Thermal, acid and alkali resistant, glucose resistant enzyme and application thereof

Abstract

The application discloses a heat-resistant, acid-and-alkali-resistant and glucose-resistant enzyme and application thereof, and belongs to the technical field of genetic engineering. The application provides a bfbg8 enzyme gene. The enzyme provided by the application has the characteristics of heat resistance, glucose resistance, superior heat stability and pH resistance, and has a wide application prospect in the food, medicine and high-temperature industrial industries. The enzyme also has a very important role in improving the degradation rate of lignocellulose and preparing ethanol through simultaneous saccharification and fermentation. According to the technical scheme of the application, the enzyme with excellent properties and suitable for industrial application can be produced by using genetic engineering means.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and more particularly to a heat-resistant, acid and alkali-resistant, glucose-resistant enzyme and its application. BACKGROUND

[0002] Cellulose is an important component of lignocellulose and widely exists in agricultural straw (rice straw, wheat straw, corn straw, etc.) and forest waste. The utilization and conversion of cellulose is of great significance to solve the global problems such as energy crisis, food shortage and environmental pollution. The conversion of lignocellulosic biomass into biofuels is now considered as a sustainable alternative energy choice to maintain global energy demand. The conversion of cellulose into biofuels relies on the synergistic hydrolysis of enzymes, including endo-1, 4-glucanase (EC 3.2.1.4), exo-1, 4-glucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). Although β-glucosidase does not directly degrade cellulose, it plays a key role in the degradation of cellulose by removing the inhibition of endo- and exo-enzymes on cellulose oligosaccharides, and is therefore considered as the rate-limiting enzyme for cellulose degradation.

[0003] β-glucosidase widely exists in nature and can catalyze the cleavage of glycosidic bond in cellulose, oligosaccharides and alkyl or aryl β-glycosides. Compared with enzymes from plants and animals, microbial enzymes have the advantages of large-scale fermentation production, high yield, substrate specificity and catalytic activity diversity. However, the thermal stability of many existing β-glucosidases is insufficient, which limits their use in high-temperature industrial applications. Although many β-glucosidases have been characterized, it is necessary to further screen new β-glucosidases with better properties (high activity, good stability, easy large-scale production, etc.) in order to reduce the cost of biorefining process. Therefore, it is of great importance to find new thermophilic and thermotolerant β-glucosidases to meet the industrial demand and study their thermophilic mechanism. Traditionally, the biological prospecting of extremozymes is completed by cultivation-dependent methods, and the activity determination after classical enrichment and isolation is a key strategy for identifying enzyme-producing microorganisms. In the past few decades, this method has successfully discovered various types of heat-stable enzymes and identified microbial species producing heat-stable enzymes (such as cellulases, xylanases and oxidoreductases). In addition to traditional techniques obtained from plants, animals and microorganisms, we can also screen new β-glucosidases through next-generation sequencing. Today, it has become a popular trend to screen new enzymes from enriched environments by using biological prospecting techniques.

[0004] Metagenomics is one of the means to mine new enzymes in biological prospecting technology. Although thermophilic bacteria can produce enzymes for industrial use, only 0.1-1% of the bacteria can be cultured by traditional techniques, which makes it challenging to isolate new enzymes with superior functions by culturing methods. In order to break through the limitations of traditional culturing techniques, metagenomics technology emerged. Metagenomics technology breaks through the limitations of traditional laboratory culturing and can identify functional genes in uncultured microbial communities from various environmental samples. However, due to the extremely wide diversity of environmental genomes, much effort is needed to select target genes. Therefore, enrichment of environmental genomes may make it possible to effectively select target genes. Enrichment culturing can efficiently select and enrich microorganisms and their enzyme resources with specific functions from complex environmental samples, and has a unique advantage in seeking enzymes resources that are resistant to heat and extreme conditions. In summary, the research on mining thermophilic enzymes is faced with problems such as low screening efficiency and difficulty in obtaining novel enzyme resources, and enrichment culturing and metagenomics technology provide strong support to solve these problems.

