth technical solution provided by the present invention is a method for producing oligogentiobiose. The method is to add the β-glucosidase mutant described in the first technical solution to a reaction system with glucose as a substrate for transformation.

[0024] In some embodiments, the reaction conditions of the reaction system are to react for 12 - 60 h under the conditions of 40 - 80 °C and pH 3.0 - 6.0.

[0025] In some embodiments, the concentration of the substrate is at least 480 g / L.

[0026] In some embodiments, the enzyme addition amount of the β-glucosidase in the reaction system is at least 50 U / g.

[0027] 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 gentiobiose oligosaccharide or products containing gentiobiose oligosaccharide.

[0028] Beneficial effects

[0029] (1) By mutating the β-glucosidase derived from Thermotoga maritima STB25, the present invention changes the key active sites of the enzyme, transforms the β-glucosidase that could not efficiently act on glucose substrates into a mutant with improved ability to produce gentiobiose oligosaccharide, expands its application scope in the field of oligosaccharide preparation, and has good use effect in the production process of preparing gentiobiose oligosaccharide with glucose-related products as substrates.

[0030] (2) The β-glucosidase mutant provided by the present invention is derived from a thermophilic bacterium. Compared with the existing β-glucosidases that are difficult to be applied 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 still maintain its activity without loss at 80°C. Therefore, it can meet the requirements for different reaction temperatures in the production of gentiobiose oligosaccharide and provides a reference solution for high-temperature industrial processes.

[0031] (3) The β-glucosidase mutant provided by the present invention can prepare gentiobiose oligosaccharide under the condition of using glucose as a substrate. Among them, the conversion rate of gentiobiose oligosaccharide in the product catalyzed by G343H is increased by 1.42 times compared with the wild type, reaching 22.12%. The high conversion rate provides a new choice for the industrial production of gentiobiose oligosaccharide and has good application prospects in the field of preparation of starch functional sugars.

[0032] In summary, by site-directed mutagenesis of the β-glucosidase gene derived from Thermotoga maritima STB25, β-glucosidase mutants G343L, G343V, G343H, I455A, A468G, G343T, and G343D with improved ability to produce gentiobiose were obtained. On the basis of the original reaction with glucose to produce gentiobiose, the yields of gentiobiose of these mutants were further increased. Among them, the yields of G343L, G343V, G343H, I455A, and A468G were increased by 1.37, 1.20, 1.42, 1.10, and 1.09 times, respectively, compared with the catalytic products of the wild-type β-glucosidase, and the industrial production effect was better. The yields of G343T and G343D were decreased by 0.35 and 0.61 times, respectively, compared with the catalytic products of the wild-type β-glucosidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 SDS-PAGE electrophoresis analysis of wild-type and mutant crude enzyme solutions. Among them, M: protein molecular weight standard, lane 1: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl, lane 2: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / G343L, lane 3: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / G343V, lane 4: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / G343H, lane 5: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / I455A, lane 6: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / A468G, lane 7: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / G343T, lane 8: extracellular crude enzyme of B. subtilis WB600 / pP43NMK / tm-bgl / G343D.

[0034] Figure 2 Yield of gentiobiose of wild-type β-glucosidase and its mutants acting on the secondary mother liquor of crystalline glucose. DETAILED DESCRIPTION OF THE INVENTION

[0035] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] Testing method:

[0037] (1) Method for determining the enzyme activity of β-glucosidase mutant (refer to the following literature: Zhu Qihui, Gao Zexin, He Laping, et al. Screening and identification of β-glucosidase-producing strains for hydrolyzing ginkgo flavonoid glycosides. China Brewing. 2017; 36(9): 5.)

[0038] Prepare a 1% (w / v) salicin solution as the substrate with 50 mM acetic acid-sodium acetate solution at pH 4.8. Pipette 900 μL into a centrifuge tube, preheat at 60 °C for 3 min, then add 100 μL of the enzyme solution diluted to an appropriate multiple, mix well and react for 10 min. Then add 1 mL of DNS reagent, boil in a boiling water bath for 5 min, and measure the absorbance at 540 nm. Use the enzyme solution inactivated at high temperature as the blank control.

[0039] Definition of enzyme activity: 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 1 enzyme activity unit (U).

