A salt-tolerant beta-glucosidase and its application

Abstract

The present invention discloses a salt-tolerant β-glucosidase and application, and relates to the field of biotechnology. The amino acid sequence of the β-glucosidase is shown in SEQ ID NO: 1. The present invention clones a novel β-glucosidase functional gene b0-bg40 belonging to the GH1 family from Karamay saline-alkali land using metagenomic technology, and realizes heterologous expression in Escherichia coli. The enzymatic properties of b0-bg40 are then characterized, and it is found that b0-bg40 is an alkali-resistant, thermally stable, and pH-wide β-glucosidase. In addition, b0-bg40 is tolerant to glucose and has a high affinity for cellobiose. These findings indicate that it has potential utilization value in the degradation of cellulose biomass and production in strong alkaline environments.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a salt-tolerant beta-glucosidase and an application thereof. Background Art

[0002] Cellulase is a general term for a class of enzymes that can degrade cellulose and eventually convert it into glucose, including endoglucanases, exoglucanases and β-glucosidases. β-Glucosidase (EC 3.2.1.21) is a group of glycosyl hydrolases that can hydrolyze terminal non-reducing β-D-glucosidic bonds. β-Glucosidase is widely used in food, feed, biomass biofuels and other industries. With the accumulation of straw crops and environmental pollution, β-glucosidase has received more and more attention. As a key enzyme for the degradation of lignocellulose, β-glucosidase plays a great role in industrial production and environmental governance.

[0003] β-Glucosidase is widely present in animals, plants, and microorganisms, but 99% of microorganisms in nature are not easy to obtain pure culture, and uncultured microorganisms are also the largest untapped biological resources on the earth. Therefore, the method of metagenomics was introduced. Metagenomics is an effective technology for mining new enzyme resources from the metagenome of uncultivated microorganisms. β-Glucosidase can promote the hydrolysis of cellulose, but the product glucose generated in the biocatalytic process and some ions, chemical reagents, salts, etc. will inhibit the activity of β-glucosidase, greatly limiting its application in industrial production. Microorganisms living in extreme environments can adapt well to conditions that microorganisms from ordinary environmental sources cannot adapt to. For example, the β-glucosidase obtained from the hot spring metagenome has higher thermal stability and tolerance to glucose and ethanol, and the β-glucosidase obtained from the marine bacterium Alteromonas sp.L82 has cold adaptability and salt tolerance. Most β-glucosidases that can tolerate glucose are not heat-resistant, and β-glucosidases with good thermal stability fail to show good glucose tolerance. my country is a large agricultural producer, and a lot of straw waste is produced every year. In addition, the degree of land salinization is increasing, and these wood celluloses are difficult to degrade naturally. Therefore, it is very important to be able to tap into β-glucosidase with strong thermal stability, good alkaline resistance, and glucose tolerance, which has great application potential in industrial applications, environmental protection, and cellulose biomass processing.

[0004] This study used metagenomic technology to extract the total microbial metagenome from soil samples brought back from the saline-alkali zone of Karamay, obtained a new β-glucosidase functional gene (b0-bg40), cloned it, heterologously expressed it, separated and purified its protein, and investigated its enzymatic properties. Finally, it was separated and purified and its enzymatic properties were analyzed. It was found that it is a β-glucosidase with strong thermal stability and good alkaline resistance, which provides a reference for the development and application of β-glucosidase from salt environment. Summary of the invention

[0005] In view of this, the present invention provides a β-glucosidase gene, a recombinant vector, a host cell, a β-glucosidase and its application, aiming to solve the problems of poor thermal stability and glucose tolerance of the existing β-glucosidase, and to provide a β-glucosidase with strong thermal stability, a wide pH tolerance range and strong glucose tolerance and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A salt-tolerant beta-glucosidase, the amino acid sequence of the beta-glucosidase is shown in SEQ ID NO: 1.

[0008] A DNA molecule encoding the β-glucosidase of claim 1, wherein the DNA molecule is (a) or (b):

[0009] (a) the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0010] (b) A DNA molecule encoding the β-glucosidase formed by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 2.

