A beta-1,4-glucanase and its application

Abstract

The present invention relates to a kind of β-1,4-glucanase and application thereof, belong to the field of biotechnology. The present invention screens and identifies a kind of novel β-1,4-glucanase in tiger milk mushroom, the β-1,4-glucanase of the present invention has wide substrate specificity, can specifically hydrolyze the β-glucan (such as sodium carboxymethyl cellulose) containing β-1,4 glycosidic bonds, can hydrolyze the β-glucan (such as barley β-glucan and oat β-glucan) containing β-1,3-1,4 glycosidic bonds again, therefore the β-1,4-glucanase of the present invention has good enzyme activity to the β-glucan containing β-1,4 glycosidic bonds and the β-glucan containing β-1,3-1,4 glycosidic bonds. In addition, the β-1,4-glucanase of the present invention is under the conditions of pH 4.0-7.0 and its enzyme activity can keep more than 70%, and at 40 DEG C, its enzyme activity can keep more than 88%, with good stability.

Description

Technical Field

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

[0002] The diverse biological activities of β-glucan, including immunomodulatory, anti-tumor, anti-inflammatory, and antioxidant activities, are closely linked to its specific structural features, including the type of glycosidic bonds, degree of polymerization, and three-dimensional molecular conformation. Naturally extracted β-glucans suffer from poor water solubility or excessive viscosity after dissolution, which hinders 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-glucans. Compared to physical and chemical methods, enzymatic treatment is a highly specific and environmentally friendly method. β-glucanase, based on its specificity for glycosidic bonds, can modify the structure of β-glucans or produce specific oligosaccharides or oligosaccharides, thereby improving the properties of β-glucans.

[0003] β-glucanases can be classified according to the type of glycosidic bond they catalyze, primarily 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 cleave β-1,4-glycosidic bonds from within the β-glucan chain, producing a series of oligosaccharides with varying degrees of polymerization; whereas exo-β-1,4-glucanases cleave from the non-reducing ends of cellulose, producing cellobiose residues or glucose. β-1,3-1,4-glucanases (also known as lichenases) are a specialized class of enzymes that specifically recognize and cleave β-1,4-glycosidic bonds adjacent to β-1,3-glycosidic bonds, generating oligosaccharides or glucose consisting of 3-5 glucose units. This type of enzyme is highly effective in degrading glucans containing a mixed structure of β-1,3 and β-1,4 glycosidic bonds (such as cereal β-glucans), and is of particular 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 relatively single origin, mainly bacterial, and mainly belong to the GH16 and GH17 families of glycoside hydrolases. To meet industrial needs, researchers often use methods such as chemical modification and rational design to improve enzyme activity and stability. However, these enzymes still have limitations in terms of substrate specificity and functional diversity.

[0004] While some existing endo-β-1,4-glucanases exhibit broad substrate specificity, a multifunctional β-glucanase capable of efficiently degrading a variety of β-glucans (such as β-1,3-1,4 mixed glycosidic bonds and β-1,4 glycosidic bonds) is still lacking. Furthermore, edible fungi, as a safe and edible natural resource, possess promising enzyme applications, but the exploration and systematic characterization of these enzyme resources remain insufficient. Therefore, there is an urgent need to identify a β-glucanase from edible fungi with a broad substrate range and diverse functionality. 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] 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 Pleurotus ostreatus, 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) as well as β-glucans containing β-1,3-1,4 glycosidic bonds (such as barley β-glucan and oat β-glucan). Therefore, 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 pH conditions of 4.0-7.0, and can maintain an enzyme activity of more than 88% at temperatures below 40°C, showing 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 which 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 a use 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 to carry out a reaction.

