Construction and application of a glucanase mutant and its gene sequence

Abstract

The present invention belongs to the field of genetic engineering and enzyme engineering technology, and specifically relates to the construction and application of a glucanase mutant and its gene sequence, wherein the glucanase mutant has an amino acid sequence as shown in SEQ ID NO.2. The glucanase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 by G214A, S249P, and D286L. Compared with the EGL without mutation, the glucanase mutant EGL62 provided by the present invention has significantly improved thermal stability; incubated at 75°C for 3min, it can still maintain more than 60% enzyme activity; treated under pH 3.0-7.0 conditions for 1h, the enzyme activity is still above 50%. The glucanase mutant EGL62 provided by the present invention has an optimum pH of 5.0, an optimum temperature of 50°C, good stability, and broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to the construction and application of a glucanase mutant and a gene sequence thereof. Background Art

[0002] In the field of biomass conversion, glucanase, as an important type of biocatalyst, plays a vital role. The enzyme can efficiently decompose the glucan in the plant cell wall into fermentable sugars, providing a basis for the subsequent bioconversion process. Glucanase is mainly divided into endoglucanase and exoglucanase, which are responsible for cutting the inside and end of glucan, respectively, thereby breaking it down into smaller sugar molecules.

[0003] Endoglucanase mainly acts on the internal segments of glucan, and by randomly cutting glycosidic bonds and generating new ends, endoglucanase can increase the accessibility and solubility of glucan, creating conditions for the further action of exoglucanase. The activity of endoglucanase increases the availability of substrates and lays the foundation for the subsequent degradation process. Exoglucanase gradually cuts from the end of glucan to produce glucose or disaccharide molecules. Exoglucanase plays a key role in the final degradation of cellulose, ensuring that glucan is completely decomposed into monosaccharides. The efficient action of exoglucanase makes the final product purer and suitable for further industrial applications. Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin, so multiple enzymes are required to participate in the degradation. Especially in the decomposition of hemicellulose, the synergistic effect of glucanase and other enzymes is particularly important. Hemicellulose, like cellulose, is one of the key polysaccharides that constitute the plant cell wall. Glucanase can effectively decompose it and improve the overall degradation efficiency.

[0004] The potential of glucanase in industrial applications is gradually being explored. The main target of glucanase is glucan, which is closely related to cellulose in structure and also exists in plant cell walls. Through its catalytic action, glucanase can effectively break down glucan into smaller sugar units, providing raw materials for the production of biofuels and other industrial processes. In the papermaking industry, glucanase can convert xylan in papermaking fertilizers and agricultural waste into xylose monomers, and then into fuel. In the textile industry, endoglucanase can be used for fiber degumming to improve the softness and spinnability of fibers.

[0005] Glucanase is widely used in the food industry and feed industry. In the food industry, glucanase can be used in the sugar industry. Due to the presence of glucan, the extraction rate of the sugar industry is reduced, and the residual sucrose in the molasses increases, resulting in a loss of 3 to 5 times the loss of sucrose converted into glucan. Some sugar factories use glucanase to treat glucan in decoction, which reduces sugar loss and achieves good results. In addition, glucanase can be used to reduce wort viscosity, improve filtration performance, increase malt dissolution rate, prevent beer turbidity, and stabilize beer quality during beer brewing. In the feed industry, glucanase can be used in feed to reduce the content of non-starch polysaccharides (NSP) and their anti-nutritional factors, improve the absorption of nutrients by livestock and poultry, and increase the growth rate and feed conversion efficiency of livestock and poultry.

[0006] Glucanase has also shown initial application prospects in the pharmaceutical field. In the oral field, Streptococcus mutans has been widely regarded as the main pathogen causing dental caries. Many researchers have discussed the efficacy of glucanase and its decomposition of glucan. Glucanase can decompose the glucan produced by Streptococcus mutans. This enzyme can prevent the formation of dental caries and dental plaque. In addition, the degradation of β-glucan into oligosaccharides by β-glucanase is beneficial to human health. Studies have shown that Bacillus spp. Bacillus sp. and Penicillium P. occitanis Pol6-derived β-glucanase was immobilized and used in the production of oligoglucose. It was found that the immobilized enzyme had the same enzymatic properties as the free enzyme and better enzyme activity stability. The obtained oligoglucose had higher antioxidant capacity and higher antibacterial activity.

[0007] With the continuous development of biotechnology, significant progress has been made in the research and improvement of glucanase. Through genetic engineering and protein engineering, scientists have developed a variety of efficient and stable glucanase mutants. These mutants have not only significantly improved activity and stability, but also performed well in heat resistance and stress resistance. This will not only promote the optimization of existing industrial processes, but also provide possibilities for the development of new bioconversion technologies.