[0011] A recombinant vector comprises the DNA molecule and a regulatory sequence for expression which is operably linked to the DNA molecule.

[0012] A host cell, wherein the host cell comprises the DNA molecule or the recombinant vector.

[0013] Application of the salt-tolerant beta-glucosidase in degrading cellulose.

[0014] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0015] The present invention predicts a new β-glucosidase functional gene (b0-bg40) from the Karamay saline-alkali soil metagenome, obtains the gene by PCR amplification, clones it, performs heterologous expression, separates and purifies its protein, and explores its enzymatic properties. It is found that the enzyme is a β-glucosidase with strong thermal stability and good alkaline resistance, and has broad application prospects in industrial production, food and other industries.

[0016] The optimum temperature and optimum pH of b0-bg40 are 45℃ and 8.6 respectively. It can maintain more than 60% relative enzyme activity at 20-55℃ and more than 50% relative enzyme activity at pH 5.6-9.6. b0-bg40 has strong thermal stability and pH stability. After incubation at 40℃ and 45℃ for 10h, its activity still remains above 85%; after incubation at 50℃ for 8h, it still has about 60% relative enzyme activity; after incubation at pH 3.0-10.0 for 12h and 24h, it can still maintain more than 100% relative enzyme activity. In addition, it has strong thermal stability and pH stability. 2+ 、Ni 2+ The relative activity of b0-bg40 is still maintained at more than 85% when the concentration of glucose is 3000mM. The Ki value of glucose is 3604.85mM, and the activity is still maintained at more than 60% when the concentration of glucose is 3000mM. In summary, the β-glucosidase b0-bg40 screened by the present invention has the excellent characteristics of strong thermal stability, good alkali resistance and glucose resistance, indicating that it is suitable for commercial cellulose utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 is the phylogenetic tree of b0-bg40 constructed by the maximum likelihood method (ML);

[0019] Figure 2 This is the SDS-PAGE gel analysis of recombinant β-glucosidase b0-bg40;

[0020] Figure 3 The effects of temperature and pH on the activity and stability of recombinant b0-bg40;

[0021] Figure 4 is the effect of glucose concentration on the activity of recombinant b0-bg40;

[0022] Figure 5It is the Lineweaver-Burk double reciprocal plot of b0-bg40 with 1% cellobiose as substrate. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1

[0025] 1 Materials and methods

[0026] 1.1 Sample collection and storage

[0027] Soil samples were collected from saline-alkali land in Karamay and stored in the Molecular Ecology Laboratory of the College of Agriculture and Biological Sciences, Dali University.

[0028] 1.2 Culture medium, strains and vectors

[0029] LB basal medium: 5 g yeast extract, 10 g trypsin, 10 g NaCl, solid separation medium supplemented with 2% agar, 1 L distilled water; kanamycin was used at 50 μg / mL.

[0030] E. coli DH5α strain was used for cloning, E. coli BL21 DE3 was used as host to express proteins, and pET28a vector was used to construct recombinant plasmids.

[0031] 1.3 Methods

[0032] 1.3.1 Sample collection and gene cloning

[0033] Soil samples from Karamay saline-alkali land were collected as experimental subjects, and metagenomic DNA was extracted using a soil DNA extraction kit (brand MOBIO, USA, item number: 12888-50). Then the samples were sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for metagenomic sequencing and annotation. Through bioinformatics analysis, a new functional gene of β-glucosidase was screened and named b0-bg40.

[0034] Design primers for this sequence:

[0035] b0-bg40-F: CAAATGGGTCGCGGATCCGAA ATGCGCATGAAGGGCCTTCGG, as shown in SEQ ID NO: 3;

[0036] b0-bg40-R: GTGCTCGAGTGCGGCCGCAAGTTATTCGAGACCGGTGATCTCGCG, as shown in SEQ ID NO: 4;

