β-glucosidase mutants and their application in the preparation of laminaribiose

Abstract

The present invention discloses β-glucosidase mutants and their use in the preparation of laminaribiose, belonging to the field of enzyme engineering technology. The present invention constructs a series of β-glucosidase mutants with improved transamination / hydrolysis ratios through site-directed mutagenesis. The optimal combination, W324M / H298F, achieves a maximum yield of 11.41% for the synthesis of laminaribiose under optimal conditions, with a product concentration of 91.3 g / L, exceeding previously reported levels and demonstrating good potential for industrial application.

Description

Technical Field

[0001] The invention relates to a beta-glucosidase mutant and application thereof in the preparation of laminaribiose, belonging to the technical field of enzyme engineering. Background Art

[0002] β-Glucosidase (BGL, EC 3.2.1.21) is a glycoside hydrolase (GH) that specifically hydrolyzes the β-D-glucosidic bond at the non-reducing end of oligosaccharide or glycoside derivative substrates, releasing glucose and the corresponding ligand. Most characterized β-glucosidases utilize a conserved acid-base double displacement mechanism, encompassing two catalytic processes: glycosylation and deglycosylation. It is generally believed that conserved glycoside hydrolases have the potential to transfer glycosyl groups to non-water-containing acceptor ligands during the deglycosylation of the glycosyl-enzyme covalent complex, thereby catalyzing transglycosidation reactions. β-glucosidases can mediate transglycosylation and condensation reactions of glucose derivatives and have activity in the synthesis of oligosaccharides, alkyl glycosides, and vitamin derivatives. The synthetic and hydrolytic potential of β-glucosidases, particularly those derived from thermophiles, has attracted considerable attention in industrial technology. For example, Le Gao et al. discovered a β-glucosidase from Penicillium that not only has the hydrolysis activity of 4-nitrophenyl β-D-pyranoglucoside (pNPG) and cellobiose, but can also use glucose as a substrate to generate sophorose through transglycosidation. Compared with glycosyltransferases, which require expensive nucleotide sugars as substrates to generate oligosaccharides, β-glucosidase can use cheaper substrates, such as monosaccharides or disaccharides to generate oligosaccharides. Therefore, β-glucosidase has great advantages in industrial synthetic production.

[0003] Laminaribiose is a disaccharide composed of two glucose units connected by a β-1,3 bond. It is generally obtained by hydrolysis or acetic acid hydrolysis of natural plant polysaccharides such as laminarin, and is widely used in the food and agricultural fields. Using β-glucosidase with high transglycosidic activity and high-concentration glucose as a substrate, disaccharides with different bond types can be synthesized, but the synthetic products are mostly based on the disaccharide gentiobiose connected by β-1,3 bonds, and the yield of laminaribiose is generally low. The present invention successfully obtained a mutant with a significantly improved ratio and yield of synthesized laminaribiose by molecularly designing the β-glucosidase TsBgl1 with high transglycosidic activity (described in the Chinese patent application with publication number CN111411117A), which has good application value. Summary of the Invention

[0004] The present invention first provides a β-glucosidase mutant, which is obtained by mutating the amino acid at position 298, position 246 and / or position 324 of the β-glucosidase with an amino acid sequence as shown in SEQ ID NO.1.

[0005] In one embodiment of the present invention, the β-glucosidase mutant is: the β-glucosidase mutant is obtained by mutating the histidine at position 298 to phenylalanine and the tryptophan at position 324 to methionine in the β-glucosidase shown in the amino acid sequence of SEQ ID NO.1, and is named W324M / H298F;

[0006] Alternatively, the amino acid sequence of β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating histidine at position 298 to methionine and mutating tryptophan at position 324 to methionine, and the result is named W324M / H298M;

[0007] Alternatively, the amino acid sequence of the β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating position 246 from threonine to arginine and position 324 from tryptophan to methionine, and the result is named W324M / T246R.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the wild-type β-glucosidase is shown as SEQ ID NO.2.

[0009] The present invention also provides a gene encoding the above-mentioned β-glucosidase mutant.

[0010] The present invention also provides an expression vector carrying the gene.

[0011] The present invention also provides a microbial cell carrying the above gene or the above expression vector.

[0012] In one embodiment of the present invention, the microbial cells include but are not limited to bacteria and fungal cells.

[0013] The present invention also provides a method for preparing laminaribiose, which comprises adding the above-mentioned β-glucosidase mutant to a reaction system containing high-concentration glucose to carry out a reaction to obtain a reaction liquid; and separating galacto-oligosaccharides from the reaction liquid.

