Beta-1, 4-glucanase and application thereof

Abstract

The invention relates to beta-1, 4-glucanase and application thereof, and belongs to the technical field of biology. A novel beta-1, 4-glucanase is screened and identified in Pleurotus tuber-regium, and the beta-1, 4-glucanase has wide substrate specificity, can specifically hydrolyze beta-glucan (such as sodium carboxymethyl cellulose) containing beta-1, 4 glucosidic bonds, and can hydrolyze beta-glucan (such as sodium carboxymethyl cellulose) containing beta-1, 3-1, 4-glucanase) containing beta-1, 3-1, 4-glucanase. The beta-1, 4-glucanase disclosed by the invention can be used for preparing beta-glucan (such as barley beta-glucan and oat beta-glucan) containing beta-1, 3-1, 4 glucosidic bonds, so that the beta-1, 4-glucanase disclosed by the invention has good enzyme activity on the beta-glucan containing the beta-1, 4 glucosidic bonds and the beta-glucan containing the beta-1, 3-1, 4 glucosidic bonds. Besides, the enzyme activity of the beta-1, 4-glucanase disclosed by the invention can be kept at 70% or above under the condition that the pH value is 4.0-7.0, the enzyme activity of the beta-1, 4-glucanase can be kept at 88% or above under the temperature of 40 DEG C, and the beta-1, 4-glucanase has good stability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a beta-1,4-glucanase and application thereof. Background Art

[0002] The immunomodulatory, anti-tumor, anti-inflammatory, and antioxidant biological activities of β-glucan are closely related to its specific structural characteristics, including the type of glycosidic bonds, the degree of polymerization, and the three-dimensional conformation of the molecule. Naturally extracted β-glucan has problems such as poor water solubility or high viscosity after dissolution, which affects food processing or brewing processes and limits industrial applications. Currently, physical and chemical methods such as ultrasound, heating, and acid-base treatment are commonly used to improve the properties of β-glucan. Compared with physical and chemical methods, enzymatic treatment is a highly specific and environmentally friendly method. β-glucanase can modify the structure of β-glucan or produce specific oligosaccharides or oligosaccharides based on its specificity for glycosidic bonds, thereby improving the properties of β-glucan.

[0003] β-glucanases can be classified according to the type of glycosidic bonds they catalyze, mainly including β-1,3-, β-1,4-, and β-1,6-glucanases, which can be further divided into exo- and endo-types. For example, endo-β-1,4-glucanases can randomly cut β-1,4-glycosidic bonds from the inside of the β-glucan chain to produce a series of oligosaccharides with different degrees of polymerization; while exo-β-1,4-glucanases cut from the non-reducing ends of cellulose to produce cellobiose residues or glucose. β-1,3-1,4-glucanases (also known as lichenases) are a special class of enzymes that can specifically recognize and cut β-1,4-glycosidic bonds next to β-1,3-glycosidic bonds to produce oligosaccharides or glucose with 3-5 glucose units. This type of enzyme has a highly efficient degradation effect on glucans containing a mixed structure of β-1,3 and β-1,4 glycosidic bonds (such as cereal β-glucans), and has special value in the fields of beer brewing, feed processing, medicine and health products. However, the currently characterized β-1,3-1,4-glucanases are of a single source, mainly bacterial, and mainly belong to the GH16 and GH17 families of glycoside hydrolases. In order to meet the needs of industrialization, researchers often use chemical modification, rational design and other methods to improve enzyme activity and stability. However, these enzymes still have limitations in substrate specificity and functional diversity.

[0004] In the existing technology, some endo-β-1,4-glucanases have shown wide substrate specificity, but there is still a lack of multifunctional β-glucanases that can efficiently degrade a variety of β-glucans (such as β-1,3-1,4 mixed glycosidic bonds and β-1,4 glycosidic bonds). In addition, as a safe and edible natural resource, edible fungi have good application potential for their enzymes, but the current exploration and systematic characterization of related enzyme resources are still insufficient. Therefore, it is urgent to seek a β-glucanase with a wide substrate range and diverse functions from edible fungi. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of narrow substrate specificity, single function and limited application range of β-glucanase in the prior art.

