Heat-resistant, acid-base-resistant and glucose-resistant enzyme and application thereof

Abstract

The invention discloses a heat-resistant, acid-base-resistant and glucose-resistant enzyme and application thereof. Belongs to the technical field of gene engineering. The invention provides a bfbg8 enzyme gene, the enzyme provided by the invention has the characteristics of thermophile, glucose tolerance and excellent thermal stability and pH tolerance, has wide application prospects in food, medicine and high-temperature industry, and also plays a very important role in improving the degradation rate of lignocellulose and preparing ethanol by simultaneous saccharification and fermentation; according to the technical scheme provided by the invention, the enzyme which is excellent in property and suitable for industrial application can be produced by utilizing a genetic engineering means.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically to a heat-resistant, acid-resistant, alkali-resistant and glucose-resistant enzyme and its application. Background Art

[0002] Cellulose is an important component of lignocellulose and is widely found in agricultural straw (rice straw, wheat straw, corn straw, etc.) and forest waste. The utilization and conversion of cellulose is of great significance to solving the world energy crisis as well as global problems such as food shortage and environmental pollution. The conversion of lignocellulosic biomass into biofuels is now considered a sustainable alternative energy option to maintain global energy demand. The conversion of cellulose into biofuels relies on the coordinated 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 inhibitory effects of cellulosic oligosaccharides on endo- and exo-enzymes, and is therefore considered to be the rate-limiting enzyme for cellulose degradation.

[0003] β-Glucosidases are widely present in nature and can catalyze the cleavage of glycosidic bonds 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 insufficient thermostability of many existing β-glucosidases limits their use in high-temperature industrial applications. Although many β-glucosidases have been characterized, in order to reduce the cost of biorefining processes, it is necessary to further screen for new β-glucosidases with better properties (strong activity, good stability, and easy large-scale production). Therefore, the search for new thermophilic and thermostable β-glucosidases is crucial to meet industrial needs and study their thermophilic mechanisms. Traditionally, bioprospecting of extreme enzymes has been accomplished by culture-dependent methods, and classical enrichment and isolation followed by activity assays are the key strategies for identifying enzyme-producing microorganisms. In the past few decades, this approach has successfully discovered various types of thermostable enzymes and identified microbial species that produce thermostable 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 by next-generation sequencing. Nowadays, it has become a popular trend to screen new enzymes from enrichment environments using bioprospecting techniques.

[0004] Metagenome is one of the means of mining new enzymes in bioprospecting technology. Although thermophilic bacteria can produce enzymes for industrial use, only 0.1-1% of bacteria can be cultured using traditional techniques, which makes it challenging to isolate new enzymes with superior functions using culture methods. In order to break through the limitations of traditional culture techniques, metagenomic technology came into being. Metagenomics technology breaks through the limitations of traditional laboratory culture and can identify functional genes in uncultured microbial communities from various environmental samples. However, due to the extremely wide diversity of environmental genomes, it takes a lot of effort to select target genes. However, due to the extremely wide diversity of environmental genomes, it takes a lot of effort to select target genes. Therefore, enrichment of environmental genomes may make it possible to effectively select target genes. Enrichment culture can efficiently select and enrich microorganisms and their enzyme resources with specific functions from complex environmental samples, especially in seeking enzyme resources that are heat-resistant and resistant to extreme conditions. In general, the research on mining thermophilic enzymes faces problems such as low screening efficiency and difficulty in obtaining novel enzyme resources, and enrichment culture and metagenomics technology provide strong support for solving these problems.

[0005] In summary, how to provide a new type of β-glucosidase is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides a heat-resistant, acid-resistant, alkali-resistant, and glucose-resistant enzyme and application thereof.

[0007] The present invention aims to explore the potential thermophilic and thermostable β-glucosidase in the sediment samples of Lotus Hot Spring in Tengchong City, Yunnan Province. First, specific microorganisms were screened out from the sediment samples by enrichment culture technology, and new β-glucosidase genes were obtained by metagenomics technology. Then, clones of thermophilic and thermostable β-glucosidase were obtained by molecular cloning technology and rapid activity screening. Based on heterologous expression and protein purification technology, recombinant β-glucosidase was obtained, and its enzymatic properties and applications were determined. The study showed that a new type of thermophilic and thermostable multifunctional β-glucosidase was successfully identified by combining enrichment culture, metagenomics technology and rapid activity detection method, which indicates that it has potential application prospects in the fields of commercial utilization of lignocellulose, bioenergy production and dairy processing.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0010] Furthermore, it has the activities of β-glucosidase and β-galactosidase.