[0005] In summary, how to provide a novel beta-glucosidase is a problem that those skilled in the art urgently need to solve. SUMMARY

[0006] Therefore, the present application provides a heat-resistant, acid and alkali-resistant, glucose-resistant enzyme and its application.

[0007] The present application aims to explore the potential thermophilic and heat-resistant beta-glucosidase in the mud sample of Hehua hot spring in Tengchong City, Yunnan Province. First, specific microorganisms are screened from the mud sample by enrichment culturing technology, and a new beta-glucosidase gene is obtained by metagenomics technology. Then, through molecular cloning technology and rapid activity screening, a clone of thermophilic and heat-resistant beta-glucosidase is obtained. Based on heterologous expression and protein purification technology, a recombinant beta-glucosidase is obtained, and its enzymatic properties and application are determined. The research shows that by combining enrichment culturing, metagenomics technology and rapid activity detection method, a new thermophilic and heat-resistant multifunctional beta-glucosidase is successfully determined, which indicates that it has potential application prospects in the fields of commercial utilization of lignocellulose, bioenergy production and dairy product processing, etc.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] A heat-resistant, acid and alkali-resistant, glucose-resistant enzyme, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] Further, it simultaneously has the activities of beta-glucosidase and beta-galactosidase.

[0011] The coding gene of the enzyme.

[0012] Further, the nucleotide is shown as SEQ ID NO: 2.

[0013] A recombinant carrier comprising the coding gene.

[0014] A recombinant bacterium comprising the recombinant carrier.

[0015] The enzyme in the application in the application of degrading corn stalks in cooperation with cellulase.

[0016] The enzyme in the application in the application of food, feed, medicine and industrial production.

[0017] The gene, the recombinant carrier or the recombinant bacterium in the application in the industrial production of heat-resistant, acid and alkali-resistant, glucose-resistant beta-glucosidase.

[0018] According to the technical solution, compared with the prior art, the application has the following beneficial effects:

[0019] In the technical solution, the enzyme gene bfbg8, the recombinant plasmid carrying the gene and the recombinant strain are provided, the bfbg8 gene can encode a beta-glucosidase / beta-galactosidase, the optimum pH of the enzyme is 6.0, the activity of the enzyme remains at 100% after incubation in a pH4.0-10.0 buffer for 24 hours, and the enzyme has very strong pH tolerance; the optimum temperature of the enzyme is 70℃, and the activity of the enzyme does not decrease after incubation at 65℃ for 2 hours, the enzyme provided in the application has the superior properties of strong pH tolerance and thermal stability, and can be applied to fuel energy, food processing, papermaking, textile and medical care industries. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below, and obviously, the drawings in the following description can be obtained by the drawings provided by the person skilled in the art without creative labor.

[0021] Figure 1 The technical roadmap of the application;

[0022] Figure 2 The phylogenetic tree of the BFBG8 protein in the embodiment 1 of the application is shown, and the bootstrap value (expressed as a percentage of 1000 replicates) is given on the node;

[0023] Figure 3SDS-PAGE electrophoretogram of BFBG8 protein in Example 4 of the present application; lane 1, protein molecular weight marker, the left side indicates the mass size; lane 2, total protein of E. coli DH5α / pSHY211-BFBG8 after induced expression; lane 3, purified BFBG8 protein;

[0024] Figure 4 Effect of temperature and pH on BFBG8 activity and stability in Example 5 of the present application, wherein a represents the effect of temperature on BFBG8 activity; b represents the effect of pH on BFBG8 activity; c represents the effect of temperature on stability; d represents the effect of pH on stability; error bars represent the mean ± SEM of three biological replicates;

[0025] Figure 5 Effect of glucose on BFBG8 activity in Example 5 of the present application, error bars represent the mean ± SEM of three biological replicates, p<0.01 (**) indicates extremely significant difference, p<0.05 (*) indicates significant difference;

[0026] Figure 6 Synergistic degradation of corn stalks by BFBG8 and commercial cellulase at 60℃ in Example 5 of the present application, error bars represent the mean ± SEM of three biological replicates, p<0.01 (**) indicates extremely significant difference, p<0.05 (*) indicates significant difference;