[0006] In order to solve the above technical problems, the present invention provides a β-1,4-glucanase and its application. The present invention screened and identified a new type of β-1,4-glucanase in the edible fungus Tiger Milk Mushroom, which was identified as belonging to the GH5 family of glycoside hydrolases. The β-1,4-glucanase of the present invention has a wide substrate range, and can specifically hydrolyze β-glucans containing β-1,4 glycosidic bonds (such as sodium carboxymethyl cellulose), and can also hydrolyze β-glucans containing β-1,3-1,4 glycosidic bonds (such as barley β-glucan and oat β-glucan), so it can efficiently degrade various types of β-glucans and has diverse functions. In addition, the β-1,4-glucanase of the present invention can maintain an enzyme activity of more than 70% under the condition of pH 4.0-7.0, and can maintain an enzyme activity of more than 88% at a temperature below 40°C, and has good stability.

[0007] The first object of the present invention is to provide a β-1,4-glucanase, the amino acid sequence of the β-1,4-glucanase is shown in SEQ ID NO.2.

[0008] Furthermore, the amino acid sequence of SEQ ID NO.2 is as follows:

[0009] MQSPIYGQCGGIGWNGATICVSGAVCTKLNDYFSQCVPGTASSVPSSTPASSSSTPSSTISAGPTPTGKLRFTGVNIAGFDFGCNSDGNCVASAAWPPLTQYYGADGAGQMQHFVNDDGFNTFRLPVGWQFLVNDVLGGPINEDNFKKYDDLVQTCLATGAFCIIDVHNYARWNGKIIGQGGPTDEQFASLWGAIA AKYASNSKILFGVMNEPHDVPDITRWAQSVQAAVTAIRNAGATSQLILLPGNNWTSAATFVSNGSADALSKVTNPDGSTTGLIFDVHKYLDYDNSGTNAECVTDNVADAWAPLADYLRAHNRQAINTETGGGNTASCVQYMCQQVAYQEANADVFLGYIGWAAGNFYPTYVLGEVPTNTGSGWTDTLLVESCLKRT

[0010] The second object of the present invention is to provide a gene encoding the above-mentioned β-1,4-glucanase, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] Furthermore, the nucleotide sequence of SEQ ID NO.1 is as follows:

[0012]

[0013] The third object of the present invention is to provide a recombinant plasmid carrying the above gene.

[0014] The fourth object of the present invention is to provide a recombinant cell expressing the above-mentioned β-1,4-glucanase.

[0015] Furthermore, the recombinant cell is a bacterium or a fungus.

[0016] A fifth object of the present invention is to provide an application of the above-mentioned β-1,4-glucanase, the above-mentioned gene, the above-mentioned recombinant plasmid or the above-mentioned recombinant cell in degrading β-glucan or β-glucan-rich materials, wherein the β-glucan contains β-1,4 glycosidic bonds.

[0017] Furthermore, the application is to add the β-1,4-glucanase or the expression system containing the β-1,4-glucanase to a system containing β-glucan for reaction.

[0018] The sixth object of the present invention is to provide a method for degrading β-glucan, wherein the above-mentioned β-1,4-glucanase is added to a substrate system containing β-glucan for reaction, wherein the substrate contains β-1,4 glycosidic bonds or β-1,3-1,4 glycosidic bonds.

[0019] Furthermore, the pH of the reaction is 4.0-7.0.

