Non-sugar tolerant beta-glucosidase, expression vector and application of non-sugar tolerant beta-glucosidase in improvement of cellulose degradation capability

Abstract

The invention provides non-sugar-tolerant beta-glucosidase, an expression vector and application of the non-sugar-tolerant beta-glucosidase in improvement of cellulose degradation capacity, and belongs to the technical field of genetic engineering. According to the invention, the non-sugar tolerance type beta-glucosidase BGLC27 is obtained from streptomyces decomposer C27, and the amino acid sequence of the BGLC27 is as shown in SEQ ID No. 1. According to the invention, the BGLC27 is used as an exogenous enzyme and is added into a degradation system of cellulose degradation bacteria, and research finds that after the BLGC27 is exogenously added into the compound bacteria system, the cellulose degradation capability can be obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a non-sugar-tolerant beta-glucosidase and an expression vector and applications thereof in improving cellulose degradation capability. Background Art

[0002] Lignocellulose is mainly composed of lignin, cellulose and hemicellulose. It is mainly composed of plant polysaccharide structures as its basic skeleton, and thus exists in large quantities in various plant organisms in nature. The efficient degradation of cellulose requires the synergistic action of different cellulose-degrading enzymes. Studies have found that many microorganisms can secrete cellulases. However, the gradual slowdown of cellulose hydrolysis rate is the main problem in the efficient cellulose degradation process. One of the main reasons for the reduction in its hydrolysis efficiency is product inhibition. During the degradation process of cellulose, high concentrations of products such as glucose, cellobiose and other cellooligosaccharides will produce high substrate loading conditions. Under such conditions, the inhibitory effect of products in the cellulose hydrolysis process is very significant.

[0003] β-glucosidase is a key rate-limiting enzyme in the cellulose hydrolysis process. Due to its low content in the cellulose degrading enzyme system and its dual inhibition by substrates and products, it often leads to low cellulose degradation efficiency, especially under conditions of high glucose concentrations. Adding exogenous β-glucosidase can alleviate the restriction of β-glucosidase on the cellulose degrading enzyme system. Current studies have found that all exogenously added β-glucosidases are sugar-tolerant, but the role of non-sugar-tolerant β-glucosidases is not clear. Summary of the Invention

[0004] The present invention provides a non-sugar-tolerant beta-glucosidase and an expression vector and application thereof in improving cellulose degradation ability. The non-sugar-tolerant beta-glucosidase can significantly improve the cellulose degradation ability of a composite bacterial system.

[0005] The present invention provides a non-sugar-tolerant β-glucosidase, the amino acid sequence of the non-sugar-tolerant β-glucosidase is shown in SEQ ID No. 1.

[0006] The present invention also provides a gene encoding the non-sugar-tolerant β-glucosidase.

[0007] In a preferred embodiment of the present invention, the nucleotide sequence of the gene is shown as SEQ ID No. 2.

[0008] The present invention also provides an expression vector comprising the above gene.

[0009] The present invention also provides an expression cell comprising the above gene and expressing the above non-glucose-tolerant β-glucosidase.

[0010] The present invention also provides the use of the non-sugar-tolerant β-glucosidase or the recombinant non-sugar-tolerant β-glucosidase produced by the expression cell in improving the degradation ability of cellulose-degrading bacteria.

[0011] In a preferred embodiment of the present invention, the cellulose-degrading bacteria include Streptomycetaceae sp. C27 or a composite bacterial strain containing Streptomycetaceae sp. C27; the deposit number of the Streptomycetaceae sp. C27 is GDMCC 66321.

[0012] The present invention also provides a method for improving the degradation ability of cellulose degrading bacteria, comprising adding the above-mentioned non-sugar-tolerant β-glucosidase or the recombinant non-sugar-tolerant β-glucosidase produced by the above-mentioned expression cells to the degradation system of cellulose degrading bacteria.

[0013] In a preferred embodiment of the present invention, the cellulose-degrading bacteria include Streptomyces C27 or a composite bacterial strain containing Streptomyces C27; the deposit number of the Streptomyces C27 is GDMCC 66321.

[0014] The present invention also provides a cellulose degradation system, comprising Streptomyces C27 or a composite bacterial system containing Streptomyces C27, and also comprising the above-mentioned non-sugar-tolerant β-glucosidase or a recombinant non-sugar-tolerant β-glucosidase produced using the above-mentioned expression cell; the deposit number of the Streptomyces C27 is GDMCC 66321.

