A dextranase mutant with improved thermal stability and high catalytic activity and its application

Abstract

The present invention discloses a dextranase mutant with improved thermal stability and high catalytic activity and its application, belonging to the technical field of enzyme engineering. The amino acid sequence of the dextranase mutant M3 with improved thermal stability disclosed by the present invention is shown in SEQ ID NO.10. The high-catalytic-activity dextranase mutant disclosed by the present invention is obtained by point mutation of the amino acids of the wild-type dextranase. This mutant can withstand high-temperature treatment above 50 °C and has very high enzyme activity under acidic and neutral pH values. In particular, it has good application potential in industrial fields such as feed, beer brewing, and food.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a high-catalytic-activity glucanase mutant with improved thermal stability and application thereof. Background Art

[0002] Dextranase is an important enzyme, typically derived from certain fungi or bacteria. It is produced through submerged fermentation and is specifically targeted at the 1,3- and 1,4-glycosidic bonds of β-glucans. β-glucanases, enzymes that break down glucose polymers linked by β-glycosidic bonds, are classified as endo- and exo-types based on their mechanisms of action. A specific enzyme, endo-β-1,3-1,4-glucanase (EC3.2.1.73), possesses unique decomposition capabilities. It precisely targets β-1,4-glycosidic bonds directly attached to β-1,3 bonds, breaking them down into smaller molecular weight fragments. This decomposition process not only reduces the hydrophilicity and viscosity of the substance but also significantly reduces the viscosity of the intestinal contents of monogastric animals. Furthermore, it has other positive effects, such as increasing the activity of endogenous digestive enzymes in animals and optimizing the intestinal microbial environment, thereby improving animal growth performance and feed conversion efficiency. In short, this special β-glucanase improves the intestinal environment of animals through specific enzymatic hydrolysis, promoting growth and improving feed utilization.

[0003] β-glucanase is widely used in the beer, bread, and biomass degradation industries. It can gently and efficiently deconstruct β-glucan specifically, reducing its negative effects such as increased difficulty in the production process due to water retention, production impact, and increased costs. In the biomass degradation process, β-glucanase needs to maintain high catalytic activity under high temperature conditions; in the beer process, β-glucanase must remain stable within a wide temperature range of 40 to 70°C, and during bread fermentation, it must maintain high activity at a specific temperature of 28 to 37°C for at least two hours. However, most of the β-glucanases reported so far lack thermal stability and are difficult to meet industrial needs.

[0004] Improving the thermal stability and catalytic activity of glucanases is crucial for industrial applications, broadening their scope of application and deepening our understanding of the relationship between enzyme structure and stability. However, endogenous β-glucanases in cereals are susceptible to inactivation during high-temperature processes such as malting, saccharification, and feed pelleting. To meet the demands of industrial production, protein engineering is needed to modify the enzyme to improve its stability and promote its industrial application. Summary of the Invention

[0005] The present invention aims to provide a high-catalytic activity glucanase mutant with improved thermal stability and its application to address the problems of the prior art. The high-catalytic activity glucanase mutant of the present invention is obtained by subjecting the wild-type glucanase to amino acid point mutations. The mutant can withstand high-temperature treatment above 50°C and has very high enzyme activity at both acidic and neutral pH levels. It has great application potential in industrial fields such as feed, beer brewing, and food.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a high-catalytic activity glucanase mutant M3 with improved thermal stability. The amino acid sequence of the high-catalytic activity glucanase mutant M3 is shown in SEQ ID NO.10.

[0008] The present invention also provides a gene encoding the above-mentioned high catalytic activity glucanase mutant M3, and the nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0009] The present invention also provides a recombinant vector comprising the above gene.

[0010] The present invention also provides a recombinant microorganism, which contains the recombinant vector.

[0011] The present invention also provides a method for constructing the above-mentioned high catalytic activity glucanase mutant M3, comprising the steps of simultaneously performing site-directed mutagenesis on the amino acids at positions 11, 209 and 228 based on the wild-type glucanase as shown in SEQ ID NO.2.

[0012] The present invention also provides the use of the above gene, the above recombinant vector or the above recombinant microorganism in producing a high-catalytic activity glucanase with improved thermal stability.

[0013] The present invention also provides the use of the high catalytic activity glucanase mutant M3 or the high catalytic activity glucanase mutant M3 constructed by the above construction method in degrading glucan, wherein the degradation temperature is 50-80° C. and the pH is 2.5-6.5.