[0020] Furthermore, the reaction temperature is 20°C-55°C.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a β-1,4-glucanase and its application. The present invention screened and identified a new type of β-1,4-glucanase in tiger milk mushroom. The β-1,4-glucanase of the present invention has a wide substrate specificity, and can specifically hydrolyze β-glucans containing β-1,4 glycosidic bonds (such as sodium carboxymethyl cellulose) and β-glucans containing β-1,3-1,4 glycosidic bonds (such as barley β-glucan and oat β-glucan). Therefore, the β-1,4-glucanase of the present invention has good enzyme activity for β-glucans containing β-1,4 glycosidic bonds and β-glucans containing β-1,3-1,4 glycosidic bonds. In addition, the enzyme activity of the β-1,4-glucanase of the present invention can be maintained at more than 70% under the condition of pH 4.0-7.0, and the enzyme activity can be maintained at more than 88% at 40°C, and has good stability. The present invention provides a β-1,4-glucanase derived from edible fungi, which solves the problems of single enzyme resources and limited functions in the prior art, has wide substrate specificity and efficient hydrolysis performance, makes up for the deficiencies in the properties and application research of β-glucanase derived from fungi, and provides a new solution for the specific hydrolysis of β-glucan. The enzyme can specifically degrade β-1,3-1,4-glucan and β-1,4-glucan to generate oligosaccharides with a specific degree of polymerization, has potential application value as a prebiotic or food functional ingredient, and provides a new solution for the efficient hydrolysis of β-glucan and the production of oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0024] Figure 1 The figure is the SDS-PAGE electrophoresis result of the expression product and the purified enzyme protein, where M: standard protein marker; 1: crude enzyme solution; 2: target protein after purification by Ni-NTA affinity column;

[0025] Figure 2 The effect of reaction pH on the relative activity of β-1,4-glucanase PTRGLT4 and the pH stability of the enzyme; A: optimal reaction pH; B: pH stability;

[0026] Figure 3 The effect of reaction temperature on the relative activity of β-1,4-glucanase PTRGLT4 and the thermal stability of the enzyme; where: A: optimal reaction temperature; B: thermal stability;

[0027] Figure 4This is the MALDI-TOF-MS analysis of oat β-glucan hydrolysate; A: MS spectra after 5min and 30min of enzyme action; B: secondary structure information of DP4 to DP7;

[0028] Figure 5 It is a three-dimensional structure diagram of the β-1,4-glucanase PTRGLT4 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] Escherichia coli JM109 and Pichia pastoris GS115 (stored in our laboratory); expression vector pPIC9K (stored in our laboratory); restriction enzymes SnaB I, EcoR I and Sac I (purchased from Thermo Fisher Scientific); LB medium (containing 5 g yeast extract, 10 g peptone, and 10 g NaCl per liter); YPD medium (containing 10 g yeast extract, 20 g peptone, and 20 g glucose per liter); BMGY medium (containing 10 g yeast extract, 20 g peptone, 13.4 g amino-free yeast nitrogen source, and 4.0 × 10 -4 g, 10% 1M pH 5.0 phosphate buffer, 10 g glycerol); BMMY medium (containing 10 g yeast extract, 20 g peptone, 13.4 g amino-free yeast nitrogen source, 4.0 × 10 -4 g, 1M pH 5 phosphate buffer 10%, methanol 1.5%); MD plate medium (containing 13.4 g of amino-free yeast nitrogen source per liter, 4.0×10 -4 g, glucose 20 g, agar powder 20 g); Geneticin G418 (purchased from Sangon Biotech); gel recovery / product purification kit, plasmid extraction kit, one-step cloning kit (purchased from Vazyme).

[0031] Example 1: Screening and Acquisition of β-1,4-Glucanase

[0032] Based on the whole genome sequencing data of Pleurotus tuber-regium (NCBI: ASM1969331v1), dbCAN and other tools were used to screen carbohydrate enzyme-related genes, and the gene of β-1,4-glucanase was mined through gene structure and conserved motif analysis. The protein domain, transmembrane region and signal peptide were analyzed. The gene sequence contained a signal peptide sequence, a fungal cellulose binding domain and a GH5 family cellulase domain, and the protein was located outside the cell membrane. The gene sequence of the β-1,4-glucanase gene of the present invention after removing the signal peptide is SEQ ID NO.1; its amino acid sequence is SEQ ID NO.2. Using the NCBI online BLAST tool analysis, it was found that the highest homology of the protein sequence of the β-1,4-glucanase of the present invention and its homologous protein was 80.53% (the homologous protein was derived from the endoglucanase of Pleurotus ostreatus, which randomly cut the β-1,4 glycosidic bonds inside the cellulose), which is a new type of β-1,4-glucanase.