[0015] Beneficial effects: The present invention obtains a non-sugar-tolerant β-glucosidase, referred to as BGLC27, from the decomposer Streptomycetaceae sp. C27. The amino acid sequence of BGLC27 is shown in SEQ ID No. 1. The present invention uses an expression vector and an expression method to express the BGLC27, and the obtained recombinant BGLC27 is added as an exogenous enzyme to the degradation system of cellulose-degrading bacteria, especially to a composite bacterial system composed of Streptomycetaceae sp. C27 and Enterobacteriaceae sp. Z23. Studies have found that the exogenous addition of the BGLC27 to the composite bacterial system can significantly improve the cellulose degradation ability.

[0016] Biological deposit information

[0017] Streptomycetaceae sp. C27 was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on May 13, 2025. The specific deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, China. The deposit number is GDMCC No: 66321.

[0018] Enterobacteriaceae sp. Z23 was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 13, 2025. The specific deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit number is GDMCCNo: 66322. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram for the construction of recombinant plasmids;

[0020] Figure 2 This is the PCR verification result diagram, where M is DNA Ladder (100 bp-5000 bp); 1 is the BGL9A gene; 2 is the BGLC27 gene; 3 is the BGLHL gene;

[0021] Figure 3 The results of colony PCR verification are shown in Figure 1. In the figure, M is DNA Ladder (100bp-5000bp); 1 is pET28a-bgl9a; 2 is pET28a-bglc27; 3 is pET28a-bglhl;

[0022] Figure 4 Figure 2 is the result of SDS-PAGE gel electrophoresis detection, in which M is a marker (10-180 kDa), 1-3 are pET28a-bgl9a uninduced, pET28a-bgl9a induced supernatant, and BGL9A purified, respectively; 4-6 are pET28a-bglc27 uninduced, pET28a-bglc27 induced supernatant, and BGLC27 purified, respectively; 7-9 are pET28a-bglhl uninduced, pET28a-bglhl induced supernatant, and BGLHL purified, respectively;

[0023] Figure 5 The results of the effect of pH on the activity of β-glucosidase are shown in Figure a. The activity of three β-glucosidases under different pH conditions is shown in Figure b. The stability of three β-glucosidases under different pH conditions is shown in Figure b.

[0024] Figure 6 The results of the effect of temperature on the activity of β-glucosidase are shown in Figure a. The activity of three β-glucosidases under different temperature conditions is shown in Figure b. The stability of three β-glucosidases under different temperature conditions is shown in Figure b.

[0025] Figure 7 This is the result diagram of the effect of glucose pressure on β-glucosidase activity;

[0026] Figure 8Figure 2 is a graph showing changes in glucose, cellobiose, and cellotriose concentrations. Figure a shows the results for BGL9A, b shows the results for BGLC27, and c shows the results for BGLHL.

[0027] Figure 9 Figure 2 is a graph showing changes in β-glucosidase hydrolysis activity and cellotriose content with glucose concentration, where a is the result for BGL9A, b is the result for BGLC27, and c is the result for BGLHL;

[0028] Figure 10 is the change of β-glucosidase activity with the addition of BGL9A;

[0029] Figure 11 The weight changes of corn straw in synthetic communities with different BGL additions. DETAILED DESCRIPTION

[0030] The present invention provides a non-sugar-tolerant β-glucosidase, the amino acid sequence of the non-sugar-tolerant β-glucosidase is shown in SEQ ID No. 1.

[0031] The non-sugar-tolerant β-glucosidase BGLC27 of the present invention is derived from Streptomycetaceae sp. C27, has strong transglycosylation activity, and its amino acid sequence is shown in SEQ ID No. 1.

[0032] The present invention also provides a gene encoding the non-sugar-tolerant β-glucosidase.

[0033] In a preferred embodiment of the present invention, the nucleotide sequence of the gene is shown as SEQ ID No. 2.

[0034] The present invention also provides an expression vector comprising the above gene.

[0035] The expression vector of the present invention can be a prokaryotic expression vector. For example, in one embodiment, the gene is inserted into the prokaryotic expression vector pET-28a(+), and the connection sites used are EcoRI and HindIII restriction sites.