[0037] The underlined sequence is the homologous recombination fragment of pET28a(+), and the plasmid has been digested by EcoR I and Hind III. The above metagenome was used as the amplification template, and the PCR amplification conditions were 94℃ pre-denaturation for 3min, 98℃ denaturation for 15s, 58℃ annealing for 30s, 72℃ extension for 60s, 32 cycles, and finally 72℃ extension for 5min. After the amplification was completed, it was identified by 1.0% agarose gel electrophoresis, and then the DNA band with a size of about 1500bp was selected for gel cutting and gel recovery. The purified PCR product was ligated with the double-digested pET28a(+) using the pEASY-Uni Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd., China, Catalog No.: CU101-01). The ligated product was introduced into the E. coli DH5α strain for cloning. The expression recombinant plasmid pET28a(+)-b0-bg40 was obtained by sequencing verification. The recombinant plasmid was transferred into the E. coli BL21(DE3) strain to obtain the recombinant strain E. coliBL21(DE3)-b0-bg40 for expression.

[0038] 1.3.2 Sequence analysis

[0039] The DNA and protein sequences of β-glucosidase functional genes b0-bg40 were analyzed using BLASTx and BLASTp online software (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and their signal peptides were predicted using SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ). The phylogenetic tree was constructed using the maximum likelihood method (ML) and Poisson correction model using MEGA 7 software.

[0040] A 1431bp β-glucosidase gene of the GH1 family was predicted in the genome and named b0-bg40. Through sequence analysis and phylogenetic tree construction, it was found that the highest similarity between b0-bg40 and the β-glucosidase from Puniceicoccaceaebacterium K14 was 56.79% ( Figure 1 ).

[0041] β-glucosidase b0-bg40 gene DNA sequence (1431 bp), as shown in SEQ ID NO: 2:

[0042]

[0043] The b0-bg40 protein sequence (476 residues) is shown in SEQ ID NO: 1:

[0044] MRMKGLRWSGFFPYRSLMSNADFFFGAGTSSYQIEGAWNADGKGPSIWDYHTRNPGAVYRGHSGDVACDHYYRYQEDVRLMQTIGLQAYRFSINWPRVLPEGTGLPNEAGLAFYDRLVD ELLEAGITPWITLYHWELPWALHVRGGWLNPDMPKHVADFAALMATRLGDRVTNWITLNEPQVFIGLGYANGVHAPGYKLSLRECLLGAHHAVLAHHAAVGAIREHALQPVQIGSAPVGV VCRPQTESAADVEAARRATYAIKKPTTHTPDDLIGCLWNSTWWIDAMVLGTYPAEGLEAFNGFLPENAQHELNEAFLPTDFVGSNIYHGRTVKATADGGFEHVDLPAGSPRTTMGWDIT PDILYWGGKYLYERYKKPMYITENGIAVPELVNDEHAVEDTVREQFIKVHLRGLQRAREEGIPYKGYFHWSLMDNFEWEQGYSQRFGIVYVDYQSQERILKRSGKAFAPIVREITGLE-;

[0045] 1.3.3 Inducible expression of recombinant protein

[0046] The strain E.coli BL21(DE3)-b0-bg40 was inoculated into an LB plate containing kanamycin resistance, and a single colony was picked out and placed in a test tube containing 5 mL LB liquid culture medium. The culture was continuously shaken at 220 rpm at 37°C overnight, and then transferred to 200 mL LB culture medium containing 50 μg / mL kanamycin as a 1% inoculum of seed liquid. The culture was continuously shaken at 220 rpm at 37°C for about 2 hours until the OD600 was about 0.6. The culture was then placed in an ice water bath for 10 minutes, and IPTG was added to a final concentration of 0.1 mM. The culture was then transferred to 25°C, 220 rpm, and incubated for another 8 hours. After the culture was completed, the cells were collected by centrifugation at 4°C and 12000 r / min for 20 minutes and stored in a -80°C refrigerator.