[0033] Example 2: Construction of recombinant plasmid

[0034] After introducing two restriction sites, SnaB I and EcoR I, at the 5' and 3' ends of the β-1,4-glucanase gene sequence, primers were designed, the target gene was cloned from the Pleurotus eryngii cDNA and the PCR product was purified, the pPIC9K vector was digested with restriction endonucleases SnaB I and EcoR I and gel recovered, and the purified target gene fragment and vector fragment were recombined and connected through homology arms using a one-step cloning kit; the connection product was mixed with the recipient bacteria E. coli JM109 competent cells and allowed to stand on ice for 30 minutes; after heat shock in a 42°C metal bath for 90 seconds, it was immediately placed on ice for cooling for 5 minutes, 1 mL of LB liquid culture medium without antibiotics was added, and after incubation at 37°C on a shaker for 1 hour, it was spread on an LB plate containing ampicillin resistance and incubated at 37°C for 12-16 hours, and several single clones were selected for colony PCR identification and sequencing verification to obtain the correct recombinant plasmid containing the target gene.

[0035] Example 3: Construction and screening of recombinant yeast strains

[0036] The restriction endonuclease Sac I was used to linearize the recombinant plasmid, and the digestion product was purified and mixed with the recipient bacteria P. pastoris GS115 competent cells and allowed to stand on ice for 10 minutes. After electric shock at 2.5 kV, 1 mL of pre-cooled 1 M sorbitol was immediately added, and after incubation at 30°C on a shaker for 2 hours, the plate was spread on an MD plate and incubated at 30°C for 2-3 days.

[0037] Several single colonies on the MD plate were selected for high-copy strain screening on YPD plates containing different concentrations (2, 4, 6 mg / mL) of Geneticin G418 resistance, and inverted culture was carried out in a 30°C incubator for 1-2 days. Several single colonies on the YPD plate with high concentration resistance (6 mg / mL G418) were selected for colony PCR verification, and the positive colonies verified by sequencing were the recombinant yeast strains containing the β-1,4-glucanase gene.

[0038] Example 4: Enzyme induced expression and purification

[0039] The recombinant yeast strain was streaked on a YPD plate (containing 6 mg / mL G418) for activation, and a single clone was picked and inoculated into a BMGY medium, and cultured at 30°C and 200 rpm for 36-48 hours; the bacteria were collected and transferred to a BMMY medium containing methanol at a final concentration of 1.5% for inducing expression, and cultured at 30°C and 200 rpm for 72 hours to obtain a fermentation broth, and methanol at a final concentration of 1.5% was added every 24 hours; the fermentation broth was centrifuged at 4°C and 12000 rpm for 5 minutes, and the supernatant was collected and filtered with a 0.22 μm sterile filter to obtain a crude enzyme solution.

[0040] Based on the sequence encoding the His-Tag protein in the pPIC9K plasmid, nickel ion metal chelate affinity chromatography (Ni-NTA) was used to purify the protein of β-1,4-glucanase PTRGLT4. The specific purification steps are as follows:

[0041] The crude enzyme solution was loaded onto a pre-equilibrated Ni-NTA affinity column for purification at a flow rate of 0.5 mL / min. The impurities were washed away with 5 column volumes of 20 mM buffer A (20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, pH 5.0), and then the target protein was eluted with 500 mM imidazole at a flow rate of 1 mL / min. After ultrafiltration to remove high concentrations of imidazole and NaCl, the pure enzyme solution of β-1,4-glucanase PTRGLT4 was obtained. The purified protein was analyzed by SDS-PAGE ( Figure 1 ), which is consistent with the predicted molecular weight of 41.45 kDa.