[0036] In the examples of the present invention, a variety of BGLs with different properties were constructed for comparison. When constructing the corresponding prokaryotic expression vectors, the corresponding insertion sequences were amplified by PCR and then ligated to the EcoRI and HindIII restriction sites in pET-28a(+).

[0037] Table 1 Target gene PCR specific primers

[0038]

[0039] In one embodiment of the present invention, the primers shown in Table 1 were synthesized by Jilin Kumei Biotechnology Co., Ltd., and then the extracted DNA template was used to perform a PCR amplification reaction of the target gene to obtain the complete coding gene of β-glucosidase required for subsequent experiments, and the PCR reaction conditions for amplifying the β-glucosidase gene of BGL9A, BGLHL and HGLC27 were the same, and the reaction system was 25 μL: 0.2 μL DNA template, 2 μL upstream primer (100 mM), 2 μL downstream primer (100 mM), 0.3 μL La Taq, 12.5 μL 2×GC Buffer II, 4 μL dNTP Mixture and 4 μL dd H2O; the reaction procedure was: 94°C pre-denaturation for 5 min; 94°C denaturation for 0.5 min, 55-60°C annealing for 0.5 min, 72°C extension for 2 min, 30 cycles; 72°C final extension for 10 min, and the PCR product was stored at -20°C. In the present invention, genes BGL9A and BGLHL are derived from metagenomic data of a cellulose-degrading microbial functional community, wherein the nucleotide sequence of BGL9A is shown as SEQ ID No. 9; and the nucleotide sequence of BGLHL is shown as SEQ ID No. 10.

[0040] The present invention also provides an expression cell comprising the above gene and expressing the above non-glucose-tolerant β-glucosidase.

[0041] The expression cells of the present invention can be hosted by prokaryotic expression bacteria, such as Escherichia coli. In one embodiment, the plasmid of the verified correct recombinant vector is transformed into Escherichia coli competent cells (SHuffle T7 Express E. coliBChemically Competent Cell, WEIDI).

[0042] The present invention does not particularly limit the method for producing the recombinant BGLC27 using the expression cells, and the cells can be transformed using a transformation method known in the art.

[0043] The present invention also provides the use of the non-sugar-tolerant β-glucosidase or the recombinant non-sugar-tolerant β-glucosidase produced by the expression cell in improving the degradation ability of cellulose-degrading bacteria.

[0044] In a preferred embodiment of the present invention, the cellulose-degrading bacteria include Streptomycetaceae sp. C27 or a composite bacterial system containing Streptomycetaceae sp. C27, wherein the composite bacterial system is a composite bacterial system constructed by C27 and Enterobacteriaceae sp. Z23, wherein the preservation number of Streptomycetaceae sp. C27 is GDMCC 66321, and the preservation number of Enterobacteriaceae sp. Z23 is GDMCC 66322.

[0045] The present invention also provides a method for constructing the above-mentioned composite bacterial system, comprising the following steps: inoculating the spore suspension of Streptomyces C27 and the bacterial liquid of Enterobacter Z23 in equal proportions into a sodium carboxymethyl cellulose liquid culture medium, and performing shaking culture to obtain the composite bacterial system. In a preferred embodiment of the present invention, the method for preparing the spore suspension comprises inoculating the Streptomyces C27 on a PDA plate for constant temperature inverted culture, and eluting with sterile water. In one embodiment, the spore suspension is counted using the dilution spread plate method and the spore count is adjusted to 1.0×10 8 The method for preparing the bacterial solution of the present invention comprises inoculating a single colony of the Enterobacter Z23 into LB culture medium and culturing on a shaker. For example, in one embodiment, a single colony of the Z23 is inoculated into LB culture medium and cultured on a shaker at 28°C and 180 r / min until the OD 600 =0.8. The present invention converts the above 1.0×10 8 Spore suspension of 500 μg / mL and OD 600 =0.8 bacterial liquid, are inoculated into sodium carboxymethyl cellulose liquid culture medium at an inoculum size of 2%, and cultured at 30°C, 120r / min shaking table. In an embodiment of the present invention, the sodium carboxymethyl cellulose liquid culture medium comprises the following components: 20g / L CMC-Na, 1g / L KNO3, 0.5g / LMgSO4, 1g / L K2HPO4, 0.01g / LFeSO4, 0.5g / LNaCl and 20g / L agar, with a pH of 7.2-7.4, is sterilized at 121°C for 30min, and can be used after cooling.