[0027] Figure 7 Effect of temperature and pH on BFBG8 activity with lactose as substrate in Example 5 of the present application, wherein a represents the effect of temperature on BFBG8 activity; b represents the effect of pH on BFBG8 activity. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0029] Genes: The genome DNA of the bottom mud collected from the Hehua hot spring (25.439621 °N, 98.460999 °E) in Tengchong County, Baoshan City, Yunnan Province of the present application embodiment was extracted by using a soil DNA extraction kit (brand MOBIO, USA, item number: 12888-50), and then sent to a sequencing company for macro-genome sequencing and annotation. Through data analysis, the functional gene of β-glucosidase was screened out and named as bfbg8. Then, the functional gene was subjected to PCR amplification, identification, clone expression, β-glucosidase purification and enzymatic property determination. The clone expression vector was a constitutive expression vector pSHY211 independently constructed.

[0030] The construction method of the pSHY211 plasmid is as follows: first, the primer TFH-F1 and THF-R1 (TFH-F1: CCCTATAGTGAGTCGTATTAATTTCGCGGGATCG, SEQ ID NO: 3; THF-R1: SEQ ID NO: 4; the bold underlined part is the Hind III restriction enzyme recognition site) was used to PCR amplify a 5194 bp TFH1

[0031] SEQ ID NO: 6, wavy part is The GH11 endoxylanase gene promoter of Bacillus subtilis AQ1. The bold underlined DNA sequences are EcoR I, BamH I and Hind III restriction enzyme recognition sites, and the double underlined part is the EGFP gene. The primer TFH-F2 and THF-R2 (TFH-F2: GGGGTACCTAGCGTGGTATTAT, SEQ ID NO: 7; THF-R2: SEQ ID NO: 8, the bold underlined part is the Hind III restriction enzyme recognition site) were used for amplification. Subsequently, the TFH1 and TFH2 fragments were double digested with Hind III, and the two fragments were ligated using T4 DNA ligase. The ligation product was transformed into E. coli DH5a, and the fluorescent positive clones were screened on LB plates containing kanamycin. Finally, the constitutive expression plasmid pSHY211 was obtained, and its correctness was verified by sequencing. This plasmid can achieve constitutive expression of the target gene without an inducer, and the positive clones can be quickly screened by fluorescence, significantly simplifying the cloning and expression process of functional genes.

[0032] Enzymes and other biochemical reagents: Restriction enzymes (EcoR I and Hind III) were purchased from Thermo Scientific Company, T4-DNA ligase was purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., and other biochemical reagents were purchased from domestic ordinary biochemical reagent companies.

[0033] Culture medium:

[0034] LB basic medium (g / L): yeast extract 5, tryptone 10, sodium chloride 10, pH 7.4; the antibiotic used was kanamycin, with a final concentration of 50 μg / mL, and 2% agar was added to the solid separation medium, which was sterilized at 121°C for 30 min.

[0035] Example 1

[0036] Extraction of soil sample DNA and gene synthesis

[0037] The mud of Hehua hot spring in Tengchong County, Baoshan City, Yunnan Province was used as the experimental sample. The genomic DNA was extracted using a soil DNA extraction kit (brand MOBIO, USA, item number: 12888-50), and the operation was performed according to the instruction manual provided by the manufacturer. The metagenomic sequencing was performed using a HiSeq 2500 sequencer of Suzhou GENWIZ Company. All the result sequences were investigated using the IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi). In order to further detect the potential functions of individual genes and ORFs, we used KEGG, COG and Pfam databases for annotation analysis. Based on this functional prediction, the β-glucosidase gene sequence was obtained from the metagenomic database and named bfbg8. The DNA and protein sequences were compared using BLASTx and BLASTp programs (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), respectively. The molecular weight of the protein BFBG8 was predicted using EXPASY (https: / / web.expasy.org / protparam / ). Multiple alignments were performed on the protein sequences closely related to it, and phylogenetic analysis was performed using MEGA 7.0.