[0042] Example 5: Enzymatic properties of β-1,4-glucanase PTRGLT4

[0043] (1) β-1,4-glucanase activity assay

[0044] The reducing sugar content was determined by the DNS method. 2.5 μL of enzyme solution and 200 μL of sodium carboxymethyl cellulose (CMC, 2 mg / mL) were mixed and reacted at 37°C for 10 min. An equal volume of DNS solution was added to the reaction system and heated at 100°C for 10 min. After cooling to room temperature, the supernatant was measured with an ELISA reader at a wavelength of 540 nm to calculate the enzyme activity (the blank control was the inactivated enzyme solution). Enzyme activity unit definition: Under the above determination conditions, the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of reducing sugar (measured in glucose) per minute is defined as one enzyme activity unit (U).

[0045] (2) Substrate specificity of β-1,4-glucanase PTRGLT4

[0046] Amylopectin, curdlan, laminarin, CMC, barley β-glucan, oat β-glucan, and yeast β-glucan were prepared at a concentration of 2 mg / mL, and 200 μL of each was taken as a substrate. 2.5 μL of enzyme solution was added, and the amount of reducing sugar generated was measured after reaction at 37°C for 10 minutes (the blank control was the inactivated enzyme solution). The relative enzyme activity of each substrate was calculated with the substrate with the highest enzyme activity as 100%. The results are shown in Table 1. PTRGLT4 acts on substrates containing β-1,4 glycosidic bonds, indicating that PTRGLT4 is a β-1,4-glucanase. At the same time, PTRGLT4 has a broad substrate specificity and has significant hydrolysis activity for barley / oat β-glucan substrates containing mixed β-1,3 and β-1,4 glycosidic bonds, which is much higher than the substrate sodium carboxymethyl cellulose (CMC) containing only β-1,4 glycosidic bonds.

[0047] Table 1 Substrate specificity results of β-1,4-glucanase PTRGLT4

[0048]

[0049] 3. Enzyme action pH range and pH stability

[0050] The enzyme activity was determined using oat β-glucan (2 mg / mL) prepared in different buffers (glycine-HCl buffer, pH 1.0-4.0; citric acid-NaH2PO4 buffer, pH 4.0-8.0; glycine-NaOH buffer, pH 8.0-12.0) as a substrate. The relative activity of the enzyme at different pH values ​​was calculated with the maximum enzyme activity as 100%. Figure 2 As shown in A, β-1,4-glucanase has high activity in citric acid-NaH2PO4 buffer, the pH working range is 4.0-7.0, and the optimal pH is 5.0.

[0051] The enzyme solution was diluted into different pH buffers and incubated for 1 h. The enzyme activity was determined according to the above method. The enzyme activity without incubation was taken as 100% and the residual enzyme activity was calculated. Figure 2 As shown in B, the enzyme has good stability in a buffer solution with a pH value of 5.0, and the enzyme activity can still be maintained at about 95% after incubation for 1 hour; in a pH value of 4.0-7.0, the enzyme activity is maintained above 70%; during the dilution of the enzyme solution, when the pH of the buffer solution exceeds 9, obvious precipitation will appear, indicating that the alkaline environment will destroy the enzyme protein.

[0052] 4. Enzyme action temperature and thermal stability

[0053] The reaction was carried out at the optimal pH value at different temperatures (20-80°C). The highest enzyme activity was set as 100%, and the relative enzyme activity at each temperature was calculated to determine the optimal reaction temperature. Figure 3 As shown in A, with the increase of temperature, the activity of β-1,4-glucanase gradually increases, and 50°C is the optimal reaction temperature. Continuing to increase the temperature will destroy the enzyme activity.

[0054] To evaluate thermal stability, equal amounts of enzyme solutions were pretreated at different temperatures for 1 h. The subsequent enzyme activity was quantified, and the activity of the enzyme without temperature pretreatment was assigned as 100% to determine the residual enzyme activity after temperature treatment. Figure 3 As shown in B, β-1,4-glucanase can maintain a high enzyme activity (more than 88%) below 40°C, while 60°C will seriously destroy the enzyme activity. Therefore, the enzyme should be stored at low temperature.