[0014] The present invention discloses the following technical effects:

[0015] The present invention utilizes enzyme engineering methods to improve the enzyme molecule of wild-type glucanase to address the shortcoming that the glucanase is easily inactivated during high temperature processes. After modification and optimization, the obtained mutant M3 has significantly improved thermal stability at high temperatures and significantly enhanced catalytic activity, which can meet the requirements of feed enzymes. Therefore, the high-catalytic activity glucanase mutant M3 with improved thermal stability in the present invention shows great application potential in the feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The results of the optimal temperature determination of the glucanase mutant M3 and wild-type PaGlu16A;

[0018] Figure 2 is the half-life of the glucanase mutant M3 and wild-type PaGlu16A at 50°C (t 1 / 2 ) measurement results;

[0019] Figure 3 is the temperature stability of the glucanase mutant M3 and wild-type PaGlu16A (T 50 ) measurement results;

[0020] Figure 4 The results of the optimal pH determination of the glucanase mutant M3 and wild-type PaGlu16A;

[0021] Figure 5 The results of pH stability test of glucanase mutant M3 and wild-type PaGlu16A. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The materials that may be used in the embodiments of the present invention are as follows:

[0028] 1. Vectors and strains: The expression vector pPIC9r plasmid and Pichia pastoris GS115 were purchased from Invitrogen, and Escherichia coli DMT competent cells were purchased from Quanshijin.

[0029] 2. Enzymes, kits, and other biochemical reagents: Taq enzyme was purchased from TaKaRa, endonucleases and point mutagenesis kits were purchased from Quanshijin, and the substrate lichen polysaccharide was purchased from Magzyme. All other domestic analytical grade reagents were purchased from Sinopharm Reagent Company.

[0030] 3. Culture medium:

[0031] Escherichia coli LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 1% agar powder (solid);

[0032] Yeast YPD medium: 2% glucose, 2% peptone, 1% yeast extract;

[0033] Yeast MD medium: 1.5% agarose, 2% glucose, biotin 4 × 10 -4 g / L, YNB 13.4 g / L;

[0034] Yeast BMGY medium: 2% peptone, 1% yeast extract, 1% glycerol (V / V), biotin 4×10 -4 g / L, YNB 13.4 g / L;

[0035] Yeast BMMY induction medium: 2% peptone, 1% yeast extract, YNB 13.4 g / L, 0.5% methanol (V / V), biotin 4×10 -4 g / L.

[0036] Example 1 Obtaining a gene encoding a high catalytic activity glucanase mutant and its recombinant expression vector

[0037] Using the GH16 family glucanase gene PaGlu16A from Polychaeton citri CBS116435 (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) as a template, site-directed mutagenesis was performed at the N11Y, S209Q, and G228P sites. The mutation and cloning methods were based on the reference (Improvement of XYL10C_N catalytic performance through loop engineering for lignocellulosic biomass utilization in feed and fuel industries; You, et al., 2021), and the gene encoding the high-catalytic activity glucanase mutant (nucleotide sequence shown in SEQ ID NO.3) and its recombinant expression vector pPIC9r-M3 were obtained.

[0038] The primer information used is shown in Table 1:

[0039] Table 1 Primer information

[0040]

[0041] SEQ ID NO.1:

[0042] CAATACACCTTGGAAGATGACTACTTGGCTAATGGTTTCTTTGATCAATTTTCTTTCTTCACCTCTGGTGACCCAACTCATGGTTTCGTTCAATACGTTGATCAAGGTACCGCTAATTCTAACGGTTTGATTTCTTCTTCCGGTTCTCAAGCTGTTATGAGAGTTGATTCTACTAACACTGCTCCAAACGGTAGAACTTCTGTTAGATTGACTTCTAATAAGGCTTACGATTCTGGTTTGGTTATTGCTGATATTGCTCATATGCCTGGTGGTATTTGTGGTGTTTGGCCAGCTTTTTGGATGGTTGGTCCTAATTGGCCATCCAACGGAGAAATCGATATTATTGAAGGTGTTAATGACCAGTCTACTAACGATATGACTTTGCATACTTCTGATGGTTGTTCTATTGGTTCTGGTGGTATGTCTGGTTACGTTGTTACTTCTAATTGTTACATTAACGCTCCAGGTCAATCATCTAACCAAGGTTGTCAAATTGGAACCGGTGATACTTCTACTTACGGTTCTGGTTTTAATGCTAACGGTGGTGGTGTTTACGCTACTGAATTTACTTCTTCTGGTGTTAAGATTTTTTTCTTTCCAAGAGGTTCTATTCCTGGAGATATTTCTTCTGGTTCTCCTAATCCATCTTCTTGGGGTCAGCCAGTTGCTTACTTTCAAGGTGGTGGTTGTGATTTTGGTTCTCATATTAAGCAGCAACAAATTGTTTTCGATACTACTTTTTGTGGTGATTGGGCTGGTGCTGTTTGGGGTAATGGTGGTTGTGCTTCTAGAGCTGGTTCTTGTAACGATTTTGTTGCTAATAACCCTTCTGCTTTTTCTGATGCTTATTGGGCTGTTAATGGTTTGAAGGTTTATCAAAATTACGGTTCTACTTCTTTCGATTTGGAATCTCCACCATCTAACTCTTCTGCTTCTTCTTCTTCTGCTTCCGTTTTGAAGGAATCCAAGAAAGAAAGATTTAGAAGACATTTGGCTGAACATAGAAACTCTGGTGCTGAATTGTTT;