[0046] In one embodiment of the present invention, BGLC27 with an enzyme activity of 80 U was added to the degradation system of the composite bacterial system. The changes in the β-glucosidase activity of the composite bacterial system were measured. It was found that the β-glucosidase activity of the BGLC27-treated group increased from the 6th day and reached a peak of approximately 42.57 U / mL on the 7th day. The cellulase (CMC) enzyme activity showed a significant upward trend within 1 to 6 days, the upward trend slowed down from 7 to 10 days, and reached a peak of 20.70 U / mL on the 10th day. The filter paper enzyme activity showed an overall upward trend from 1 to 7 days, reaching a peak of 13.12 U / mL on the 7th day. The corn straw degradation rate continued to increase from 1 to 10 days, reaching a maximum of 50.9% on the 10th day. This proves that BGLC27 of the present invention has the effect of promoting the cellulose degradation ability of the composite bacterial system.

[0047] The present invention also provides a method for improving the degradation ability of cellulose degrading bacteria, comprising adding the above-mentioned non-sugar-tolerant β-glucosidase or the recombinant non-sugar-tolerant β-glucosidase produced by the above-mentioned expression cells to the degradation system of cellulose degrading bacteria.

[0048] In a preferred embodiment of the present invention, the cellulose degrading bacteria include Streptomycetaceae sp. C27 or a composite bacterial strain containing Streptomycetaceae sp. C27.

[0049] In the composite bacterial system of the present invention, the volume ratio of the bacterial solution of C27 and Z23 is 1:1. In the embodiment of the present invention, the preparation method of the C27 bacterial solution includes inoculating the C27 strain on a PDA plate for constant temperature inversion culture, eluting with sterile water, and counting the spore suspension by the dilution spread plate method to adjust the spore count to 1.0×10 8 The preparation method of the Z23 bacterial solution includes inoculating a single colony of the strain into 10 mL of LB culture medium and culturing it in a shaker at 28°C and 180 rpm until the OD 600 =0.8, which is Z23 bacterial solution.

[0050] The present invention also provides a cellulose degradation system, comprising Streptomycetaceae sp. C27 or a composite bacterial system containing Streptomycetes C27, and also comprising the above-mentioned non-sugar-tolerant β-glucosidase or a recombinant non-sugar-tolerant β-glucosidase produced by the above-mentioned expression cell.

[0051] To further illustrate the present invention, the following describes in detail a non-sugar-tolerant β-glucosidase and expression vector provided by the present invention and its application in improving cellulose degradation ability in combination with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] In the present embodiment, soil samples were taken from the main campus of Northeast Agricultural University and its surrounding bushes at a depth of 10 to 15 cm rich in humus. 1 g of soil sample was mixed with 25 mL of sterile water to prepare a soil suspension, and 5 mL of the soil suspension was inoculated into 50 mL of MSM liquid culture medium. The suspension was cultured in a shaker at 180 r / min and 28°C for 3 days, and then 1 mL of the culture medium was diluted to 10 -2 to 10 -6 Five gradients, each with three replicates, were plated onto selective media containing sodium carboxymethyl cellulose and cellobiose as the sole carbon source and inverted for culture. The strains were observed daily for growth, and single colonies of varying morphology were selected from each medium for subsequent isolation and purification. Purification and verification revealed that Streptomycetaceae sp. C27 could grow on media containing sodium carboxymethyl cellulose as the sole carbon source, while Enterobacteriaceae sp. Z23 could grow on media containing cellobiose as the sole carbon source but lacked the ability to degrade cellulose.

[0054] Selective culture medium with sodium carboxymethyl cellulose as the sole carbon source: 20 g / L CMC-Na, 1 g / L KNO3, 0.5 g / LMgSO4, 1 g / L K2HPO4, 0.01 g / L FeSO4, 0.5 g / L NaCl and 20 g / L agar, pH 7.2-7.4, sterilized at 121°C for 30 min.

[0055] Selective medium with cellobiose as the sole carbon source: M9 medium supplemented with 4 g / L cellobiose.

[0056] M9 culture medium stock solution: 64g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, adjust the volume to 1L.