[0055] 5. Enzyme catalytic mechanism

[0056] The hydrolysis products were analyzed by MALDI-TOF-MS, which further proved that PTRGTL4 not only has a hydrolysis effect, but also a transglycosidic effect. PTRGTL4 can not only catalyze the hydrolysis of β-glucan to generate oligosaccharides, but also play a transglycosidic role in the generated oligosaccharides, generating new glycosidic bonds between oligosaccharides. After characterization of the enzymatic hydrolysis products, there are mainly three types of ions: a, b, and c ( Figure 4 A): type a is [M+Na] + Molecular ion, with a mass difference of 162 Da, indicating a hexose (Hex) unit; b-type is [M+K] + The hexose chain molecular ion; type c is [M+Na] on the hexose chain + The ions (Hex) produced by losing a molecule of water n -18). After 5 min of PTRGLT4 action, the products included oligosaccharides with various degrees of polymerization (DP) with relatively uniform distribution, indicating that the enzyme has endo-hydrolytic activity.

[0057] As the hydrolysis time increased, the proportions of oligosaccharides of DP4, 7, 10, 13, 16 and 19 increased significantly. The secondary structure of DP4-DP7 in the a-type ions was characterized ( Figure 4 B), it was found that the glycosidic bond cleavage produced C-type (non-reducing end part) and Z-type (reducing end part) ions. In addition, it was observed that O,2 A n (n = 3, 4, 5, 6, 7) breaks, which is consistent with the intra-ring cleavage rule of 1→4 connection. For tetrasaccharides, specific 2,4 A4 intra-ring cleavage (m / z 569.282) corresponds to 1→4 or 1→3 linkage; 3,5 The A3 ring fragmentation (m / z 421.316) corresponds to 1→4 or 1→6 linkage. These features are consistent with the repeating unit structure of oat glucan and indicate the existence of a new sugar linkage mode. 2,4 A5 (m / z 731.416) and 2,4 A6 (m / z 893.555) was cleaved, and three types of intra-ring cleavage were again identified in the heptasaccharide ( O,2 A n , 2,4 A n , 3,5 A n ), indicating the ability of the enzyme to form new glycosidic bonds in oligosaccharides with specific DPs.

[0058] Structural prediction analysis shows that ( Figure 5 ), PTRGLT4 is similar to the typical GH5 family protein, with a core of (β / α)8TIM-barrel domain, 8 parallel β-folds forming an inner barrel, 8 α-helices surrounding the inner barrel, and the other 2 helices at the top of the barrel. Among them, E235 and E347 are active sites, and the presence of the groove is conducive to the entry and binding of the substrate. In addition, the enzyme has a fungal-type cellulose-binding domain (fCBD), which plays an important role in recognizing and binding specific polysaccharides. Compared with CMC, the molecular conformation of barley / oat β-glucan is more complex, and it is easier to match and "get stuck" with the groove structure of PTRGLT4, so the enzyme has a higher affinity for barley / oat β-glucan.

[0059] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A β-1,4-glucanase, characterized in that The amino acid sequence of the β-1,4-glucanase is shown in SEQ ID NO.

2.

2. A gene encoding the β-1,4-glucanase according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2. A recombinant cell expressing the β-1,4-glucanase according to claim 1.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell is a bacterium or a fungus.

6. Use of the β-1,4-glucanase according to claim 1, the gene according to claim 2, the recombinant plasmid according to claim 3 or the recombinant cell according to claim 4 or 5 in degrading β-glucan, characterized in that: The β-glucan contains β-1,4 glycosidic bonds or β-1,3-1,4 glycosidic bonds.

7. The use according to claim 6, characterized in that: The β-1,4-glucanase or the expression system containing the β-1,4-glucanase is added to a system containing β-glucan to carry out a reaction.

8. A method for degrading β-glucan, characterized in that: The method comprises adding the β-1,4-glucanase according to claim 1 to a substrate system containing β-glucan for reaction, wherein the β-glucan contains β-1,4 glycosidic bonds or β-1,3-1,4 glycosidic bonds.

9. The method according to claim 8, characterized in that The pH of the reaction is 4.0-7.

0.

10. The method according to claim 8, characterized in that The reaction temperature is 20°C-55°C.

Images