[0008] In the future, with the in-depth research and extensive application of glucanase, this field is expected to see more innovative biomass conversion technologies and products emerge. Further improving the activity and stability of glucanase will make greater contributions to sustainable development and environmental protection. Summary of the invention

[0009] Based on the above shortcomings, the present invention provides a construction and application of a glucanase mutant and its gene sequence. The glucanase mutant provided by the present invention has an amino acid sequence as shown in SEQ ID NO.2. The glucanase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 to G214A, S249P, and D286L. Compared with the unmutated EGL, the glucanase mutant EGL62 provided by the present invention has significantly improved thermal stability, and can still maintain more than 60% enzyme activity after incubation at 75°C for 3min; and the enzyme activity is still above 50% when treated for 1h under pH 3.0-7.0 conditions. The glucanase mutant EGL62 provided by the present invention has an optimum pH of 5.0 and an optimum temperature of 50°C, good stability, and broad application prospects.

[0010] The technical solution of the present invention includes:

[0011] In a first aspect, the present invention provides a glucanase mutant, wherein the glucanase mutant has an amino acid sequence as shown in SEQ ID NO.2.

[0012] SEQ ID NO.2:

[0013] Question QLASKYASQSRVWFAIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFIPLPGNDWQSAGAFISDGSAAALSQVTNPDGSTNLIFLVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK.

[0014] Specifically, the glucanase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1.

[0015] SEQ ID NO.1:

[0016] Question QLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK.

[0017] Preferably, the mutation is to mutate the glycine at position 214 of SEQ ID NO.1 to alanine, the serine at position 249 to proline, and the aspartic acid at position 286 to leucine.

[0018] In a second aspect, the present invention provides a gene sequence, wherein the gene sequence encodes the above-mentioned glucanase mutant.

[0019] Preferably, the gene sequence has a nucleotide sequence as shown in SEQ ID NO.3.

[0020] SEQ ID NO.3:

[0021]

[0022] In a third aspect, the present invention provides a recombinant vector, wherein the recombinant vector expresses the above gene sequence.

[0023] In a fourth aspect, the present invention provides a recombinant strain, wherein the recombinant strain comprises the above-mentioned recombinant vector.

[0024] In a fifth aspect, the present invention provides a method for preparing the above-mentioned glucanase mutant, the preparation method comprising the following steps:

[0025] S1. The above gene sequence is fused with an expression vector to construct a recombinant vector, and the recombinant vector is used to transform a host cell to obtain a recombinant strain;

[0026] S2, culturing the recombinant strain to induce the expression of the glucanase mutant;

[0027] S3. After recovery and purification, the glucanase mutant is obtained.

[0028] Specifically, the expression vector described in step S1 is selected from one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45.

[0029] Preferably, the expression vector described in step S1 is pPIC9K.

[0030] Specifically, the host cell in step S1 is a eukaryotic cell or a prokaryotic cell.

[0031] Preferably, the host cell may be Saccharomyces cerevisiae, Phaffia rhodozyma or Pichia pastoris.

[0032] More preferably, the host cell is Pichia pastoris.

[0033] More preferably, the host cell is Pichia pastoris GS115.

[0034] In a sixth aspect, the present invention provides the use of the above-mentioned glucanase mutant, gene sequence, recombinant vector or recombinant strain in food, feed, pulp and paper making, textile or pharmaceutical.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides an acid glucanase mutant EGL62 with excellent properties, an optimum pH of 5.0, an optimum temperature of 50°C, and good stability. Compared with EGL without mutation, the glucanase mutant EGL62 provided by the present invention has significantly improved thermal stability; it can still maintain more than 60% enzyme activity when incubated at 75°C for 3 minutes; and it can still maintain more than 50% enzyme activity when treated at pH 3.0-7.0 for 1 hour.

[0037] The glucanase mutant EGL62 provided by the present invention can be applied to the feed industry to reduce or eliminate the anti-nutritional effects caused by increased viscosity and improve animal production capacity; in the production of beverages and alcoholic beverages, it can effectively reduce the viscosity of malt juice and improve the filtration efficiency of fermentation liquid; in the papermaking industry, xylan in papermaking industry fertilizers and agricultural waste can be converted into xylose monomers and then converted into fuel; in the field of biomass conversion, it will help improve its efficiency and stability in the process of glucan decomposition. This will not only promote the optimization of existing industrial processes, but also provide possibilities for the development of new bioconversion technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the optimal pH for EGL, EGL62, and EGL62M.

[0039] Figure 2 pH stability of EGL, EGL62, and EGL62M.