[0057] Take 200 mL of M9 medium stock solution, add 700 mL of water, add 2 mL of 1 mol / L sterilized MgSO4 (to prevent precipitation, the M9 medium and MgSO4 should be sterilized separately), add 4 g of cellobiose, and adjust the volume to 1 L. Sterilize at 115°C for 20 min.

[0058] A composite bacterial strain, C27 and Z23, was combined to construct a cellulose-degrading system. The degradation capacity of the cellulose-degrading strain C27 and the composite strain was measured after seven days. The C27×Z23 composite system achieved a 47.45% weight loss rate for corn straw, 92.96% higher than the weight loss rate achieved by C27 alone. Enzyme activity was also verified, showing that the filter paper enzyme activity reached its peak at 19.69 U / mL on the 10th day of cellulose degradation in the C27×Z23 composite system, exceeding the 14.18 U / mL achieved by C27 alone. Furthermore, CMCase activity peaked at 24.88 U / mL on the 9th day of cellulose degradation in the C27×Z23 composite system, exceeding the 21.96 U / mL peak achieved by C27 alone at day 10.

[0059] Example 2 Gene cloning and recombinant expression of sugar-tolerant and sugar-intolerant BGL enzymes

[0060] The total DNA of the cellulose-degrading microbial community in the soil suspension in Example 1 was used as a template to amplify the target gene using the specific primers in Table 1. The target gene was detected by 1% agarose gel electrophoresis and gel recovery was performed according to the size of the gene fragment. The amplified target gene was double-digested and ligated to the empty plasmid pET-28a(+) to construct Figure 1 The recombinant plasmids shown are pET28a-bgl9a, pET28a-bglhl and pET28a-bglc27.

[0061] The recombinant plasmid was transformed into cloning competent cells E. coli DH5α by heat shock method and cultured overnight on LB solid medium containing kanamycin. The plasmid was extracted and verified by PCR using specific primers. The positive transformants were detected by 1% agarose gel electrophoresis if a single bright band with a fragment size of about 1300 bp was found. The test results are as follows: Figure 2 The positive transformants screened were sequenced, and the sequencing results showed that the recombinant plasmid was successfully constructed and transformed.

[0062] The successfully transformed clones were expanded and cultured, and the plasmids were extracted and transformed into expression competent cells SHuffle T7 Express E.coli B Chemically Competent Cells using the heat shock method. After overnight screening and culture in LB solid medium containing kanamycin, colonies were picked and verified by colony PCR using specific primers. A single bright band was detected by agarose gel electrophoresis, indicating that the recombinant plasmid was successfully transformed into the expression competent cells and the genetically engineered bacteria were successfully constructed. The test results are as follows: Figure 3 shown.

[0063] The successfully constructed genetically engineered bacteria were activated and cultured until the concentration of the bacterial solution reached OD 600=0.6, add appropriate concentration of IPTG to induce expression, collect the bacteria and ultrasonically disrupt them, collect the supernatant, and use SDS-PAGE gel electrophoresis to detect the expression of the recombinant enzyme. The results show that the recombinant enzyme is present in the supernatant, indicating that the three recombinant enzymes can be soluble expressed in the cells. The recombinant enzyme containing 6×His tag in the supernatant is purified by Ni-NTA affinity chromatography, and the impurity protein is gradient eluted using 20mM, 50mM, 80mM, and 100mM imidazole. Then, the target protein is eluted using 500mM imidazole, and finally, it is detected by SDS-PAGE gel electrophoresis again. The detection results are as follows. Figure 4 Gel electrophoresis results showed the presence of a single band with the same size as the predicted result, indicating that the three recombinant enzymes were successfully expressed and purified.

[0064] Example 3 Enzymatic properties of the recombinant β-glucosidase constructed in Example 2

[0065] 3.1 Effect of pH on the activity of recombinant β-glucosidase

[0066] The activity changes of the three recombinant β-glucosidase under different pH conditions are as follows Figure 5 As shown in the figure, the optimal pH for BGL9A and BGLHL is around 6.5, with BGL9A exhibiting an activity of approximately 26.24 U / mg and BGLHL exhibiting an activity of approximately 24.74 U / mg. The optimal pH for BGLC27 is around 7.0, with an activity of approximately 40.03 U / mg. The enzymatic activity of BGLC27 is higher than that of BGL9A and BGLHL at all pH conditions. pH stability results indicate that BGLC27 remains stable between pH 5.0 and 8.5, and maintains approximately 60% of its maximum activity at pH 10.0 after 30 minutes of stasis. BGLHL remains stable between pH 5.0 and 7.5, and maintains over 80% of its maximum activity at pH 7.5 and 8.0 after 30 minutes of stasis. BGL9A maintains over 80% of its maximum activity at pH 5.5 and 8.0 after 30 minutes of stasis.