[0038] The nucleotide sequence of the bfbg8 gene is as follows:

[0039] ATGAACGACAACCCGTTCCCCCCCAACTTCCTCTGGGGTGTCGCGACCGCCGCC

[0040] TACCAGATCGAAGGAGCGGTCAACGAAGACGGTCGCGGCCCCTCAATTTGGGACAC

[0041] CTTTAGCCACACGCCGGGCAAGACCCATAACGGCGACACCGGCGACGTCGCTTGCG

[0042] ACCACTACCACCGCTGGCAAGAGGACCTGGCGCTGATGGCGCAGCTCGGCATCCAA

[0043] AGCTATCGCTTTTCCATCGCCTGGCCGCGCGTGCTACCGTCGGGCACCGGTAAGGTC

[0044] AACGCCAAGGGGCTTGACTTCTACGAGCGGCTGGTCGACGAGCTCCTTGCGCGCAA

[0045] CATCGTCCCCAATGCCACGCTGTACCACTGGGACCTCCCCCAGGCGCTCCAGGACAA

[0046] AGGTGGCTGGGGGAACCGCGATACCGCCCATGCCTTTGTCGAGTACGCCGACGCCG

[0047] TGACCCGGCGCCTCGGTGATCGGGTGGCGCTTTATGCCACCTTCAATGAGCCCTGGT

[0048] GTATCGCCATCCTGGGCCACGAGTCCGGCGAGCACGCCCCGGGCTTTACCGACCGC

[0049] AAGCTCGCCCTGCAGACCGCCCATCACGTCCTGCTAGCGCACGGCATGGCCTTGCCG

[0050] GTGTTGCGCGAGAACGCGCCCGGCGCCAAGCACGGCATCGTCCTCAACTTTACCCCT

[0051] ACCTACGCTCTTGACGACGCCGAGGCTACCTTGCAGGCGGTAACGATGATGGACGG

[0052] CACCTTTAACCGCTGGTTTGCCGATCCGCTCTTTTTGGGACGTTACCCCGAAGATATC

[0053] TGGACGCTTTACGGCGAGTCCGTCCCCGAAGTGCTTGATGGCGACTTCGACGTCATC

[0054] CGGCGCCCGATCGACTTCTTGGGGGTGAACTACTACACCCGCCTCACCTTTGGCGGC

[0055] GACCCAGGACCGGTCGAGCGGACCGCCATGGGTTGGGAGGTCTTCCCGCAGGGGCT

[0056] CTATGACCTGCTGCTGAGGCTGCAGCGCGACTACAAGCCCCCGCTGATGTACGTGAC

[0057] CGAAAACGGCGCAGCGTATGACGACGTGCTCATCGACGGGCAGGTGCACGACCCCG

[0058] AGCGCGTGCATTACCTTGAGCGCCACATCGCCGCCCTGGCCGAAGCGATCGCCGAA

[0059] GGGGCCAACGTGCAGGGGTATTACCTATGGAGCTTAATGGACAACTTCGAATGGGC

[0060] CAAGGGCTACAGCAAGCGCTTCGGCATCATCTACGTGGACTACCCCACTCAACGGC

[0061] GCATCCTCAAGACGAGTGGGCGCTGGTATGCCGATCTGATCGCAGCCGGCGGCCGG

[0062] GTGCCGTCGCTAACCTGA, SEQ ID NO: 2.

[0063] The amino acid sequence of the BFBG8 protein is as follows:

[0064] MNDNPFPPNFLWGVATAAYQIEGAVNEDGRGPSIWDTFSHTPGKTHNGDTGDVAC

[0065] DHYHRWQEDLALMAQLGIQSYRFSIAWPRVLPSGTGKVNAKGLDFYERLVDELLARNI

[0066] VPNATLYHWDLPQALQDKGGWGNRDTAHAFVEYADAVTRRLGDRVALYATFNEPWC

[0067] IAILGHESGEHAPGFTDRKLALQTAHHVLLAHGMALPVLRENAPGAKHGIVLNFTPTYA

[0068] LDDAEATLQAVTMMDGTFNRWFADPLFLGRYPEDIWTLYGESVPEVLDGDFDVIRRPID

[0069] FLGVNYYTRLTFGGDPGPVERTAMGWEVFPQGLYDLLLRLQRDYKPPLMYVTENGAA

[0070] YDDVLIDGQVHDPERVHYLERHIAALAEAIAEGANVQGYYLWSLMDNFEWAKGYSKR

[0071] FGIIYVDYPTQRRILKTSGRWYADLIAAGGRVPSLT, SEQ ID NO: 1.