[0043] SEQ ID NO.2:

[0044] QYTLEDDYLANGFFDQFSFFTSGDPTHGFVQYVDQGTANSNGLISSSGSQAVMRVDSTNTAPNGRTSVRLTSNKAYDSGLVIADIAHMPGGICGVWPAFWMVGPNWPSNGEIDIIEGVNDQSTNDMTLHTSDGCSIGSGGMSGYVVTSNCYINAPGQSSNQGCQIGTGDTSTYGSGFNANGGGVYATEFTSSGVKIFFFPRGSIPGDISSGSPNPSSWGQPVAYFQGGGCDFGSHIKQQQIVFDTTFCGDWAGAVWGNGGCASRAGSCNDFVANNPSAFSDAYWAVNGLKVYQNYGSTSFDLESPPSNSSASSSSASVLKESKKERFRRHLAEHRNSGAELF;

[0045] SEQ ID NO.3:

[0046] CAATACACCTTGGAAGATGACTACTTGGCTTACGGTTTCTTTGATCAATTTTCTTTCTTCACCTCTGGTGACCCAACTCATGGTTTCGTTCAATACGTTGATCAAGGTACCGCTAATTCTAACGGTTTGATTTCTTCTTCCGGTTCTCAAGCTGTTATGAGAGTTGATTCTACTAACACTGCTCCAAACGGTAGAACTTCTGTTAGATTGACTTCTAATAAGGCTTACGATTCTGGTTTGGTTATTGCTGATATTGCTCATATGCCTGGTGGTATTTGTGGTGTTTGGCCAGCTTTTTGGATGGTTGGTCCTAATTGGCCATCCAACGGAGAAATCGATATTATTGAAGGTGTTAATGACCAGTCTACTAACGATATGACTTTGCATACTTCTGATGGTTGTTCTATTGGTTCTGGTGGTATGTCTGGTTACGTTGTTACTTCTAATTGTTACATTAACGCTCCAGGTCAATCATCTAACCAAGGTTGTCAAATTGGAACCGGTGATACTTCTACTTACGGTTCTGGTTTTAATGCTAACGGTGGTGGTGTTTACGCTACTGAATTTACTTCTTCTGGTGTTAAGATTTTTTTCTTTCCAAGAGGTTCTATTCCTGGAGATATTCAGTCTGGTTCTCCTTATCCATCTTCTTGGGGTCAGCCAGTTGCTTACTTTCAAGGTCCAGGTTGTGATTTTGGTTCTCATATTAAGCAGCAACAAATTGTTTTCGATACTACTTTTTGTGGTGATTGGGCTGGTGCTGTTTGGGGTAATGGTGGTTGTGCTTCTAGAGCTGGTTCTTGTAACGATTTTGTTGCTAATAACCCTTCTGCTTTTTCTGATGCTTATTGGGCTGTTAATGGTTTGAAGGTTTATCAAAATTACGGTTCTACTTCTTTCGATTTGGAATCTCCACCATCTAACTCTTCTGCTTCTTCTTCTTCTGCTTCCGTTTTGAAGGAATCCAAGAAAGAAAGATTTAGAAGACATTTGGCTGAACATAGAAACTCTGGTGCTGAATTGTTT.