[0067] 3.2 Effect of temperature on recombinant β-glucosidase activity

[0068] Under the optimal pH conditions, the activities of the three β-glucosidases were measured in the range of 30-80°C, and the temperature stability of the three recombinant enzymes was measured by standing at different temperatures for 30 minutes. The results are as follows: Figure 6As shown in the figure, the optimal temperature for BGL9A, BGLHL, and BGLC27 is around 50°C, and the activity of BGLC27 is consistently higher than that of BGL9A and BGLHL within the 30-80°C range. BGLHL and BGLC27 retain trace activity at 80°C, while BGL9A exhibits no detectable enzyme activity at 80°C, likely due to denaturation and complete inactivation of BGL9A caused by high temperatures. BGL9A, BGLHL, and BGLC27 all maintain high stability up to 50°C, retaining maximum enzyme activity after 30 minutes of incubation. Compared to BGL9A and BGLHL, BGLC27 exhibits higher temperature stability, maintaining approximately 70% of its maximum activity after 30 minutes of incubation at 65°C. When the temperature exceeds 60°C, the stability of BGL9A, BGLHL, and BGLC27 begins to decline.

[0069] 3.3 Effects of metal ions and reagents on the activity of recombinant β-glucosidase

[0070] 5mmol / L K + , Ca 2+ 、Mn 2+ 、Fe 3+ 、Zn 2+ Mg 2+ 、Cu 2+ , EDTA and 10% anhydrous ethanol were added to the reaction system to determine the effects of the three metal ions and reagents on the three β-glucosidases. The enzyme activity without adding metal ions and reagents was defined as 100%, and the relative enzyme activity of each treatment group was determined. The results are shown in Table 2. 2+ 、Mn 2+ Mg 2+ It has a promoting effect on three enzymes, K + 、Fe 3+ 、Zn 2+ 、Cu 2+ , EDTA and 10% anhydrous ethanol had inhibitory effects on the three enzymes, among which Fe 3 + 、Zn 2+ , EDTA has a strong inhibitory effect on the three enzymes. 2+ The strongest promoting effect was on BGLC27. 2+ The inhibitory effect on BGL9A and BGLC27 was small, while the inhibitory effect on BGLHL was close to 40%, and EDTA could inhibit the enzyme activity of BGLHL by nearly 75%. All three enzymes could maintain high activity under 10% anhydrous ethanol conditions.

[0071] Table 2 Effects of metal ions and reagents on β-glucosidase activity

[0072]

[0073] 3.4 Effect of glucose on recombinant β-glucosidase activity

[0074] Under the optimal reaction conditions, glucose was added at final concentrations of 100mM, 250mM, 500mM, and 1000mM, respectively. The activity changes of the three β-glucosidases under the pressure of glucose addition were as follows: Figure 7 As shown. Under the condition that the glucose concentration does not exceed 1M, the activity of BGL9A will not be inhibited by glucose and thus remain at a high level. At a glucose concentration of 250mM, its activity reaches a maximum of about 42U / mg, and it can still maintain its original activity at a glucose concentration of 1M. Within the range of glucose concentration not exceeding 1M, the activity of BGLC27 will be inhibited, and the inhibitory effect will increase with the increase of glucose concentration. At a glucose concentration of 1M, its activity will be reduced to the lowest. The activity of BGLHL will be enhanced by the stimulation of low concentrations of glucose. When the glucose concentration is 100mM, its activity is only slightly enhanced. However, as the glucose concentration further increases, the activity of BGLHL will be inhibited. When the glucose concentration reaches 1M, its activity can only retain 15%.