[0040] Figure 3 It is the optimum temperature for EGL, EGL62 and EGL62M.

[0041] Figure 4 Thermal stability of EGL, EGL62, and EGL62M. DETAILED DESCRIPTION

[0042] The present invention is described below in conjunction with specific embodiments. The following embodiments are not intended to limit the present invention but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp.

[0043] 1. Strains and vectors: glucanase gene of the present invention egl , egl62 and egl62m It was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the Pichia pastoris expression vector pPIC9K and strain GS115 were purchased from Invitrogen.

[0044] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases were purchased from Invitrogen, mannan was purchased from Sigma, and the others were domestic reagents (all available from common biochemical reagent companies).

[0045] 3. Culture medium:

[0046] (1) Yeast medium YPD: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0047] (2) Escherichia coli culture medium LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0048] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, and 1% glycerol.

[0049] (4) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, and 0.5% methanol.

[0050] The molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0051] Example 1 Trichoderma reesei ( Trichoderma reesei ) Glucanase encoding gene egl62 Synthesis

[0052] The present invention uses the glucanase from Trichoderma reesei egl As a reference, the glucanase egl The gene has an amino acid sequence as shown in SEQ ID NO.1. egl The amino acid sequence of the gene was mutated to G214A, S249P, and D286L, and the 5' and 3' ends of the mutated sequence were added EcoR I and Not I The restriction enzyme cutting site was added, and the sequence was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized glucanase mutant EGL62 is shown in SEQ ID NO.2, and has a nucleotide sequence shown in SEQ ID NO.3, that is, egl62 Gene.

[0053] Example 2 Cloning of glucanase gene egl62

[0054] The synthesized target gene egl62The vector was stored in the form of puncture bacteria. The puncture bacteria were picked up with a sterile toothpick in the clean bench and placed in an LB shaking tube containing Amp (working concentration: 100 μg / ml) antibiotics. The tubes were cultured overnight at 37°C and 220 rpm. The next day, the vector containing the gene was extracted according to the instructions of the PurePlasmid Mini Kit (CW0500).

[0055] According to the glucanase gene sequence, the primers shown in Table 1 were designed and synthesized:

[0056] Table 1

[0057]

[0058] The extracted vector was used as a template for PCR amplification. The PCR reaction parameters were: 94℃ denaturation for 5 min; then 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 75 sec, and 72℃ incubation for 10 min after 30 cycles. A fragment of about 1191 bp was obtained, which was recovered and connected to the pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 42.2 kDa.

[0059] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence was compared with egl62 The sequences were compared and confirmed to be correct.

[0060] Example 3 Preparation of recombinant glucanase EGL62

[0061] The expression vector pPIC9K Double enzyme digestion ( Eco R I+ Not I) and the gene encoding the glucanase mutant EGL62 egl62 Gene (SEQ ID NO.3) double enzyme digestion ( Eco R I+ Not I), enzymatically cut out the glucanase encoding egl62 Gene fragments and expression vectors pPIC9K Connect to obtain recombinant plasmid pPIC9K-egl62 Pichia pastoris GS115 , obtain recombinant Pichia pastoris strains GS115 / EGL62 .

[0062] Recombinant Pichia pastoris strain GS115 / EGL62 Compared with the control strain (i.e., the non-mutated strain GS115 / EGL , the preparation method is the same as GS115 / EGL62), inoculated in 300 mL BMGY culture medium, cultured at 30°C 200rpm with shaking for 48h, and then centrifuged to collect the cells. Then resuspended in 150 mL BMMY medium, cultured at 30°C 200rpm with shaking. After 72h of induction, the supernatant was collected by centrifugation to obtain the recombinant glucanase EGL62 and the unmutated glucanase EGL, and the enzyme activity was measured.

[0063] Example 4 Activity analysis of recombinant glucanase EGL62

[0064] The present invention adopts the DNS method to determine the enzyme activity, and the specific method is as follows: under the conditions of pH 5.0 and 37°C, 1 ml of the reaction system includes 100 μl of appropriately diluted enzyme solution and 900 μl of substrate, react for 30 min, add 1.5 ml of DNS to terminate the reaction, boil in boiling water for 5 min, and measure the OD value at 540 nm after cooling. 1 enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

[0065] The expression level of the recombinant glucanase EGL62 was 240 U / mL, and the expression level of the non-mutated glucanase EGL was 210 U / mL.