[0011] Genes encoding the above enzymes.

[0012] Furthermore, the nucleotide sequence is shown in SEQ ID NO:2.

[0013] A recombinant vector comprises the above-mentioned coding gene.

[0014] A recombinant bacterium comprises the above-mentioned recombinant vector.

[0015] The enzyme is used in the synergistic degradation of corn stalks with cellulase.

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

[0017] The application of the above gene, the above recombinant vector or the above recombinant bacteria in the industrial production of heat-resistant, acid-resistant, alkali-resistant and glucose-resistant β-glucosidase.

[0018] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0019] The technical scheme of the present invention provides an enzyme gene bfbg8, a recombinant plasmid carrying the gene, and a recombinant strain. The bfbg8 gene can encode a β-glucosidase / β-galactosidase, the optimum pH of the enzyme is 6.0, and the activity of the enzyme after incubation in a pH 4.0-10.0 buffer for 24 hours is still maintained at 100%, and the enzyme has very strong pH tolerance; the optimum temperature of the enzyme is 70°C, and the activity of the enzyme does not decrease after incubation at 65°C for 2 hours. The enzyme provided by the present invention has the superior properties of pH tolerance and strong thermal stability, and can be applied to industries such as fuel energy, food processing, papermaking, textiles, and health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 It is the technical roadmap of the present invention;

[0022] Figure 2 This is the phylogenetic tree of the BFBG8 protein in Example 1 of the present invention, with bootstrap values ​​(expressed as percentage of 1000 replications) given at the nodes;

[0023] Figure 3The SDS-PAGE electrophoresis diagram of the BFBG8 protein in Example 4 of the present invention; 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 The effects of temperature and pH on the activity and stability of BFBG8 in Example 5 of the present invention, wherein a represents the effect of temperature on the activity of BFBG8; b represents the effect of pH on the activity of BFBG8; c represents the effect of temperature on stability; d represents the effect of pH on stability; the error bars represent the mean ± SEM of three biological replicates;

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

[0026] Figure 6 The synergistic degradation of corn stover by BFBG8 and commercial cellulase at 60°C in Example 5 of the present invention, the error bars represent the mean ± SEM of three biological replicates, p < 0.01 (**) indicates an extremely significant difference, and p < 0.05 (*) indicates a significant difference;

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

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Gene: In the present invention, the soil DNA extraction kit (brand MOBIO, USA, item number: 12888-50) was used to extract genomic DNA from the sediment collected from Lotus Hot Spring, Tengchong County, Baoshan City, Yunnan Province (25.439621°N, 98.460999°E), and then sent to a sequencing company for metagenomic sequencing and annotation. The functional gene of β-glucosidase was screened out through data analysis and named bfbg8. The functional gene was then PCR amplified, identified, cloned and expressed, β-glucosidase was purified and the enzymatic properties were determined. The cloned expression vector was the self-constructed constitutive expression vector pSHY211.

[0030] The construction method of pSHY211 plasmid is as follows: First, primers TFH-F1 and THF-R1 (TFH-F1: CCCTATAGTGAGTCGTATTAATTTCGCGGGATCG, SEQ ID NO: 3; TFH-R1: SEQ ID NO:4; the bold underlined part is the HindⅢ restriction enzyme recognition site) was PCR amplified from the pET28a template to obtain a 5194 bp TFH1

[0031] SEQ ID NO:6, the wavy line portion is The promoter of the GH11 endoxylanase gene of Bacillus subtilis AQ1., the bold underlined DNA sequences are the EcoRⅠ, BamHⅠ and HindⅢ restriction enzyme recognition sites, and the double underlined part is the EGFP gene), and the primers TFH-F2 and THF-R2 (TFH-F2: GGGGTACCTAGCGTGGTATTAT, SEQ ID NO: 7; TFH-R2: SEQ ID NO:8, the bold underlined part is the HindⅢ restriction enzyme recognition site) for amplification. Subsequently, the TFH1 and TFH2 fragments were double-digested with Hind III, and the two fragments were connected using T4 DNA ligase. The ligation product was transformed into Escherichia coli DH5α, and the fluorescence-positive clones were screened on the LB plate containing kanamycin resistance, and 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 under the condition of no inducer, and the positive clones can be quickly screened by fluorescence, which significantly simplifies the cloning and expression process of the functional gene.