[0047] 1.3.4 Isolation and purification of recombinant protein

[0048] The prepared bacteria were suspended in a centrifuge tube with PBS solution (containing 10mM imidazole), then placed in an ice-water mixture and crushed with an ultrasonic cell disruptor for 30 minutes, and centrifuged at 12000r / min at 4°C using a high-speed refrigerated centrifuge for 20 minutes. The supernatant after centrifugation was filtered using a 0.45μm filter membrane, and then separated and purified using a Ni-NTA affinity chromatography column. First, the nickel column was cleaned and the column balance was adjusted, and then the preserved supernatant was added to the nickel column for column filtration. Finally, about 1.0mL was collected and retained in a 1.5mL centrifuge tube. The protein concentration was measured using the Bradford method, and the protein size of the enzyme was determined and analyzed using SDS-PAGE electrophoresis.

[0049] After SDS-PAGE electrophoresis analysis, it was found that the molecular weight of the recombinant β-glucosidase b0-bg40 was basically consistent with the theoretically predicted protein molecular weight, indicating that it was the recombinant target protein. Figure 2 As shown (1. Protein Marker; 2. E. coli BL21 (DE3) -pET-28a-b0-bg40 cells after IPTG induction; 4. Purified b0-bg40 protein).

[0050] 1.4 Analysis of the enzymatic properties of recombinant β-glucosidase B0-BG40

[0051] 1.4.1 Enzyme activity assay

[0052] The activity of β-glucosidase was determined using cellobiose as a substrate. 10 μL of enzyme solution was added to 90 μL of buffer containing 1% (w / v) cellobiose, reacted at the preset optimal temperature for 10 minutes, and then frozen at -80°C for 2-3 minutes to terminate the reaction. Then 10 μL of the reaction mixture was placed on a 96-well culture plate, and 200 μL of glucose oxidase-peroxidase detection kit buffer was added respectively, incubated at 37°C for 10 minutes, and the absorbance value was measured at 492 nm using an ELISA reader. Each group of experiments was repeated 3 times.

[0053] 1.4.2 Optimum reaction temperature and thermal stability

[0054] The relative enzyme activity of the purified β-glucosidase was measured at different temperatures (4-75°C), and the optimal reaction temperature was determined with a gradient of 5°C. For thermal stability analysis, the pure enzyme solution was incubated at different temperatures (40°C, 45°C and 50°C) for 10 h, and the residual enzyme activity of B0-BG40 was determined with a gradient of 1 h.

[0055] The recombinant β-glucosidase showed the highest activity at 45°C. In addition, it was relatively adaptable to lower temperatures and maintained more than 20% of its enzyme activity at 4°C. Figure 3A). It is worth noting that the recombinant β-glucosidase showed high thermal stability within 10 hours, was inactivated at 50°C for 9 hours, and its relative enzyme activity remained above 85% after incubation at 40°C and 45°C for 10 hours, indicating that it has good thermal stability ( Figure 3 B).

[0056] 1.4.3 Optimal reaction pH and pH stability

[0057] The enzymatic reaction was carried out with B0-BG40 at the optimal temperature using citric acid-sodium hydrogen phosphate buffer with different pH values ​​(3, 3.6, 4, 4.6, 5, 5.6, 6, 6.6, 7, 7.6, 8) and glycine-sodium hydroxide buffer with pH values ​​(8, 8.6, 9, 9.6, 10, 10.6, 11), and the optimal reaction pH was recorded. To analyze pH stability, the enzyme was incubated at 4°C in buffers with different pH values ​​(3.0-10.0) for 12 and 24 hours to measure the residual enzyme activity of purified B0-BG40 at each pH value to evaluate its pH stability.

[0058] The optimum pH of the recombinant β-glucosidase is 8.6. The recombinant β-glucosidase exhibits a relative enzyme activity of more than 55% at pH 5.6-9.6, and has a wide pH adaptability range ( Figure 3 C). The enzyme activity of the enzyme after incubation for 12h and 24h showed consistency. After incubation in pH 8.0-10.0 buffer for 12h and 24h, its activity was activated to a certain extent, showing good pH tolerance ( Figure 3 D).