[0066] Example 5 Determination of properties of recombinant glucanase EGL62

[0067] The enzymatic properties of the recombinant glucanase EGL62 and the unmutated glucanase EGL were determined and compared. GS115 / EGL62 The enzymatic properties were measured and compared with those of the unmutated glucanase EGL. At the same time, the recombinant glucanase EGL62M was added for enzymatic property comparison, and EGL62M had an amino acid sequence as shown in SEQ ID NO.6 (G134A mutation was performed on the basis of SEQ ID NO.1), and the specific preparation methods of the EGL62 mutant and the recombinant glucanase EGL62M were as described in the above examples.

[0068] SEQ ID NO.6:

[0069] QQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNDDGMTIFRLPVAWQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWS QLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK.

[0070] 1. Optimal pH determination

[0071] Purified unmutated glucanase EGL, recombinant glucanase EGL62 and recombinant glucanase EGL62M were subjected to enzymatic reaction at 37℃ for 30min at different pH to determine their optimal pH. The substrate glucan was tested for glucanase activity at 40℃ in 0.1mol / L citric acid-sodium hydrogen phosphate buffer at different pH.

[0072] result( Figure 1 ) showed that the optimum pH of the unmutated glucanase EGL, recombinant glucanase EGL62, and recombinant glucanase EGL62M was 5.0. Among them, EGL62 could achieve more than 60% relative enzyme activity at pH 4.0-8.0, while EGL and EGL62M had less than 60% enzyme activity at pH 8.0, indicating that the pH application range of the recombinant glucanase EGL62 was significantly improved.

[0073] 2. pH stability determination

[0074] Purified unmutated glucanase EGL, recombinant glucanase EGL62, and recombinant glucanase EGL62M were treated in 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH values ​​at 40°C for 1 h, and then the enzyme activity was measured in a pH 5.0 buffer system at 40°C to study the pH tolerance of the enzyme.

[0075] result( Figure 2) showed that the recombinant glucanase EGL62 was very stable between pH 3.0 and 7.0. After treatment for 1 h in this pH range, the remaining enzyme activity was above 50%, and the relative enzyme activity was significantly improved compared with EGL and EGL62M. This indicates that the recombinant glucanase EGL62 has good pH stability in a wider pH range.

[0076] 3. Optimum temperature determination and thermal stability determination

[0077] The optimal temperature was determined by subjecting the purified unmutated glucanase EGL, recombinant glucanase EGL62, and recombinant glucanase EGL62M to an enzymatic reaction in a citric acid-disodium hydrogen phosphate buffer (pH 5.0) buffer system at different temperatures for 30 min, and then determining the enzyme activity at 40°C.

[0078] The temperature resistance test was performed by treating the purified non-mutated glucanase EGL, recombinant glucanase EGL62, and recombinant glucanase EGL62M at 75°C for 3 min, and then measuring the enzyme activity at 40°C.

[0079] Results of determination of the optimal reaction temperature of recombinant glucanase EGL62 ( Figure 3 ) showed that its optimum temperature was 50℃, and it maintained high enzyme activity at 40-60℃. Figure 4 ) showed that EGL62 had good thermal stability and could maintain more than 60% of the enzyme activity after incubation at 75°C for 3 minutes. However, the remaining enzyme activity of EGL was only 20%, and that of EGL62M was only 40%. Compared with the two, EGL62 had significantly improved thermal stability and good heat resistance.

[0080] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

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

2.

2. The glucanase mutant according to claim 1, characterized in that The dextranase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1; the mutation is to mutate the 214th glycine in SEQ ID NO.1 to alanine, the 249th serine to proline, and the 286th aspartic acid to leucine.

3. A gene molecule, characterized in that The gene molecule encodes the glucanase mutant according to any one of claims 1-2.

4. The gene molecule according to claim 3, characterized in that The nucleotide sequence of the gene molecule is shown as SEQ ID NO.

3.

5. A recombinant vector, characterized in that: The recombinant vector comprises the gene molecule according to any one of claims 3-4.

6. A recombinant strain, characterized in that: The recombinant strain comprises the recombinant vector according to claim 5.

7. The method for preparing the glucanase mutant according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Fusing the gene molecule according to any one of claims 3 to 4 with an expression vector to construct a recombinant vector, and transforming a host cell with the recombinant vector to obtain a recombinant strain; S2, culturing the recombinant strain to induce the expression of the glucanase mutant; S3. After recovery and purification, the glucanase mutant is obtained.

8. The preparation method according to claim 7, characterized in that: The expression vector described in step S1 is selected from one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZα, and pYAM75P.

9. The preparation method according to claim 8, characterized in that: The host cell described in step S1 is a eukaryotic cell or a prokaryotic cell.

10. Use of the glucanase mutant according to any one of claims 1 to 2, the gene molecule according to any one of claims 3 to 4, the recombinant vector according to claim 5 or the recombinant strain according to claim 6 in the fields of food, feed, pulp and paper making or textile.

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