[0072] The molecular weight of the BFBG8 protein is predicted to be 49 kDa.

[0073] Figure 2 For the phylogenetic relationship of the BFBG8 protein and related proteins, by constructing the phylogenetic tree of the BFBG8 protein and β-glucosidase from other sources, it can be known that the amino acid sequence of the BFBG8 protein has 79.95% homology with the amino acid sequence of β-glucosidase from Truepera sp. (NCBI: MBS3933596.1).

[0074] Example 2

[0075] Amplification of functional genes and construction of recombinant expression vectors

[0076] PCR amplification of functional genes was performed using the above genes as templates, and the primer pairs used were as follows:

[0077] bfbg8-F: CAAATGGGTCGCGGATCCGAA ATGAACGACAACCCGTTCCCC, SEQ ID NO: 9;

[0078] bfbg8-R: GTGCTCGAGTGCGGCCGCAAG TCAGGTTAGCGACGGCACCCG, SEQ ID NO: 10.

[0079] The underlined sequence represents a homologous recombination fragment with the pSHY211 vector.

[0080] PCR amplification was performed using high-fidelity DNA polymerase Gold Medal Mix (Beijing Prime Tech Biotechnology Co., China). The PCR program included 98°C pre-denaturation for 3 min, then 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 60 s, 30 cycles, and 72°C final extension for 5 min. Identification was performed using 1.0% agarose gel electrophoresis. Then the PCR products were observed under ultraviolet light, the correct size band was cut off, and the gel recovery kit (Beijing Tsingke Biotech Co., Ltd) was used for gel recovery.

[0081] The PCR product and the constitutive expression vector pSHY211 were double-digested with restriction enzymes (EcoRI and Hind III), and then electrophoresed using a 1.0% agarose gel, and the resulting digestion product was recovered by gel excision. The digested PCR product and the vector were ligated with T4-DNA ligase at 25°C for 2 h, and the ligation product was transformed into E. coli DH5α by the chemical transformation method for 60 s. Positive clones were obtained by colony PCR and sequencing. Ten clones were picked from each functional gene clone plate, added to 500 mL of LB liquid medium in a high-temperature sterilized EP tube, and incubated at 37°C for 24 h. Then, 50 μL of the bacterial solution and 10 μL of lysozyme were added to two 96-well plates and reacted at 37°C for 30 min or more, and then 50 μL of 1% cellobiose substrate was added to each plate and reacted at 50°C and 70°C for 1 h, respectively. Finally, the colorimetric primary screening was performed using a glucose oxidase-peroxidase assay kit, and the clones with the deepest color were used as the objects of subsequent experiments. 2+ The ligation product was introduced into E. coli DH5α by the chemical transformation method for 60 s. Positive clones were obtained by colony PCR and sequencing. Ten clones were picked from each functional gene clone plate, added to 500 mL of LB liquid medium in a high-temperature sterilized EP tube, and incubated at 37°C for 24 h. Then, 50 μL of the bacterial solution and 10 μL of lysozyme were added to two 96-well plates and reacted at 37°C for 30 min or more, and then 50 μL of 1% cellobiose substrate was added to each plate and reacted at 50°C and 70°C for 1 h, respectively. Finally, the colorimetric primary screening was performed using a glucose oxidase-peroxidase assay kit, and the clones with the deepest color were used as the objects of subsequent experiments.

[0082] Example 3

[0083] Induced expression of recombinant protein

[0084] The recombinant vector pSHY211-bfbg8 constructed in Example 2 was transformed into E. coli DH5α (DE3) to obtain recombinant E. coli DH5α / pSHY211-BFBG8. A single colony was picked from LB medium containing 50 μg / mL kanamycin and inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin, and incubated at 37°C at 220 rpm overnight. The above strain was incubated in 200 mL of LB medium containing 50 μg / mL kanamycin at an inoculation amount of 1% at 37°C at 220 rpm for 5-8 h, and then transferred to 25°C and incubated at 220 rpm for 8 h. Finally, the cells were centrifuged at 4°C at 7000 rpm for 20 min, and the supernatant was removed to obtain E. coli cells.