[0075] 3.5 Determination of kinetic constants of recombinant β-glucosidase

[0076] Under the optimal reaction conditions, the enzymatic reaction was carried out using pNPG at final concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 mM, respectively. The reaction rates of the enzymes at different pNPG concentrations were calculated. The three β-glucosidases were fitted with the "Michaelis-Menten" equation by nonlinear regression using OriginPro, and the Km and Kcat values ​​of each enzyme were calculated. The results are shown in Table 3. The Km value of BGLC27 was the smallest, indicating that BGLC27 had a high affinity for the substrate, that is, the enzyme could achieve a higher reaction rate at a lower substrate concentration, and the Kcat / Km value of BGLC27 reached 48.58S. -1 ·mM -1 , indicating that BGLC27 has a high catalytic efficiency.

[0077] Table 3 β-glucosidase kinetic constants

[0078]

[0079] 3.6 Transglycosylation activity of recombinant β-glucosidase

[0080] BGL9A, BGLHL, and BGLC27 were incubated with 50 mM cellobiose as the reaction substrate under the optimal reaction conditions for 5, 10, 15, 20, and 30 min, respectively. The reaction solutions were inactivated at high temperature, and the content of the reaction products was detected by high pressure liquid chromatography (HPLC).

[0081] The results are as follows Figure 8 As shown, BGL9A, BGLHL, and BGLC27 all possess transglycosylation activity and produce cellotriose. The accumulation of transglycosylation products is a combined result of β-glucosidase hydrolysis and transglycosylation. The non-glucose-tolerant β-glucosidases BGLHL and BGLC27 produce more transglycosylation products. In the BGLC27 reaction, transglycosylation activity outweighs hydrolysis activity from 0 to 15 minutes, leading to a significant increase in the concentration of the transglycosylation product, cellotriose. As the reaction proceeds, increasing glucose concentration inhibits the transglycosylation activity of BGLC27. Around 15 minutes, BGLC27's transglycosylation activity equals its hydrolysis activity for the transglycosylation product, resulting in maximum cellotriose production. After 15 minutes, the transglycosylation activity of BGLC27 is less than its hydrolysis activity for cellotriose, resulting in a slight decrease in cellotriose content. Unlike BGLC27, BGLHL's transglycosylation and hydrolysis activities reach equilibrium around 10 minutes and remain constant for 30 minutes. In the reaction of BGL9A, the production rate of the transglycosylation product cellotriose is the fastest at 1 to 5 minutes, mainly because the transglycosylation activity of BGL9A is greater than the hydrolysis activity of cellotriose during this period. As the reaction proceeds, due to the accumulation of the transglycosylation product cellotriose and the sugar tolerance of BGL9A, its transglycosylation activity and hydrolysis activity reach equilibrium at about 5 minutes, resulting in a significant decrease in the production rate of the transglycosylation product cellotriose in the BGL9A reaction after 5 minutes. As the reaction proceeds, the cellobiose consumption rate and glucose production rate in the reactions of BGLHL and BGLC27 gradually decrease, which may be related to the decrease in substrate concentration and the inhibitory effect of increased glucose concentration. BGL9A still has a certain level of hydrolysis activity at a reaction time of 30 minutes because of its own glucose tolerance. The changes in the hydrolysis activity of β-glucosidase and the cellotriose content after adding different concentrations of glucose pressure to the reaction system are shown in the figure. Figure 9 The results show that BGL9A, BGLHL, and BGLC27 are all affected by different glucose concentrations. The hydrolysis activity of BGL9A is less inhibited by glucose, indicating that BGL9A is a glucose-tolerant β-glucosidase, while BGLHL is most strongly inhibited by glucose. The transglycosylation activities of all three β-glucosidases are inhibited by glucose.

[0082] Example 4 Effect of adding exogenous β-glucosidase on the activity of synthetic cellulase

[0083] The BGL9A, BGLHL, and BGLC27 added in the experiment are the β-glucosidases expressed and purified in Example 2, and BGLA and BGLB are β-glucosidases disclosed in the literature (Zhihua Fan, Jingxue Kang, Kaice Lang, Guangxin Chen, Xinyue Zhang, Hongtao Li, Bo Ma. Characterization of glucose / non-glucose-tolerant β-glucosidases from the metatranscriptome in compost. Process Biochemistry. 2024, 143: 198-209.), wherein BGLA is a sugar-tolerant β-glucosidase and BGLB is a non-sugar-tolerant β-glucosidase.