[0059] 1.4.4 Effects of metal ions and chemical reagents on recombinant enzymes

[0060] 1mM and 10mM metal ions (K + Mg 2+ , Fe 3+ , Ca 2+ 、Zn 2+ 、Co 2+ , Cu 2+ 、Ag + , Mn 2+ , Pb 2+ and Ni 2+ ) and 1%, 10% chemical reagents [disodium ethylenediaminetetraacetate (EDTA), phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), Tween-80 (Tween-80), ethanol, sodium dodecyl sulfate (SDS)], the enzymatic reaction was carried out under the optimal reaction conditions, and the effects of these additives on the recombinant enzyme activity were recorded, as shown in Table 1.

[0061] Table 1: Effects of metal ions and chemical reagents on the activity of recombinant b0-bg40;

[0062]

[0063] The results in Table 1 show that the 11 metal ions and 5 chemical reagents involved in the test all have a certain effect on the activity of the enzyme. 1mM and 10mM metal ion Mg 2+ , Fe 3+ , Ca 2+ 、Ni 2+ When Zn 2+ 、Ag + The effect on enzyme activity is the greatest, and the enzyme activity is completely inhibited at a concentration of 10mM. Except for SDS, which has a strong inhibitory effect on enzyme activity, the other organic solvents involved in the test have relatively small effects on enzyme activity, and the activity can be maintained above 80%, so the b0-bg40 recombinant enzyme has a certain tolerance to these ions or chemical reagents.

[0064] 1.4.5 Effect of glucose concentration on enzyme activity

[0065] Using p-nitrophenyl-β-D-pyranoglucoside (pNPG) as substrate, 10 μl of b0-bg40 was added to 125 μl of glucose solution of different concentrations (0-4000 mM) containing 10 mM pNPG (15 μl), with no enzyme added as the control group. After incubation at the optimal temperature for 10 minutes, the reaction was stopped by adding 450 μl of Na2CO3. The inhibition constant (Ki) of b0-bg40 was determined as the glucose concentration at which the enzyme activity was inhibited by half.

[0066] The glucose tolerance of b0-bg40 was determined using 10mM pNPG as a substrate. When the added glucose concentration was 500mM, its activity was still maintained at about 100%. When the glucose concentration was lower than 2000mM, b0-bg40 was able to maintain more than 60% of its activity, and its Ki value was determined to be 2566mM ( Figure 4 ). This indicates that b0-bg40 is a glucose-tolerant β-glucosidase.

[0067] 1.4.6 Determination of substrate specificity and kinetic parameters

[0068] Different substrates (Cellobiose, Corn cob xylan, Avicel, Wheatbran xylan, CMC, Bagasse xylan, Beech xylan) were added to the enzyme reaction system and placed under optimal reaction conditions to determine the amount of hydrolysis product produced.

[0069] Different concentrations of cellobiose (0.2%-2%) were used to react at the optimal temperature and pH for 5 min and 10 min, respectively. The kinetic parameters (Vmax and Km) of b0-bg40 were calculated using the Michaelis Menten equation of cellobiose, and the Lineweaver-Burk double reciprocal plot was drawn.

[0070] The substrate specificity of b0-bg40 is shown in Table 2.

[0071] Table 2: Effects of different substrates on the activity of recombinant b0-bg40;

[0072]

[0073] The results in Table 2 show that it has the highest hydrolysis activity against cellobiose, while it has no hydrolysis activity against the other substrates (Corn cob xylan, Avicel, Wheat bran xylan, CMC, Bagasse xylan, Beech xylan).

[0074] The kinetic parameters of the recombinant β-glucosidase were calculated based on the Michaelis Menten equation for cellobiose. At the optimum temperature and pH, the Km and Vmax were calculated to be 0.025 mM and 370.37 μmol / min / mg, respectively. Figure 5 ).

[0075] 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 salt-tolerant β-glucosidase, characterized in that The amino acid sequence of the β-glucosidase is shown in SEQ ID NO:

1.

2. A DNA molecule encoding the β-glucosidase according to claim 1, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that: The recombinant vector comprises the DNA molecule of claim 2 and a regulatory sequence for expression operably linked to the DNA molecule.

4. A host cell, characterized in that The host cell comprises the DNA molecule according to claim 2 or the recombinant vector according to claim 3.

5. Use of the salt-tolerant β-glucosidase according to claim 1 in degrading cellulose.

Images