[0014] In one embodiment of the present invention, the added enzyme amount of the β-glucosidase mutant is: 300-700 U / g glucose.

[0015] In one embodiment of the present invention, in the reaction system, the concentration of the substrate lactose is 500-1000 g / L.

[0016] In one embodiment of the present invention, the pH of the reaction is 5.8-6.2.

[0017] In one embodiment of the present invention, the pH of the reaction is 6.0.

[0018] In one embodiment of the present invention, the reaction temperature is 75-85°C

[0019] In one embodiment of the present invention, the reaction temperature is 80°C.

[0020] In one embodiment of the present invention, the reaction time is 2-8 hours.

[0021] In one embodiment of the present invention, the reaction time is 6 hours.

[0022] The present invention also provides use of the mutant, the gene, the expression vector or the microbial cell in preparing laminaribiose.

[0023] Beneficial effects

[0024] The β-glucosidase mutants of the present invention produce laminaribiose with significantly higher yields and purity than the wild type. The optimal mutant combination, W324M / H298F, achieves a maximum yield of 11.41% and a product concentration of 91.3 g / L under optimal conditions, exceeding previously reported levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Analysis of β-glucosidase mutant W324M / H298F enzyme conversion products. A, Effect of enzyme dosage on laminaribiose production; B, Time course curves of accumulation of each product. DETAILED DESCRIPTION

[0026] 4-Nitrophenyl β-D-glucopyranoside and glucose involved in the following examples were purchased from Shanghai Vita Chemical Reagent Co., Ltd., and Escherichia coli JM109 competent cells were purchased from Shanghai Sangon.

[0027] The culture medium involved in the following examples is as follows:

[0028] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0029] LB solid medium: Add 10 g / L agar to LB liquid medium.

[0030] TB liquid fermentation medium: yeast powder 24 g / L, glycerol 5 g / L, tryptone 12 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.31 g / L.

[0031] The detection methods involved in the following embodiments are as follows:

[0032] Determination of β-glucosidase activity:

[0033] The reaction system consists of 1 mL of acetic acid buffer (pH 5.0), 960 μL of acetate buffer, 20 μL of a moderately diluted crude enzyme solution (ideally, the absorbance at 405 nm should be between 0.2 and 1.2 at the time of reaction termination) and 20 μL of 100 mmol / L pNPG. The reaction is incubated in a 60°C water bath for 10 minutes. Immediately after 10 minutes, 200 μL of 1 mol / L Na₂CO₃ solution is added to terminate the reaction. The reaction is then incubated on ice for 5 minutes, and absorbance is measured at 405 nm. A blank is prepared by treating the enzyme solution inactivated by heat in the same manner.

[0034] Definition of enzyme activity unit: One enzyme activity unit is the enzyme activity that hydrolyzes pNPG to produce 1 μmol of p-nitrophenol per minute per milliliter of enzyme solution.

[0035] Relative enzyme activity calculation method: enzyme activity = (A 405 +0.002)*reaction system*dilution factor / (0.0074*reaction time*enzyme amount)

[0036] β-glucosidase-catalyzed synthesis of laminaribiose and gentiobiose reaction system and product content detection method:

[0037] Different concentrations of glucose substrate were dissolved in phosphate-citrate buffer at pH 6.0, and the enzyme solution was added, followed by 10 mL of buffer. The reaction tubes were placed in a shaker at a constant temperature and allowed to react at 1500 rpm. Samples were taken at various time points, inactivated by boiling, and centrifuged to obtain the supernatant. The supernatant was diluted with ultrapure water and analyzed by high-performance liquid chromatography.

[0038] Detection method: Agilent 1200 liquid chromatograph with differential detection was used for detection. The column specifications were HYPERSILAPS2 column (250×4.6 mm, 5 μm), the column temperature was set to 35°C, the flow rate was 0.8 mL·min-1, and the mobile phase was 78% acetonitrile / water solution.

[0039] Calculation of laminaribiose yield: laminaribiose yield = laminaribiose mass concentration / substrate glucose mass concentration * %

[0040] Example 1: Expression of wild-type β-glucosidase gene

[0041] The recombinant Bacillus subtilis strain B. subtilis / pBSMμL3-Tsbgl1A containing wild-type β-glucosidase was constructed in the laboratory (the construction method is described in Example 2 of Chinese patent application publication number CN111411117A, [paragraphs 0071-0077]).