[0085] Example 4

[0086] Isolation and purification of recombinant protein

[0087] The E. coli cells were resuspended in a centrifuge tube containing 10 mM imidazole in PBS solution (pH 7.6), and were subjected to ultrasonic disruption in an ice-water mixture for 30 min, and then were centrifuged at 4°C and 7000 r / min for 20 min. The supernatant was collected after centrifugation. The supernatant was purified by using a Ni-NTA column. The Ni-NTA column was equilibrated with 5 column volumes of equilibration buffer (10 mM imidazole in PBS solution, pH 7.6). The supernatant was loaded into the Ni-NTA column, and the loading was repeated once. Then, the column was washed with 10 column volumes of equilibration buffer. The target protein was eluted with 3 column volumes of elution buffer (250 mM imidazole in PBS solution, pH 7.6), and the protein solution was collected in 1.5 mL centrifuge tubes, 1.0 mL per tube. The protein concentration was determined by using the Bradford reagent with bovine serum albumin as a standard. The absorbance was determined at OD 595 The purified target protein was analyzed by SDS-PAGE.

[0088] Figure 3 The SDS-PAGE electrophoretogram of the BFBG8 protein is shown in FIG. 1. The purified BFBG8 protein showed a single band on SDS-PAGE, and the molecular weight was about 50 kDa, which was consistent with the theoretically predicted molecular weight (49 kDa).

[0089] Example 5

[0090] Enzymatic property of BFBG8 protein

[0091] (1) Determination of β-glucosidase enzyme activity

[0092] The determination was performed by using a glucose oxidase-peroxidase assay kit. Cellobiose was used as a substrate to determine the β-glucosidase activity. 10 μL of 10-fold diluted purified enzyme solution was added to 90 μL of a buffer containing 1% (w / v) cellobiose, and the reaction was performed at an optimal temperature for 30 min. The reaction was terminated by freezing at -80°C for 2-3 min. 10 μL of the reaction mixture was added to a 96-well plate, and 200 μL of a glucose oxidase-peroxidase assay kit buffer was added. After incubation at 37°C for 10 min, the absorbance was determined at OD 492 nm by using a microplate reader. One unit (U) was defined as the amount of enzyme required to release 2 μmol of glucose per minute from cellobiose.

[0093] (2) Determination of optimal temperature and temperature stability of β-glucosidase

[0094] According to the activity assay, the purified β-glucosidase was determined at the optimum reaction pH condition at different temperatures (30-85°C) with 5°C as a gradient to determine the optimum reaction temperature of BFBG8. The thermal stability analysis was performed by incubating the enzyme solution at different temperatures (65°C, 70°C and 75°C) for different time (0-120 min), and the residual activity was determined every 20 min. The 0 min was the control group, and the residual activity was determined to determine the thermal stability of BFBG8.

[0095] The activity of BFBG8 reached the highest value at 70°C. After 70°C, the enzyme activity gradually decreased, and at 80°C, there was still about 50% activity Figure 4 a) After incubation at 65°C for 120 min, it still maintained 100% residual activity, and after incubation at 70°C for 80 min, it maintained more than 50% residual activity, and t 1 / 2 83 min Figure 4 c).

[0096] (3) Optimum pH and pH stability determination of β-glucosidase

[0097] The purified β-glucosidase was subjected to enzymatic reaction at different pH to determine the optimum pH. The buffer used was a series of citric acid-disodium hydrogen phosphate buffer with pH 3.0-8.0, and glycine-hydrochloric acid buffer with pH 8.0-10, with a gradient of every 0.6 pH. The purified β-glucosidase was determined at the optimum pH in different pH buffer systems at 70°C. The residual activity was determined after the purified enzyme solution was placed at 4°C for 12 h and 24 h, respectively, with a gradient of every 1 pH, to determine the pH stability of BFBG8.