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

[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 from tiger milk mushrooms. The β-1,4-glucanase of the present invention has broad 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 enzymatic activity against both β-glucans containing β-1,4 glycosidic bonds and β-glucans containing β-1,3-1,4 glycosidic bonds. In addition, the β-1,4-glucanase of the present invention can maintain an enzyme activity of more than 70% at a pH of 4.0-7.0 and an enzyme activity of more than 88% at 40°C, showing good stability. The present invention provides a β-1,4-glucanase derived from edible fungi. This enzyme addresses the existing issues of limited enzyme resources and functional limitations. It exhibits broad substrate specificity and efficient hydrolysis, addressing deficiencies in the properties and application research of fungal β-glucanases and providing a new solution for the specific hydrolysis of β-glucans. The enzyme can specifically degrade β-1,3-1,4-glucans and β-1,4-glucans to produce oligosaccharides with a specific degree of polymerization. This enzyme has potential application as a prebiotic or functional food ingredient, providing a new solution for the efficient hydrolysis of β-glucans 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 based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0024] Figure 1 The figure shows the SDS-PAGE electrophoresis results of the expression product and the purified enzyme protein, where M is the standard protein marker; 1 is the crude enzyme solution; 2 is the 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; where: 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 a MALDI-TOF-MS analysis of oat β-glucan hydrolysates; A: MS spectra after 5 and 30 minutes of enzyme treatment; B: secondary structure information of DP4 to DP7;

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

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, 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 ThermoFisher 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, and 13.4 g amino-free yeast nitrogen base, and 4.0 × 10 -4 g, 1 M pH 5.0 phosphate buffer 10%, glycerol 10 g); BMMY medium (containing yeast extract 10 g, peptone 20 g, amino-free yeast nitrogen source 13.4 g, biotin 4.0 × 10 -4 g, 1 M pH 5 phosphate buffer 10%, methanol 1.5%; MD plate medium (containing 13.4 g of amino-free yeast nitrogen source per liter, biotin 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), tools such as dbCAN were used to screen for carbohydrate enzyme-related genes. Gene structure and conserved motif analysis revealed a β-1,4-glucanase gene. Analysis of the protein domains, transmembrane region, and signal peptide revealed that the gene sequence contains a signal peptide sequence, a fungal cellulose-binding domain, and a GH5 family cellulase domain, with the protein localized 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. Analysis using the NCBI online BLAST tool revealed that the protein sequence of the present β-1,4-glucanase shared a maximum homology of 80.53% with its homologous protein (the homologous protein is derived from the endoglucanase of Pleurotus ostreatus, which randomly cleaves β-1,4 glycosidic bonds within cellulose), indicating that it is a novel 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, respectively, primers were designed. The target gene was cloned from Pleurotus ostreatus cDNA and the PCR product was purified. The pPIC9K vector was digested with restriction endonucleases SnaB I and EcoR I and recovered on gel. The purified target gene fragment and vector fragment were recombinantly ligated via homology arms using a one-step cloning kit. The ligation product was mixed with competent cells of the recipient bacterium E. coli JM109 and allowed to stand on ice for 30 minutes. After heat shock in a 42°C metal bath for 90 seconds, the cells were immediately cooled on ice for 5 minutes, 1 mL of antibiotic-free LB liquid medium was added, and the cells were incubated on a shaker at 37°C for 1 hour. The cells were then plated on LB plates containing ampicillin resistance and incubated at 37°C for 12-16 hours. 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 recombinant plasmid was linearized by restriction endonuclease Sac I. The digested product was purified and mixed with competent cells of the recipient bacterium P. pastoris GS115 and allowed to stand on ice for 10 minutes. After electroporation at 2.5 kV, 1 mL of pre-cooled 1 M sorbitol was immediately added. After incubation at 30°C on a shaker for 2 hours, the product was spread on MD plates and incubated at 30°C for 2-3 days.

[0037] Several single colonies from the MD plates were plated on YPD plates containing different concentrations (2, 4, and 6 mg / mL) of Geneticin G418 resistance for high-copy strain screening. The plates were incubated upside down at 30°C for 1-2 days. Several single colonies from the YPD plates containing high-resistance concentration (6 mg / mL G418) were selected for colony PCR verification. Positive colonies confirmed by sequencing were the recombinant yeast strains containing the β-1,4-glucanase gene.

[0038] Example 4: Enzyme expression and purification

[0039] The recombinant yeast strain was streaked onto a YPD plate (containing 6 mg / mL G418) for activation, and a single colony was picked and inoculated into BMGY medium and cultured with shaking at 30°C and 200 rpm for 36-48 hours. The cells were collected and transferred to BMMY medium containing methanol at a final concentration of 1.5% to induce expression. The culture was shaken at 30°C and 200 rpm for 72 hours to obtain a fermentation broth, and methanol was supplemented at a final concentration of 1.5% every 24 hours. The fermentation broth was centrifuged at 4°C and 12,000 rpm for 5 minutes, and the supernatant was collected and filtered through a 0.22 μm sterile filter to obtain a crude enzyme solution.