[0042] With reference to the Chinese patent application with publication number CN111411117A, the inducible expression of wild-type β-glucosidase was performed. The recombinant Bacillus subtilis strain B.subtilis / pBSMμL3-Tsbgl1A was inoculated from a glycerol tube stored in the laboratory in the early stage and grown in LB liquid medium (containing 100 mg / L ampicillin) for 8 h to obtain a seed solution. The seed solution was inoculated into TB liquid fermentation medium (containing 10 mg / L tetracycline) at a 5% (v / v) inoculation amount, cultured at 37°C for 2 h, and then transferred to a 33°C shaker for further culture and fermentation for 24 h to obtain a fermentation broth; the fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the cells were collected. 50 mL of 50 mM pH 6.0 citric acid-disodium hydrogen phosphate buffer was added to the cells. After the cells were fully resuspended, the cells were broken using a high-pressure homogenizer. After centrifugation at 10000 rpm for 20 min, the broken cell supernatant was collected as the crude enzyme solution, and the OD 600 The enzyme activity of the crude enzyme solution with a concentration of 5 was 10.41 U / mL.

[0043] Example 2: Construction and expression of β-glucosidase single mutants

[0044] (1) Preparation of mutants

[0045] According to the gene sequence of the wild-type β-glucosidase as shown in SEQ ID NO.2, H298F, H298M, T246Q, T246R, W324F, W324M and W324L mutant primers were designed and synthesized, and site-directed mutagenesis of the wild-type β-glucosidase was performed. The coding genes of the β-glucosidase mutants were respectively sequenced to confirm whether they were correct. The vector carrying the mutant gene was introduced into Bacillus subtilis for expression to obtain the β-glucosidase mutant.

[0046] PCR amplification of site-directed mutant encoding genes: Using rapid PCR technology, the expression vector pBSMμL3-tsbgl carrying the β-glucosidase gene (recorded in the Chinese patent application with publication number CN111411117A) was used as a template, and the designed mutation primers were used to obtain the mutant plasmids pBSMμL3-tsbgl-H298F, pBSMμL3-tsbgl-H298M, pBSMμL3-tsbgl-T246Q, pBSMμL3-tsbgl-T246R, pBSMμL3-tsbgl-W324F, pBSMμL3-tsbgl-W324M and pBSMμL3-tsbgl-W324L by PCR.

[0047] Using the mutant plasmid pBSMμL3-tsbgl-W324M as a template, PCR was performed using H298F, H298M, and T246R mutant primers, respectively, to obtain the double mutant plasmids pBSMμL3-tsbgl-W324M / H298F, pBSMμL3-tsbgl-W324M / H298M, and pBSMμL3-tsbgl-W324M / T246R.

[0048] The PCR reaction system was as follows: 5×PS buffer 10 μL, dNTPs Mix (2.5 mmol·L -1 )4μL, forward primer (10μmol·L-1)1μL, reverse primer (10μmol·L -1 ) 1 μL, template DNA 1 μL, PrimerStar HS (5 U / μL) 0.5 μL, and distilled water to 50 μL.

[0049] The PCR amplification program was as follows: initial denaturation at 94°C for 5 minutes; followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and extension at 72°C for 7 minutes 50 seconds; and finally, extension at 72°C for 10 minutes, followed by incubation at 4°C. PCR products were detected by electrophoresis on 1% agarose gels.

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] Dpn I was added to the verified PCR product and incubated at 37°C in a water bath for 2 hours to degrade the template. The product was then transformed into competent E. coli JM109 cells. The transformation product was spread onto LB solid medium containing 100 mg / L ampicillin and incubated at 37°C for 10-12 hours. Positive clones were selected and inoculated into LB liquid medium and incubated at 37°C for 8-10 hours. The correctly sequenced recombinant bacteria were inoculated from the glycerol tube into LB liquid medium and cultured overnight. The plasmid was extracted and transformed into competent expression host B. subtilis WS11 cells to obtain recombinant strains capable of expressing mutants H298F, H298M, T246Q, T246R, W324F, W324M, and W324L.