[0040] (2) Method for analyzing reaction products

[0041] Centrifuge the sample diluted to an appropriate multiple, then take the supernatant and filter it through a 0.22 μm aqueous filter membrane. Analyze the carbohydrate components of the sample using a high-performance anion-exchange chromatograph equipped with a pulsed amperometric detector. The analysis conditions are as follows: Thermo Fisher Scientific ICC-5000, chromatographic column CarboPac PA 200, gradient elution with 100 mM NaOH solution and 500 mM NaAc solution, flow rate 0.5 mL / min, column temperature 35 °C, injection volume 10 μL, and quantitative analysis by external standard method. The calculation method for the yield of oligogentiobiose in the product is as follows:

[0042] Yield of oligogentiobiose = (total mass of oligogentiobiose / dry mass of substrate) × 100%.

[0043] Raw materials used in the examples:

[0044] 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 miniprep kit, super competent cell preparation kit, ampicillin, kanamycin, etc. were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; yeast powder and tryptone were purchased from Oxoid Company, UK; sodium chloride, agar powder, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, glycerol were purchased from Shanghai Reagent Co., Ltd., Sinopharm Chemical Reagent Co., Ltd.; crystalline glucose secondary mother liquor was purchased from Heilongjiang Jingxiang Biochemical Co., Ltd.; STAR Primer GXL kit was purchased from TaKaRa Company.

[0045] LB liquid medium: 0.5% (w / v) yeast extract, 1% (w / v) tryptone, 1% (w / v) sodium chloride, pH 7.0.

[0046] LB solid medium: Based on the LB liquid medium, add 1.5% (w / v) agar.

[0047] 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.

[0048] Both Escherichia coli JM109 and Bacillus subtilis WB600 are commercial strains.

[0049] Plasmids pP43NMK, pHT01, pHT43, and pHCMC05 are all commercial plasmids.

[0050] The secondary mother liquor of crystalline glucose contains 60% - 80% glucose, 5% - 10% maltose, 1% - 5% isomaltose, 0.1% - 5% maltotriose, 0.5% - 5% isomaltotriose, 1% - 5% panose, and 1% - 5% maltotetraose by mass fraction.

[0051] Example 1: Preparation of β-glucosidase mutant

[0052] (1) Using the protein sequence of β-glucosidase from Thermotoga maritima STB25 publicly available on NCBI (SEQ ID NO.1) as a template, it was constructed into the vector pP43NMK using the restriction enzyme sites NcoI and XhoI. GC bases were added at the NcoI restriction enzyme site to prevent frameshift, and no stop codon was added at the end of the target gene, resulting in the recombinant expression vector pP43NMK / tm-bgl.

[0053] (2) According to the above steps, the plasmid pP43NMK was replaced with pHT01, pHT43, and pHCMC05 to obtain the recombinant expression vectors pHT01 / tm-bgl, pHT43 / tm-bgl, and pHCMC05 / tm-bgl.

[0054] (3) The recombinant expression vector obtained in the above steps was transformed into Bacillus subtilis WB600, and the LB plate containing antibiotics was coated. The transformants were picked for sequencing and colony PCR verification, and the recombinant plasmid containing the β-glucosidase gene was extracted. The recombinant plasmids were respectively transformed into B. subtilis WB600 to obtain the genetically engineered bacteria B. subtilis WB600 / pP43NMK / tm-bgl, B. subtilis WB600 / pHT01 / tm-bgl, B. subtilis WB600 / pHT43 / tm-bgl, B. subtilis WB600 / pHCMC05 / tm-bgl.

[0055] (4) Based on the gene sequence of β-glucosidase as shown in SEQ ID NO.2, primers introducing mutations of G343L, G343V, G343H, G343T, G343D, I455A and A468G were designed and synthesized to perform site-directed mutagenesis on the β-glucosidase gene. The DNA coding sequence was determined, and it was respectively identified that the Gly codon at the 343rd position was changed to the Leu codon, the Gly codon at the 343rd position was changed to the Val codon, the Gly codon at the 343rd position was changed to the His codon, the Gly codon at the 343rd position was changed to the Thr codon, the Gly codon at the 343rd position was changed to the Asp codon, the Ile codon at the 455th position was changed to the Ala codon, and the Ala codon at the 468th position was changed to the Gly codon. The mutant gene was placed in an expression vector and introduced into Bacillus subtilis for expression to obtain single mutant β-glucosidase. Using the rapid PCR technique, with the recombinant plasmid pP43NMK-tm-bgl as the template, single mutants G343L, G343V, G343H, G343T, G343D, I455A and A468G were constructed. The complementary primer strands required for the experiment were designed (see Table 1). The primers were synthesized by Genewiz Biotechnology Co., Ltd., and site-directed mutagenesis was carried out according to the method described in the TaKaRa STAR Primer GXL kit instruction manual. To reduce the possibility of PCR errors, the upstream and downstream primers designed were only complementary in the middle part and staggered at both ends.