[0040] Based on the His-Tag protein sequence encoded in the pPIC9K plasmid, nickel ion metal chelate affinity chromatography (Ni-NTA) was used to purify the protein, which is the 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. 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 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, a pure enzyme solution of β-1,4-glucanase PTRGLT4 was obtained. The purified protein was subjected to SDS-PAGE analysis ( 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] Reducing sugar content was determined using the DNS method. 2.5 μL of enzyme solution was mixed with 200 μL of sodium carboxymethyl cellulose (CMC, 2 mg / mL) and incubated at 37°C for 10 minutes. An equal volume of DNS solution was added to the reaction system and heated at 100°C for 10 minutes. After cooling to room temperature, the supernatant was measured using a microplate reader to measure absorbance at 540 nm and calculate enzyme activity (the blank control was inactivated enzyme solution). One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of reducing sugar (measured as glucose) per minute under the above assay conditions.

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

[0046] A 200 μL aliquot of each of pullulan, curdlan, laminarin, CMC, barley β-glucan, oat β-glucan, and yeast β-glucan was prepared at a concentration of 2 mg / mL. 2.5 μL of enzyme solution was added and the reaction was carried out at 37°C for 10 minutes. The amount of reducing sugar produced was measured (the blank control was inactivated enzyme solution). The relative enzyme activity of each substrate was calculated, with the substrate with the highest activity being considered 100%. As shown in Table 1, PTRGLT4 acts on substrates containing β-1,4 glycosidic bonds, indicating that PTRGLT4 is a β-1,4-glucanase. PTRGLT4 also exhibits broad substrate specificity, showing significant hydrolysis activity against barley / oat β-glucans containing mixed β-1,3 and β-1,4 glycosidic bonds, and significantly higher than that of sodium carboxymethylcellulose (CMC), a substrate 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, with a pH working range of 4.0-7.0 and an optimum pH of 5.0.

[0051] The enzyme solution was diluted into different pH buffers and incubated for 1 hour. 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 Figure B, the enzyme is relatively stable 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 at more than 70%; during the dilution of the enzyme solution, when the buffer pH 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, as the temperature rises, 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 hour. The subsequent enzyme activity was quantified, and the activity of the enzyme without temperature pretreatment was assigned to 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 the enzyme activity will be seriously damaged at 60°C. Therefore, the enzyme should be stored at low temperature.

[0055] 5. Enzyme catalytic mechanism

[0056] MALDI-TOF-MS analysis of the hydrolysis products further demonstrated that PTRGTL4 not only has a hydrolysis function but also a transglycosidic function. 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 the oligosaccharides. After characterization of the enzymatic hydrolysis products, the main ions were a, b, and c ( Figure 4 A): type a is [M+Na] + Molecular ion, with a mass difference of 162 Da, indicates a hexose (Hex) unit; b-type is [M+K] + The hexose chain molecular ion; type C is [M+Na] on the hexose chain + Ions (Hex) produced by losing a water molecule n After 5 min of PTRGLT4 treatment, the product included oligosaccharides with various degrees of polymerization (DP) with relatively uniform distribution, indicating that the enzyme has endo-hydrolytic activity.

[0057] With the extension of hydrolysis time, the proportion of oligosaccharides of DP4, 7, 10, 13, 16 and 19 increased significantly. The secondary structure of DP4-DP7 in 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 1→4 linking intra-ring cleavage rule. For tetrasaccharides, specific 2,4 A4 ring cleavage (m / z 569.282), corresponding to 1→4 or 1→3 linkage; also identified 3,5 The fragmentation within the A3 ring (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 fragmented, and three types of intra-ring fragmentation 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. Eight parallel β-sheets form an inner barrel, eight α-helices surround the inner barrel, and the other two helices are at the top of the barrel. Among them, E235 and E347 are active sites, and the presence of the groove facilitates 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 to specific polysaccharides. Compared with CMC, the molecular conformation of barley / oat β-glucan is more complex, which is easier to match and "get stuck" in the groove structure of PTRGLT4. Therefore, the enzyme has a higher affinity for barley / oat β-glucan.

[0059] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present 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 a β-1,4 glycosidic bond or a β-1,3-1,4 glycosidic bond.

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.

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