[0054] (2) Expression of mutants

[0055] Single colonies of the recombinant strains expressing mutants H298F, H298M, T246Q, T246R, W324F, W324M and W324L obtained in step (1) were picked and inoculated into LB liquid medium (containing 100 mg / L ampicillin) for 8 h to obtain seed liquid; the seed liquid was inoculated into TB liquid fermentation medium (containing 10 mg / L tetracycline) at a 5% (v / v) inoculation amount, cultured at 37°C for 2 h, and then transferred to a 33°C shaker for further culture and fermentation for 24 h to obtain fermentation liquid; the fermentation liquid was centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected. 50 mL of 50 mM pH 6.0 citric acid-disodium hydrogen phosphate buffer was added to the bacteria, the bacteria were fully resuspended, and the cells were broken using a high-pressure homogenizer. After centrifugation at 10000 rpm for 20 min, the broken cell supernatant was collected as the crude enzyme liquid, and the OD was detected. 600 The activity of β-glucosidase in the crude enzyme solution was 5. The activity of β-glucosidase in the crude enzyme solution obtained by fermentation of the recombinant strains of each mutant was 3.9-13.6 U / mL, indicating that the recombinant strains all successfully expressed the β-glucosidase mutants.

[0056] Table 2 Enzyme activities of β-glucosidase mutants

[0057]

[0058]

[0059] Example 3: Purification of β-glucosidase

[0060] The specific steps are as follows:

[0061] (1) Add 175 g of solid ammonium sulfate to 500 mL of the crude enzyme solution obtained in Example 2 and salt out for 12 h;

[0062] (2) The crude enzyme solution after salting out obtained in step (1) was centrifuged at 4°C and 10,000 rpm for 20 min, and the precipitate was dissolved in an appropriate amount of buffer A containing 20 mM sodium phosphate, 0.5 M sodium chloride, 20 mM imidazole, pH 7.4, and dialyzed in buffer A for 10 h. The sample was then filtered through a 0.22 μm membrane to prepare a loading sample;

[0063] (3) After the Ni affinity column is equilibrated with buffer A, the sample obtained in step (2) is aspirated into the Ni column and completely adsorbed. The column is then eluted with 100 mL of buffer A, 100 mL of buffer A containing 60 mM imidazole, and 100 mL of buffer A containing 480 mM imidazole, respectively, at a flow rate of 1 mL / min. The target protein β-glucosidase is eluted with buffer A containing 480 mM imidazole, and the eluate is collected.

[0064] (4) The eluate obtained in step (3) (buffer A containing 480 mM imidazole) was dialyzed for 10 h against a 50 mM sodium phosphate buffer at pH 6.0 to obtain a purified β-glucosidase enzyme product.

[0065] After purification, the wild-type and mutant β-glucosidase enzyme products reached electrophoretic purity with an apparent molecular weight of 45,000 Daltons.

[0066] Example 4: Transamination / hydrolysis ratio (R s / R h )

[0067] The transamination / hydrolysis ratio (R) of the enzyme was determined using pNPG as substrate. s / R h ), among the reaction product components, pNP is the total reaction product, glucose is the hydrolysis reaction product, pNPG2 is the transglycoside reaction product, and the transglycoside / hydrolysis ratio (R s / R h ) is the ratio of pNPG2 production to glucose production.

[0068] Transglycosidase hydrolysis reaction system (100 μL): To the water solvent, add 90 μL of pNPG substrate and 10 μL of the purified β-glucosidase preparation obtained in Example 3. The final concentration of pNPG substrate is 50 mM, and the final concentration of the enzyme solution is 5 μM. Transglycosidase hydrolysis reaction conditions: pH 6.0, 80°C, 10 min.

[0069] To determine the molar amount of pNP produced: Add 160 μL of reaction solution to 40 μL of 1 M NaCO₃, and measure the absorbance of 180 μL on a microplate. To determine the molar amount of glucose produced: Add 300 μL of glucose oxidase (GOD) to 18 μL of reaction solution, react for 10 minutes, and measure the absorbance of 200 μL on a microplate. The molar amount of transglycosidation product produced is the difference between the total reaction product and the hydrolysis product.

[0070] The results are shown in Table 3. It was found that without considering the secondary hydrolysis, the transglycosidation / hydrolysis ratio (R s / R h ) were significantly increased compared with the wild type.

[0071] Table 3 Transamination / hydrolysis ratio of mutant enzymes (Rs / Rh)

[0072]

[0073] Example 5: Application of β-glucosidase mutants in the preparation of laminaribiose

[0074] The specific steps are as follows:

[0075] Using 800 g / L glucose as substrate, the reaction was carried out at pH 6 and 90℃ for 24 h. The enzyme addition amount was set to 500 U / g glucose. The yield of laminarin synthesized by wild-type and mutant β-glucosidase using reverse hydrolysis activity using high concentration glucose as substrate was investigated.