[0098] The results showed that the optimum pH of the β-glucosidase was 6.0. It maintained more than 60% relative activity at pH 5.0-9.0 Figure 4 b) After pre-incubation at 4°C for 24 h, the activity of BFBG8 was maintained at about 90% of the initial activity at pH 3.0-11.0 Figure 4 d) The results showed that BFBG8 could function under a wide range of acid-base conditions and maintain its activity stability.

[0099] (4) Effect of glucose on the activity of β-glucosidase

[0100] Using p-nitrophenyl-β-d-glucopyranoside (pNPGlc) as a substrate, 10 μg of protein was added to a 150 μL reaction mixture containing 1 mM pNPGlc (Sigma, St. Louis, MO, USA) at pH 6.0. After incubation at the optimal temperature for 5 min, the reaction was stopped by adding 450 μL of 1 M Na₂CO₃. The release rate of p-nitrophenol was measured by monitoring the absorbance at 405 nm using p-nitrophenol (Sigma, USA) as a standard. To investigate the effect of D-glucose on the catalytic activity of BFBG8, different concentrations of D-glucose (0–5 M) were added to a test mixture containing 1 mM pNPGlc under standard conditions. The control group was the one without added glucose.

[0101] Measurement results ( Figure 5 The results show that BFBG8 exhibits unique activity changes under different glucose concentrations. When the glucose concentration is in the range of 250 mM to 3000 mM, the relative activity of BFBG8 is stimulated. The stimulation effect is most significant at a glucose concentration of 1500 mM, where the activity of BFBG8 is 1.4 times that in the glucose-free environment. When the glucose concentration exceeds 3000 mM, although the hydrolytic activity of BFBG8 is gradually inhibited with further increases in glucose concentration, its relative enzyme activity remains above 80%. Unlike most β-glucosidases, glucose does not significantly inhibit the activity of BFBG8, demonstrating its strong glucose tolerance.

[0102] (5) Determination of the hydrolysis of corn straw by β-glucosidase

[0103] 10g of corn stalks were ground and sieved through an 80-mesh sieve, then boiled in 100mL of hot water for 30min. The corn stalks were filtered through a paper filter and dried at 80℃. 0.2g of the pretreated corn stalks were added to 1mL of buffer solution (pH 6.0), along with 0.2mg of *Trichoderma reesei* cellulase (Sangon Biotech, China) and 0.05mg of BFBG8. The mixture was then incubated at 60℃. Samples were taken 0–7h after the start of the reaction, with 1h intervals between each sample. The glucose concentration in the reaction solution was determined using a glucose oxidase assay kit according to the manufacturer's instructions. The control condition was a reaction solution without enzyme. Each group was performed in triplicate, and all subsequent analyses used the average value. The glucose concentration in the hydrolyzed corn stalks was tested at different time points.

[0104] The results showed that the addition of β-glucosidase BFBG8 to commercial cellulase significantly improved the hydrolysis efficiency, with the degradation rate increasing by approximately 50% after 7 hours. Figure 6). The above results show that BFBG8 has potential application in lignocellulose degradation and can be used in synergy with commercial cellulases.

[0105] (6) Determination of the effect of different metal ions and organic reagents on the activity of β-glucosidase

[0106] To determine the effect of metal ions on β-glucosidase, different metal ions (K + , Mg 2+ , Fe 3+ , Fe 2 + , Zn 2+ , Co 2+ , Cu 2+ , Ag + , Mn 2+ , Pb 2+ and Ni 2+ ) were added to the enzyme solution at 60°C, with the final concentration of metal ions being 1 mM and 10 mM, respectively. To determine the effect of inhibitors on β-glucosidase, different inhibitors [ethylenediaminetetraacetic acid disodium salt (EDTA), sodium dodecyl sulfate (SDS), phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT)] were added to the enzyme solution at 60°C, with the final concentration of inhibitors being 0.1% and 1%, respectively. To determine the effect of ethanol on β-glucosidase, ethanol was added to the enzyme solution at 60°C, with the final concentration of ethanol being 1% and 10%, respectively. The residual enzyme activity was determined after 10 min of incubation. The reaction mixture without additives under standard conditions was used as a control (100%).