[0047] Example 2 Preparation of high catalytic activity glucanase mutants

[0048] The recombinant expression vector pPIC9r-M3 obtained in Example 1 was linearized and digested with DMT enzyme, and then transformed into Escherichia coli DMT competent cells. Colony PCR was performed to verify the recombinant expression vector with target site mutation. The vector was linearized with endonuclease BglⅡ and then electroporated into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / M3.

[0049] The yeast strain GS115 / M3 containing the recombinant plasmid was inoculated into 2 mL of BMGY medium in a 10 mL test tube and cultured at 30°C, 220 rpm, and shaker for 48 hours. The culture was then centrifuged at 4500 rpm for 5 minutes, the supernatant discarded, and the pellet resuspended in 2 mL of BMMY medium containing 0.5% methanol. The culture was again induced at 30°C, 220 rpm for 48 hours. The supernatant was collected for enzyme activity assay, and the yeast clone with the highest enzyme activity was selected.

[0050] Wild-type and selected mutant yeast strains were scaled up to induce enzyme production. First, 30 mL of YPD medium was inoculated into a shaker at 30°C, 220 rpm, and then a seed culture was obtained. A 1% inoculum was then inoculated into a 1-L Erlenmeyer flask containing 300 mL of BMGY medium and incubated at 30°C, 220 rpm, and then shaken for 48 hours. The culture was then centrifuged for 5 minutes, the supernatant discarded, and the pellet resuspended in 200 mL of BMMY medium containing 0.5% methanol. The culture was then incubated again at 30°C, 220 rpm, for another 48 hours. 1 mL of methanol was added every 12 hours, and the supernatant was collected for enzyme activity assays. Finally, the supernatant was concentrated to 30 mL, desalted with sodium hydrogen phosphate-citrate buffer (pH 6.0, 10 mM), and purified using anion exchange for enzymatic property analysis and comparison. The expressed enzyme, designated M3, had a protein content exceeding 98% of the total protein after purification. The amino acid sequence of this glucanase is shown in SEQ ID NO. 10.

[0051] SEQ ID NO.10:

[0052] QYTLEDDYLAYGFFDQFSFFTSGDPTHGFVQYVDQGTANSNGLISSSGSQAVMRVDSTNTAPNGRTSVRLTSNKAYDSGLVIADIAHMPGGICGVWPAFWMVGPNWPSNGEIDIIEGVNDQSTNDMTLHTSDGCSIGSGGMSGYVVTSNCYINAPGQSSNQGCQIGTGDTS TYGSGFNANGGGVYATEFTSSGVKIFFFPRGSIPGDIQSGSPNPSSWGQPVAYFQGPGCDFGSHIKQQQIVFDTTFCGDWAGAVWGNGGCASRAGSCNDFVANNPSAFSDAYWAVNGLKVYQNYGSTSFDLESPPSNSSASSSSASVLKESKKERFRRHLAEHRNSGAELF.

[0053] Example 3 Comparative analysis of enzymatic properties of glucanase mutants and wild type

[0054] 1. DNS method for enzyme activity determination

[0055] The specific method is as follows: Under the optimal pH and temperature conditions, 100 μL of diluted enzyme solution (1 μg / mL) and 900 μL of substrate (0.5% lichen polysaccharide) were mixed, reacted for 10 minutes, and then 1.5 mL of DNS was added to terminate the reaction. The mixture was boiled in water for 5 minutes. After cooling, the OD value was measured. 540 Under specific experimental conditions, the ability of an enzyme to hydrolyze glucan can be measured by the amount of reducing sugars produced. One unit (U) of enzyme activity is defined as the amount of enzyme that can hydrolyze glucan and produce 1 micromole (μmol) of reducing sugars per minute.

[0056] 2. Determination of properties of mutant and wild-type glucanase

[0057] 1. Optimum temperature and thermal stability determination method

[0058] Method for determining the optimal temperature of glucanase: dissolve the substrate in 0.1 mol / L sodium hydrogen phosphate-citrate buffer and perform enzymatic reaction at pH 4.0 and different temperatures (30-80°C). Figure 1 As shown, the optimum temperature of mutant M3 was 5°C higher than that of the wild type (50°C), and at high temperatures of 60°C to 80°C, the relative enzyme activity of mutant M3 (15.2%-79.9%) was 1.1-25.4 times higher than that of wild type PaGlu16A (0.7%-37.3%).