[0076] The experimental results, shown in Table 4, show that the wild-type enzyme produced only 2.18% laminariose, while some mutants showed improved yields. The single-point mutations H298M, T246R, and W324M produced laminariose yields of 5.18%, 5.95%, and 9.18%, respectively. Combining these mutants, the double mutants W324M / H298F, W324M / H298M, and W324M / T246R, produced laminariose yields of 10.78%, 6.26%, and 7.81%, respectively, representing 4.9, 2.9, and 3.6 times that of the wild-type enzyme. In addition, the ratio of laminaribiose (β-1,3 bond product) to gentiobiose (β-1,6 bond product) in the products of the three double mutants was also greatly improved, and the purity of laminaribiose in the products increased from 15.2% in the wild type to 46.7%, 27.8% and 30.8%, respectively.

[0077] Table 4 Yield, purity and β-1,3 / β-1,6 bond product ratio of laminarin synthesized by mutant enzymes

[0078]

[0079]

[0080] aPurity is the percentage of laminarin in the total disaccharide products

[0081] b The β-1,3 bond product is laminaribiose, the β-1,6 bond product is gentiobiose, and the β-1,3 / β-1,6 ratio is the ratio of the yields of the two products.

[0082] Example 6: Application of β-glucosidase mutant W324M / H298F in the preparation of laminaribiose

[0083] The specific embodiment of preparing laminaribiose is as in Example 5, except that the enzyme dosage of the purified β-glucosidase mutant W324M / H298F was adjusted to 300 U / g, 400 U / g, 500 U / g, 600 U / g, and 700 U / g glucose, respectively. The results showed that when the enzyme dosage of the β-glucosidase mutant W324M / H298F was 700 U / g glucose, the yield of laminaribiose production was as high as 11.41%, and the product concentration reached 91.3 g / L, which is higher than the currently reported ( Figure 1 A).

[0084] The time course analysis of the accumulation of each product found that as the reaction time progressed, the substrate glucose was gradually digested and disaccharide products with different bond types were synthesized. When the reaction was carried out for 36-48h, the yield of each product reached equilibrium ( Figure 1 B).

[0085] Comparative Example 1:

[0086] The specific implementation method for preparing laminariabiose is as described in Example 5, except that the β-glucosidase is adjusted to a wild β-glucosidase enzyme derived from Talaromycespiceae (described in the Chinese patent application publication No. CN111411117A) and a wild β-glucosidase enzyme derived from Talaromyces cellulolyticus (GenBank: GAM40530.1). The results are as follows: the yields of laminariabiose production are 2.6% and 4.3%, respectively.

[0087] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A β-glucosidase mutant, characterized in that The mutant is obtained by mutating the 324th position of the β-glucosidase with the amino acid sequence shown in SEQ ID NO. 1 from tryptophan to methionine.

2. A β-glucosidase mutant, characterized in that The amino acid sequence of β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating the 298th histidine to phenylalanine and the 324th tryptophan to methionine.

3. A β-glucosidase mutant, characterized in that The amino acid sequence of β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating the 298th histidine to methionine and the 324th tryptophan to methionine.

4. A β-glucosidase mutant, characterized in that The amino acid sequence of β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating the 246th position of threonine to arginine and the 324th position of tryptophan to methionine.

5. A gene encoding the β-glucosidase mutant according to any one of claims 1 to 4.

6. An expression vector carrying the gene according to claim 5.

7. A microbial cell carrying the gene according to claim 5 or the expression vector according to claim 6.

8. The microbial cell according to claim 7, characterized in that The microbial cells include bacterial cells or fungal cells.

9. A method for preparing laminariabiose, characterized in that: The beta-glucosidase mutant according to any one of claims 1 to 4 is added to a reaction system containing high-concentration glucose to carry out a reaction to obtain a reaction liquid; and laminaribiose is separated from the reaction liquid.

10. The method according to claim 9, characterized in that In the reaction system, the added enzyme amount of the β-glucosidase mutant is 300-700 U / g glucose, and the concentration of substrate glucose is 500-1000 g / L.

11. The method according to claim 9, characterized in that The reaction pH is 5.8-6.2 and the temperature is 75-85°C.

12. Use of the β-glucosidase mutant according to any one of claims 1 to 4, the gene according to claim 5, the expression vector according to claim 6, or the microbial cell according to claim 7 or 8 in the preparation of laminaribiose.

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