[0084] 4.1 Determination of the amount of exogenous β-glucosidase added

[0085] 0U, 20U, 40U, 80U, and 100U of BGL9A were added to the reaction system, and samples were taken at 1 to 10 days. The changes in β-glucosidase activity within 10 days were as follows: Figure 10 As shown in the figure, with the increase of BGL9A addition, the β-glucosidase activity in the fermentation broth of the reaction system showed an overall upward trend within 10 days. When the BGL9A addition amount increased from 80U to 100U, the upward trend of β-glucosidase activity in the fermentation broth began to slow down. Based on the measurement results of this experiment and the purification efficiency of the recombinant enzyme, it was determined that 80U of exogenous β-glucosidase should be added to the cellulose-degrading composite bacterial consortium reaction system.

[0086] 4.2 Effect of exogenous β-glucosidase addition on cellulose degradation rate

[0087] The degradation rate of corn straw by the C27×Z23 synthetic community with 80U of enzyme activity of BGL9A, BGLHL, BGLC27, BGLA and BGLB added respectively within 10 days was as follows: Figure 11The CK group represents the control group treated with an equal volume of water. The results show that the corn straw degradation rate in the CK group increased significantly from nearly 0% to 40.5% over the first 7 days, remaining at around 42% for 8-10 days. The addition of different exogenous β-glucosidases had varying effects on the degradation capacity of the synthetic community. The corn straw degradation rate in the BGL9A-treated group increased rapidly over the first 6 days, from nearly 0% to 41.3%, then slowed down over the next 6-10 days, reaching a maximum of 48.1% on the 10th day. The corn straw degradation rate in the BGLC27-treated group increased continuously over the first 10 days, reaching a maximum of 50.9% on the 10th day, 8.6% higher than the corn straw degradation rate in the CK group over the same period. The corn straw degradation rate in the BGLHL-treated group increased rapidly over the first 6 days, exceeding that of the CK group. The degradation rate remained stable over the next 7-10 days, reaching a maximum of 41.5% on the 10th day, lower than that of the CK group over the same period. The degradation rate of the BGLA-treated group continued to rise over 10 days, falling slightly below that of the CK group on day 3 and exceeding that of the CK group at all other times. The upward trend slowed from day 8 until it reached a maximum of 51.4% on day 10, 10.9% higher than the CK group during the same period. The degradation rate of the BGLB-treated group continued to rise over 10 days, approaching that of the CK group from days 1 to 5. It then fell below that of the CK group from day 6 and reached a maximum of 40.5% on day 10, 1.8% lower than the CK group during the same period.

[0088] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A non-sugar-tolerant β-glucosidase, characterized in that The amino acid sequence of the non-sugar-tolerant β-glucosidase is shown in SEQ ID No.

1.

2. A gene encoding the non-glucose-tolerant β-glucosidase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

2.

4. An expression vector comprising the gene according to claim 2 or 3. 5 . An expression cell comprising the gene according to claim 2 or 3 and expressing the non-glucose-tolerant β-glucosidase according to claim 1 .

6. Use of the non-sugar-tolerant β-glucosidase according to claim 1 or the recombinant non-sugar-tolerant β-glucosidase produced by the expression cell according to claim 5 in improving the degradation ability of cellulose-degrading bacteria.

7. The use according to claim 6, characterized in that The cellulose degrading bacteria include Streptomycetaceae sp. C27 or a composite bacterial system containing Streptomycetaceae C27; the preservation number of the Streptomycetaceae sp. is GDMCC 66321.

8. A method for improving the degradation capacity of cellulose-degrading bacteria, characterized in that: The method comprises adding the non-sugar-tolerant β-glucosidase according to claim 1 or the recombinant non-sugar-tolerant β-glucosidase produced by the expression cell according to claim 5 to the degradation system of cellulose-degrading bacteria.

9. The method according to claim 8, characterized in that The cellulose degrading bacteria include Streptomycetaceae sp. C27 or a composite bacterial system containing Streptomycetaceae C27; the preservation number of the Streptomycetaceae sp. is GDMCC 66321.

10. A cellulose degradation system, characterized in that: The invention comprises Streptomycetaceae sp. C27 or a composite bacterial system containing Streptomycetes C27, and further comprises the non-sugar-tolerant β-glucosidase according to claim 1 or the recombinant non-sugar-tolerant β-glucosidase produced by the expression cell according to claim 5; the deposit number of the Streptomycetes C27 is GDMCC66321.

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