[0056] Table 1 Introduction of β-glucosidase mutation sites

[0057]

[0058] Note: 1 The underlined bases correspond to the corresponding mutant amino acids.

[0059] This mutation uses the two-step PCR method. The PCR reaction system follows the conditions set in the STAR Primer kit instructions: 5×PrimeSTAR Buffer (Mg 2+ Plus) 10 μL, template DNA 1 μL, forward and reverse primers (10 μM) are both 1 μL, PrimeSTAR HS DNA Polymerase (2.5 U / μL) 1 μL, dNTPs (each 2.5 mM) 4 μL, and finally add 32 μL of ultrapure water.

[0060] The conditions for the first PCR amplification are: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 68°C for 4 min 30 s, cyclic amplification for 35 cycles, and incubation at 68°C for 10 min.

[0061] The conditions for the second PCR amplification are: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 68°C for 9 min, cyclic amplification for 35 cycles, and incubation at 68°C for 10 min.

[0062] Example 2: Construction of genetically engineered bacteria

[0063] At 37°C, the PCR product obtained in Example 1 was treated with Dpn I for more than 2 h. Subsequently, the treated PCR product was transformed into Escherichia coli JM109, spread on LB agar medium containing 100 μg / mL ampicillin, and cultured overnight for 12 h in an incubator at 37°C. Single colonies were selected and inoculated into LB liquid medium containing 100 μg / mL ampicillin, cultured overnight at 37°C and 200 r / min, and the plasmid was extracted and identified by sequencing according to the method described in the plasmid extraction kit instructions. The plasmids with correct sequencing and containing the β-glucosidase mutant gene, as well as the recombinant plasmid pP43NMK-tm-bgl, were transferred into the competent cells of the expression host Bacillus subtilis WB600, and finally the genetically engineered strains Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343L, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343V, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343H, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343T, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343D, Bacillus subtilis WB600 / pP43NMK / tm-bgl / I455A, Bacillus subtilis WB600 / pP43NMK / tm-bgl / A468G, and Bacillus subtilis WB600 / pP43NMK / tm-bgl were obtained.

[0064] Example 3: Expression of β-glucosidase mutants

[0065] (1) The single colonies Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343L, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343V, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343H, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343T, Bacillus subtilis WB600 / pP43NMK / tm-bgl / G343D, Bacillus subtilis WB600 / pP43NMK / tm-bgl / I455A, and Bacillus subtilis WB600 / pP43NMK / tm-bgl / A468G prepared in Example 2 were respectively inoculated into 50 mL of LB liquid medium containing 20 μg / mL kanamycin, and cultured in a shaking flask at 37°C and 200 rpm for 8 - 12 h to prepare seed solutions.

[0066] (2) Transfer the seed solution prepared above to 50 mL of TB liquid medium containing 20 μg / mL kanamycin at an inoculation amount of 2% - 4% (v / v), and then place it in a shaker at 25 - 30 °C and 200 rpm for shaking culture for 48 - 96 h to prepare a fermentation broth.

[0067] (3) Centrifuge the fermentation broth at 4 °C and 10,000 rpm for 20 min, and collect the supernatant, which is the crude enzyme solution of the β-glucosidase mutant.