[0032] Enzymes and other biochemical reagents: Restriction endonucleases (EcoRⅠ and HindⅢ) were purchased from Thermo Scientific, T4-DNA ligase was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and other biochemical reagents can be purchased from ordinary domestic biochemical reagent companies.

[0033] Culture medium:

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

[0035] Example 1

[0036] DNA extraction and gene synthesis from soil samples

[0037] The bottom mud of Lotus 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, catalog number: 12888-50) and the instructions provided by the merchant were followed. Metagenomic sequencing was performed using the HiSeq 2500 sequencer of Suzhou GENWIZ. All result sequences were investigated using the IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi). To further detect the potential functions of individual genes and ORFs, we used the 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 BLASTx and BLASTp programs (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) were used to compare its DNA and protein sequences, respectively. The molecular weight of protein BFBG8 was predicted using EXPASY (https: / / web.expasy.org / protparam / ). The closely related protein sequences were aligned multiple times 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] AGCTATCGCTTTTCCATCGCCTGGCCGCGTGCTACCGTCGGGCACCGGTAAGGTC

[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] YDDVLIDGQVHDPERVHYLERHIAALAEIAAEGANVQGYYLWSLMDNFEWAKGYSKR

[0071] FGIIYVDYPTQRRILKTSGRWYADLIAAGGRVPSLT, SEQ ID NO: 1.

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

[0073] Figure 2 The phylogenetic relationship between BFBG8 protein and related proteins was established. By constructing a phylogenetic tree of BFBG8 protein and β-glucosidase from other sources, it was found that the amino acid sequence of BFBG8 protein had 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] The above gene was used as a template for PCR amplification of the functional gene. 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 sequences represent homologous recombinant fragments with the pSHY211 vector.

[0080] PCR amplification was performed using high-fidelity DNA polymerase GoldMedal Mix (Beijing Prime Tech Biotechnology Co., China). The PCR program included 98°C pre-denaturation for 3 min, followed by 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 60 s, 30 cycles, and a final extension of 72°C for 5 min. 1.0% agarose gel electrophoresis was used for identification. The PCR product was then observed under ultraviolet light, the band of the correct size was cut, and the DNA 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 endonucleases (EcoRⅠ and HindⅢ), and then electrophoresed using 1.0% agarose gel, and the resulting digestion products were recovered by cutting the gel. The PCR product after digestion was ligated with the vector using T4-DNA ligase at 25°C for 2h, and then heated to 42°C with CaCl. 2+ The chemical transformation method was used to introduce the ligation product into Escherichia coli DH5α for 60 seconds. Positive clones were obtained by colony PCR and sequencing. Ten clones were picked from each functional gene cloning plate, placed in a high-temperature sterilized EP tube, added with 500 mL of LB liquid culture medium, and cultured at 37°C for 24 hours. Then, 50 μL of bacterial solution and 10 μL of lysozyme were taken and placed in two 96-well plates for reaction at 37°C for more than 30 minutes. At the same time, 50 μL of 1% cellobiose substrate was added and placed at 50°C and 70°C for reaction for 1 hour. Finally, the glucose oxidase-peroxidase assay kit was used for color development and initial screening, and the clone with the darkest color was used as the object of subsequent experiments.

[0082] Example 3

[0083] Inducible expression of recombinant proteins

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

[0085] Example 4

[0086] Isolation and purification of recombinant proteins

[0087] Resuspend the above E. coli cells in a centrifuge tube containing 10mM imidazole in PBS solution (pH 7.6), place in an ice-water mixture for ultrasonic disruption for 30 minutes, and then centrifuge in a high-speed refrigerated centrifuge at 4°C and 7000r / min for 20 minutes. Collect the supernatant after centrifugation. Use a Ni-NTA chromatography column for purification. Equilibrate the Ni-NTA column with 5 times the column volume of equilibrium solution (PBS solution containing 10mM imidazole, pH7.6). Add the above supernatant to the Ni-NTA column for loading, repeat once, and then wash the column with 10 times the column volume of equilibrium solution. Elute the target protein with 3 times the column volume of eluent (PBS solution containing 250mM imidazole, pH 7.6), and collect the protein solution in 1.5mL centrifuge tubes, 1.0mL per tube. Determine the protein concentration using Bradford reagent, using bovine serum albumin as the standard. At OD 595 The absorbance was measured and analyzed by SDS-PAGE electrophoresis to purify the target protein.