[0107] The results show that the effect of various metal ions, chemical reagents and ethanol on the activity of BFBG8 was studied using cellobiose as a substrate. The enzyme activity without the addition of metal ions / chemical reagents / ethanol was defined as 100%, and the relative enzyme activities of others were calculated (Table 1). When the concentration of metal ions was 1 mM, Mg 2+ , Fe 3+ , Fe 2+ , Zn 2+ , Cu 2+ , Mn 2+ , Pb 2+ and Ni 2+ had no effect on the activity of BFBG8, while K + , Co 2+ and Ag + were observed to significantly inhibit the activity of BFBG8, among which Ag +Inhibition of more than 95% of the activity; when the concentration increased to 10 mM, the metal ions significantly inhibited their activity. Organic solvents, except SDS, retained more than 90% of the activity at a concentration of 0.1%, while the activity decreased and was significantly inhibited when the concentration increased to 1%. BFBG8 was not affected by 1% ethanol, but was inhibited by 10% ethanol.

[0108] Table 1 Effect of different metal ions and chemicals on the activity of BFBG8

[0109]

[0110] (7) Determination of the substrate specificity of BFBG8

[0111] To determine the substrate specificity of BFBG8, cellobiose, lactose, p-nitrophenyl-β-D-glucopyranoside (pNPGlc), gentiobiose, maltose, melibiose, laminaribiose, raffinose, sucrose, wheat bran xylan, corn cob xylan, bagasse xylan, carboxymethylcellulose sodium (CMC-Na) and Avicel were used as substrates (1%, w / v) to measure the activity of the enzyme, and the activity of cellulase and xylanase was determined by the 3,5-dinitrosalicylic acid (DNS) assay. One unit (U) was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute.

[0112] Table 2 Substrate specificity of BFBG8

[0113]

[0114] From the determination of the substrate specificity, it was found that BFBG8 had hydrolytic activity on most of the substrates (Table 2), such as p-nitrophenyl-β-D-glucopyranoside (92.53 ± 0.84 U / mg), and it could also hydrolyze substrates with β-(1→4) glycosidic bonds, such as cellobiose (40.39 ± 1.44 U / mg), lactose (80.78 ± 2.89 U / mg) and gentiobiose (3.07 ± 0.03 U / mg); however, it could not hydrolyze substrates with α-glycosidic bonds, such as sucrose and maltose, nor could it hydrolyze polysaccharides, such as xylan, carboxymethylcellulose sodium and Avicel, due to the complexity of the substrates, which hindered the activity of the β-glucosidase.

[0115] To further determine the hydrolytic activity of BFBG8 on lactose, the optimal temperature (70°C) and the optimal pH (6.0) of BFBG8 were determined using lactose as the substrate, and the results were consistent with those determined using cellobiose as the substrate Figure 7 ). In summary, BFBG8 is a multifunctional enzyme that has the activity of both β-glucosidase and β-galactosidase.

[0116] The various embodiments described in this specification are presented by way of example, and each embodiment is not necessarily composed of all features described with respect to other embodiments. Each embodiment describes a distinct aspect of the present application, and each aspect can be used in combination with one or more other aspects.

[0117] The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An enzyme that is heat-resistant, acid- and alkali-resistant, and glucose-resistant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the enzyme according to claim 1.

3. The encoding gene as described in claim 2, characterized in that, Its nucleotides are shown in SEQ ID NO:

2.

4. A recombinant vector, characterized in that, It includes the encoding gene as described in claim 3.

5. A recombinant bacterium, characterized in that, It includes the recombinant vector as described in claim 4.

6. The application of the enzyme according to claim 1 in the synergistic degradation of corn stalks with cellulase.

7. The application of the enzyme according to claim 1 in food and feed production.

8. The application of the gene of claim 2 or 3, the recombinant vector of claim 4, or the recombinant bacteria of claim 5 in the industrial production of the heat-resistant, acid- and alkali-resistant, and glucose-resistant enzyme of claim 1.

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