[0059] Half-life determination method: wild-type PaGlu16A and mutant M3 were treated at 50°C for a certain time (0-300 minutes). The protein concentration of all mutants and wild-type was 100 μg / mL during treatment, and the volume was 100 μL. Samples were taken at different time points and quickly placed on ice. The residual enzyme activity was measured at 50°C and pH 4.0. Figure 2 As shown in the figure, in terms of thermal stability, the half-life of mutant M3 at 50°C was 232 min, which was 158 min longer than that of the wild type (74 min).

[0060] 2. Temperature stability T 50 Temperature stability is the activity remaining at half the value after treatment at the corresponding temperature for 30 minutes (T 50 The wild type and mutants were incubated at a specific temperature of 45-75°C without substrate for 30 min. Figure 3 As shown, the T of wild-type PaGlu16A was determined. 50 The value was 55.0℃, while the T of mutant M3 was 50 The value increased by 10.0°C compared with the wild type, reaching as high as 65.0°C. Moreover, when treated at 60°C, the wild type lost more than 90% of its activity, while the mutant M3 retained 83.4% of its initial activity.

[0061] 3. Optimal pH and pH stability determination method

[0062] The pH properties of wild-type PaGlu16A and its mutant M3 were evaluated in terms of their optimal pH and pH stability. Enzymatic reactions were performed at different pH levels (1.0-7.0) to determine their optimal pH. Enzyme activity was determined by dissolving the substrate lichen polysaccharide at 50°C in 0.1 mol / L sodium dihydrogen phosphate-citrate buffer at different pH values. The enzyme solution was incubated in buffers at different pH values ​​(1.0-12.0) at 37°C for 1 hour, and the residual enzyme activity was measured to demonstrate the enzyme's pH stability.

[0063] The results are as follows Figure 4 As shown in Figure 2, the optimum pH for both the wild type and the mutant is 4.0, and the mutant can maintain more than 40% relative enzyme activity between pH 2.5 and 6.5. Figure 5 As shown, the pH stability of the mutant in the solution environment at pH 6.0-9.0 was significantly better than that of the wild type. For example, at pH 9.0, the relative enzyme activity of the mutant was above 80%, while that of the wild type was only 32%.

[0064] 4. Determination of kinetic parameters and specific activity of glucanase

[0065] The detection method was referred to the literature (Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp.X4 by error-prone PCR and structural insights of mutated residues.DOI 10.1007 / s00253-017-8145-4,Baek et al.,2017) to determine the first-order reaction time of the reaction. m Value and V max The reaction time was 5 min. Different concentrations of dextran (0.625, 0.5, 0.4, 0.2, 0.1, 0.075 and 0.05 mg / mL) were used as substrates. The enzyme activity was measured under standard conditions (50°C, pH 4.0), and the corresponding reaction rate was calculated. K was calculated using GraFit7 software. m Value and V max .

[0066] As shown in Table 2, compared with the wild type, the binding ability of mutant M3 to substrate was significantly enhanced (K m The values ​​were 37.9% and 45.2% lower than those of the wild type, respectively. When barley glucan was used as substrate, the catalytic efficiency (k cat / K m ) increased by 29% compared to the wild type. The specific activity of mutant M3 was not significantly decreased compared to the wild type. When using lichen polysaccharide as a substrate, the catalytic efficiency of mutant M3 (3100 mL / s·mg) was 48% higher than that of the wild type (2100 mL / s·mg).

[0067] Table 2 Specific activity and catalytic efficiency of wild-type and mutant glucanase

[0068]

[0069] Note: Kinetic values ​​are shown as mean ± standard deviation (n=3).

[0070] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A high catalytic activity glucanase mutant M3 with improved thermal stability, characterized in that: The amino acid sequence of the high catalytic activity glucanase mutant M3 is shown in SEQ ID NO.

10.

2. A method for constructing the high catalytic activity glucanase mutant M3 according to claim 1, characterized in that: The method comprises the steps of performing site-directed mutation on the amino acids at positions 11, 209 and 228 based on the wild type of glucanase as shown in SEQ ID NO.

2.

3. Use of the high catalytic activity dextranase mutant M3 according to claim 1 or the high catalytic activity dextranase mutant M3 constructed by the construction method according to claim 2 in degrading dextran, characterized in that: The temperature during the degradation is 50-80°C, and the pH is 2.5-6.5; the glucan is barley glucan.

4. Use of the high catalytic activity glucanase mutant M3 according to claim 1 or the high catalytic activity glucanase mutant M3 constructed by the construction method according to claim 2 in the degradation of lichen polysaccharide.

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