[0088] Figure 3 The SDS-PAGE electrophoresis diagram of BFBG8 protein shows that the purified BFBG8 protein is a single band with a molecular weight of about 50 kDa, which is consistent with the theoretically predicted molecular weight (49 kDa).

[0089] Example 5

[0090] Study on the enzymatic properties of BFBG8 protein

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

[0092] The glucose oxidase-peroxidase detection kit was used for detection. Cellobiose was used as a substrate to determine the activity of β-glucosidase. 10 μL of pure enzyme solution diluted 10 times was added to 90 μL of buffer containing 1% (w / v) cellobiose, reacted at the optimal temperature for 30 minutes, and frozen at -80°C for 2 to 3 minutes to terminate the reaction. 10 μL of the reaction mixture was added to a 96-well culture plate, and 200 μL of glucose oxidase-peroxidase detection kit buffer was added. After incubation at 37°C for 10 minutes, a microplate reader was used to read the OD 492 The absorbance was measured at 400 nm. One unit (U) was defined as the amount of enzyme required to release 2 μmol of glucose from cellobiose per minute.

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

[0094] According to the activity assay, the purified β-glucosidase was tested under the optimal reaction pH conditions at different temperatures (30-85°C), and the optimal reaction temperature of BFBG8 was determined with a gradient of 5°C. For the thermal stability analysis, the enzyme solution was incubated at different temperatures of 65°C, 70°C, and 75°C for different time periods (0-120min), and the residual activity was measured every 20min. The residual activity of 0min was used as the control group, and its residual activity was measured to determine the thermal stability of BFBG8.

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

[0096] (3) Determination of the optimal pH and pH stability of β-glucosidase

[0097] The purified β-glucosidase was subjected to enzymatic reaction at different pH to determine its optimum pH. The buffer used was a pH 3.0-8.0 citric acid-sodium hydrogen phosphate series buffer, a pH 8.0-10 glycine-hydrochloric acid buffer, and a gradient of 0.6 pH. The optimum pH of the purified β-glucosidase in a buffer system of different pH was determined at 70°C. Using a buffer of pH (3.0-11.0), a gradient of 1 pH, the pure enzyme solution was placed at 4°C for 12h and 24h, and then the residual activity was measured to determine the pH stability of BFBG8.

[0098] The results showed that the optimal pH value 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 approximately 90% of its initial activity in the pH range of 3.0 to 11.0 ( Figure 4 d). The results showed that BFBG8 can function under a wide range of acid-base conditions and maintain the stability of its activity.

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

[0100] Using p-nitrophenyl-β-d-pyranoglucoside (pNPGlc) as the substrate, 10 μg of protein was added to 150 μL of reaction mixture containing 1 mM pNPGlc (Sigma, St. Louis, MO, USA) at pH 6.0. After incubation at the optimal temperature for 5 min, 450 μL of 1 M Na 2 CO 3 Stop the reaction. Using p-nitrophenol (Sigma, USA) as a standard, monitor the absorbance at 405 nm to determine the release of p-nitrophenol. In order to study the effect of D-glucose on the catalytic activity of BFBG8, different concentrations of D-glucose (0-5M) were added to the assay mixture containing 1mM pNPGlc under standard conditions. The control group was used without glucose.

[0101] The results of the test ( Figure 5 ) showed that: under different glucose concentration conditions, BFBG8 showed unique activity change characteristics. When the glucose concentration was in the range of 250mM to 3000mM, the relative activity of BFBG8 was stimulated. Among them, when the glucose concentration reached 1500mM, the stimulation effect was most significant, and the activity of BFBG8 was 1.4 times that in the absence of glucose. When the glucose concentration exceeded 3000mM, although the hydrolysis activity of BFBG8 was gradually inhibited with the continuous increase in glucose concentration, its relative enzyme activity remained above 80%. Unlike most β-glucosidases, glucose did not significantly inhibit the enzyme activity of BFBG8, which fully demonstrated that BFBG8 has strong glucose tolerance.

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

[0103] 10 g corn straw was ground and sieved through 80 mesh and boiled in 100 mL hot water for 30 min. The corn straw was filtered through a paper filter and dried at 80 °C. 0.2 g of corn straw pretreated with hot water was added to 1 mL of buffer (pH 6.0), and 0.2 mg of Trichoderma reesei cellulase (Sangon Biotech, China) and 0.05 mg of BFBG8 were added. The mixture was then incubated at 60 °C. Samples were taken 0 to 7 h after the start of the reaction, with an interval of 1 h between each sampling. 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 addition. Three biological replicates were performed for each group, and the average value was used for all subsequent analyses. The glucose concentration in the straw hydrolysis at different times was tested.