[0068] Example 4: Expression and enzyme activity determination of wild-type and mutant β-glucosidases

[0069] Take out the glycerol tubes of the recombinant strains 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-bgl obtained in Example 2 from the refrigerator. Respectively, pipette 100 μL of the bacterial solution and inoculate it into 50 mL of LB liquid medium containing 20 μg / mL kanamycin, and shake culture it in a shaker at 37 °C for 10 - 12 h to obtain a culture solution. Then, respectively pipette 2 mL of the culture solution and transfer it to 50 mL of TB medium containing 20 μg / mL kanamycin. After culturing at 30 °C for 48 h, centrifuge it at 4 °C and 10,000 rpm for 20 min, and take the supernatant, which is the crude enzyme solution containing wild-type and mutants G343L, G343V, G343H, G343T, G343D, I455A, A468G. Perform SDS-PAGE electrophoresis analysis on the crude enzyme solution respectively. The results are as Figure 1 shown. The results show that both the wild-type and mutant β-glucosidases are expressed, and, from Figure 1 the SDS-PAGE electrophoresis results of the wild-type and mutant crude enzyme solutions, it can be seen that the expression level of the mutant has been correspondingly increased.

[0070] The enzyme activities of the above-mentioned crude enzyme solutions catalyzing hydrolysis reactions were measured separately, and the results are shown in Table 2. It can be seen that the enzyme activities of G343L, G343V, G343H, and A468G are all improved compared with the wild type. Among them, the enzyme activity of the mutant G343H is increased by 220.9% compared with the wild type, while the enzyme activities of the mutants G343T, G343D, and I455A are decreased. Among them, the enzyme activity of the mutant I455A is decreased to 72.1% of the wild type.

[0071] Table 2 Enzyme activities of different β-glucosidases in the crude enzyme solution

[0072]

[0073] Example 5: Preparation of oligogentiobiose by reacting β-glucosidase with the secondary mother liquor of crystalline glucose

[0074] In this example, the β-glucosidases prepared from the wild type and its mutants were used to prepare oligogentiobiose with the secondary mother liquor of crystalline glucose as the substrate. The specific steps are as follows.

[0075] (1) Adjust the pH of the secondary mother liquor of crystalline glucose with a glucose content of about 480 g / L to 6.0.

[0076] (2) Place the substrate solution in a water bath shaker at 60 °C and preheat it for 10 min. Then, add the β-glucosidases produced by the wild type (amino acid sequence as shown in SEQ ID NO.1) and its mutants G343L, G343V, G343H, G343T, G343D, I455A, and A468G according to the enzyme addition amount of 50 U / g glucose. React in a water bath shaker at 60 °C for 24 h, and finally transfer it to boiling water to inactivate the enzyme for 30 min to terminate the reaction.

[0077] (3) After the enzyme inactivation treatment, centrifuge the reaction solution at 10000 rpm for 10 min, take the supernatant and dilute it to an appropriate multiple, and then pass it through a 0.22 μm aqueous membrane to remove impurities. That is, use ion chromatography to analyze the sugar composition and content in the reaction product. The results are shown in Table 3 and Figure 2 as shown. It can be seen that using the secondary mother liquor of crystalline glucose as the substrate, the β-glucosidase prepared by the mutant has a significant increase in the yield of oligogentiobiose catalyzed compared with the wild type. Among them, G343H has the best effect, reaching 22.12%, which is increased by 66.44% compared with the wild type.

[0078] Table 3 Yields of oligogentiobiose of wild-type β-glucosidase and its mutants acting on the secondary mother liquor of crystalline glucose

[0079]

[0080] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A β-glucosidase mutant, characterized in that, The β-glucosidase mutant is obtained by performing any one 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) The alanine at position 468 is mutated to glycine.

2. A gene encoding the mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. The recombinant vector according to claim 3, wherein The recombinant vector uses plasmid pP43NMK, pHT01, pHT43 or pHCMC05 as an expression vector.

5. A recombinant cell expressing the mutant according to claim 1, or containing the gene according to claim 2, or transformed with the recombinant vector according to claim 3 or 4.

6. A method for improving the ability of β-glucosidase to produce gentiobiose oligosaccharides, characterized in that, The method is to perform any one 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) The alanine at position 468 is mutated to glycine.

7. A method for producing gentiobiose oligosaccharide, characterized in that, The method is: adding the β-glucosidase mutant according to claim 1 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 to react for 12 to 60 h under the conditions of 40 to 80 °C and pH 3.0 to 6.

0.

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

10. Use of the β-glucosidase mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3 or 4, or the recombinant cell according to claim 5, or the method according to claim 6, or the method according to any one of claims 7 to 9 in the preparation of oligogentiobiose or a product containing oligogentiobiose.

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