[0104] The results showed that the addition of β-glucosidase BFBG8 to commercial cellulase significantly improved the hydrolysis efficiency, and the degradation rate increased by about 50% after 7 hours ( Figure 6 These results suggest that BFBG8 has potential applications in lignocellulose degradation and can work synergistically with commercial cellulases.

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

[0106] In order 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, respectively, so that the final concentration of metal ions was 1mM and 10mM, respectively. In order to determine the effect of inhibitors on β-glucosidase, different inhibitors [disodium ethylenediaminetetraacetate (EDTA), sodium dodecyl sulfate (SDS), phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT)] were added to the enzyme solution at 60°C, respectively, so that the final concentration of the inhibitors was 0.1% and 1%, respectively. In order to determine the effect of ethanol on β-glucosidase, ethanol was added to the enzyme solution at 60°C, respectively, so that the final concentration of ethanol was 1% and 10%. After standing for 10 minutes, the residual enzyme activity was determined. The reaction mixture without additives under standard conditions was used as the control (100%).

[0107] The results showed that: using cellobiose as substrate, the effects of various metal ions, chemical reagents and ethanol on the activity of BFBG8 were studied. The enzyme activity without adding metal ions / chemical reagents / ethanol was defined as 100%, and other relative enzyme activities were calculated (Table 1). When the metal ion concentration was 1mM, Mg 2+ , Fe 3+ , Fe 2+ 、Zn 2+ , Cu 2+ , Mn 2+ , Pb 2+ 、Ni 2+ No effect was observed on BFBG8 activity, whereas K + 、Co 2+ 、Ag + Significantly inhibited the activity of BFBG8, among which Ag +The activity of BFBG8 was inhibited by more than 95%; when the concentration increased to 10mM, metal ions significantly inhibited its activity. When the concentration of organic solvents was 0.1%, except for SDS, which significantly inhibited the activity of BFBG8, the activities of other organic solvents were retained at more than 90%, and when the concentration increased to 1%, the activity decreased and was significantly inhibited. BFBG8 was not affected by 1% ethanol, but was inhibited by 10% ethanol.

[0108] Table 1 Effects of different metal ions and chemicals on BFBG8 enzyme activity

[0109]

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

[0111] To determine the substrate specificity of BFBG8, cellobiose, lactose, p-nitrophenyl-β-D-pyranoglucopyranoside (pNPGlc), gentiobiose, maltose, melibiose, trehalose, raffinose, sucrose, wheat bran xylan, corncob xylan, bagasse xylan, carboxymethylcellulose sodium (CMC-Na) and microcrystals (Avicel) were used as substrates (1%, w / v) to measure the enzyme activity, and the activities of cellulase and xylanase were determined using 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] Substrate specificity assays showed that BFBG8 had hydrolysis activity on most substrates (Table 2), such as p-nitrophenyl-β-D-pyranoglucoside (92.53±0.84U / mg). In addition, it could hydrolyze substrates with β-(1→4) glycosidic bonds, such as cellobiose (40.39±1.44U / mg), lactose (80.78±2.89U / mg) and gentiobiose (3.07±0.03U / mg). However, it could not hydrolyze substrates connected by α-glycosidic bonds, such as sucrose and maltose. It also could not hydrolyze polysaccharides, such as xylan, sodium carboxymethyl cellulose and microcrystals, because the complexity of the substrates and the long chains of polysaccharides would hinder the activity of β-glucosidase.

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

[0116] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0117] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

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

1.

2. The enzyme according to claim 1, characterized in that It also has the activity of β-glucosidase and β-galactosidase.

3. A gene encoding the enzyme according to claim 1.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence is shown in SEQ ID NO:

2.

5. A recombinant vector, characterized in that: Comprising the coding gene described in claim 4.

6. A recombinant bacterium, characterized in that: Comprising the recombinant vector described in claim 5.

7. Use of the enzyme according to claim 1 in synergistic degradation of corn stalks with cellulase.

8. Use of the enzyme according to claim 1 in food, feed, medicine and industrial production.

9. Use of the gene according to claim 3 or 4, the recombinant vector according to claim 5 or the recombinant bacteria according to claim 6 in the industrial production of heat-resistant, acid-resistant, alkali-resistant and glucose-resistant β-glucosidase.

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