Structural peptide for improving high temperature resistance and / or alkali resistance of cellulase and preparation method and application of recombinant cellulase EglS-ML

Abstract

The invention relates to a structural peptide for improving high temperature resistance and / or alkali resistance of cellulase and a preparation method and application of recombinant cellulase EglS-ML, and belongs to the technical field of bioengineering. The invention provides a structural peptide capable of improving high temperature resistance and / or alkali resistance of cellulase. The amino acid sequence of the structural peptide is shown as SEQ ID NO. 1. The MGCGNL structural peptide has the function of improving the structural stability of cellulase, and the enzyme activity stability of the cellulase under the high-temperature condition and the alkaline environment can be remarkably improved by optimizing existing cellulase through the MGCGNL structural peptide.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a structural peptide for improving the high temperature resistance and / or alkali resistance of cellulase and a preparation method and application of recombinant cellulase EglS-ML. Background Art

[0002] Cellulase is a complex enzyme system that can degrade cellulose molecules into oligosaccharides or glucose. According to the substrate, action site and product of cellulase, it is mainly divided into endoglucanase (EC3.2.1.4), exoglucanase (EC.3.2.1.91) and β-glucanase (EC.3.2.1.21). As the first step of cellulose degradation, endoglucanase can randomly cut the amorphous region in the cellulose polysaccharide chain, break the β-1,4-glycosidic bond of the cellulose molecule from the inside, and produce shorter sugar chains or cellooligosaccharides. Endoglucanase is widely used and plays an important role in industrial fields such as pulp and paper, food brewing, feed production, and washing.

[0003] At present, most of the commonly used cellulases are derived from Trichoderma reesei or Aspergillus niger, but these cellulases have low thermal stability, and the enzymatic hydrolysis temperature is usually 37-60°C, and they can only work in an acidic environment with a pH of 4-6. In industrial applications such as papermaking, washing, and feed, cellulases with high temperature resistance and alkaline resistance are required. For example, the initial pH of pulp bleaching is 11-12, and the final bleaching pH is not less than 8.5; the optimal pH of washing enzymes is 7-10, and the enzyme activity is required to remain basically unchanged after being kept at 50°C for 30 minutes, and it has a certain tolerance to surfactants and metal ions.

[0004] The cellulases disclosed in the prior art cannot meet the requirements for application under high temperature and / or alkaline conditions. Summary of the invention

[0005] The purpose of the present invention is to provide a structural peptide for improving the high temperature and / or alkali resistance of cellulase and a preparation method and application of recombinant cellulase EglS-ML, so as to solve the problem in the prior art that cellulase cannot degrade cellulose under high temperature and / or alkaline conditions.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a structural peptide for improving the high temperature resistance and / or alkali resistance of cellulase. The amino acid sequence of the structural peptide is shown in SEQ ID NO.1.

[0008] The invention also provides the use of the structural peptide in improving the high temperature resistance and / or alkali resistance of cellulase.

[0009] The present invention also provides a nucleic acid molecule encoding the structural peptide, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0010] The present invention also provides a recombinant cellulase EglS-ML containing the structural peptide. The amino acid sequence of the recombinant cellulase EglS-ML is shown as SEQ ID NO.3.

[0011] The present invention also provides a gene encoding the recombinant cellulase EglS-ML, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.4.

[0012] The present invention also provides a method for preparing the recombinant cellulase EglS-ML, wherein the IGCGNV structural peptide in the amino acid sequence of the cellulase EglS is replaced with the MGCGNL structural peptide to obtain the recombinant cellulase EglS-ML;

[0013] The amino acid sequence of the cellulase EglS is shown in SEQ ID NO.5.

[0014] The present invention also provides a recombinant plasmid, which comprises the coding gene of the recombinant cellulase EglS-ML and an empty vector;

[0015] The empty vector is a pET28a vector.

[0016] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant plasmid and an empty vector bacterium;

[0017] The empty-load bacteria are Escherichia coli.

[0018] The present invention also provides the use of the recombinant cellulase EglS-ML prepared according to the preparation method in preparing products with high temperature resistance and / or alkali resistance.

[0019] The present invention also provides the use of the recombinant plasmid and the recombinant bacteria in preparing products with high temperature resistance and / or alkali resistance.

[0020] The present invention has the following technical effects and advantages:

[0021] The MGCGNL structural peptide of the present invention has the function of improving the structural stability of cellulase. The MGCGNL structural peptide of the present invention is used to optimize existing cellulase using a synthetic biology method, which can significantly improve the enzyme activity stability of cellulase under high temperature conditions and alkaline environments, and can be used for functional optimization of commercial cellulase. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The SDS-PAGE electrophoresis results of cellulase BvEgl, wherein lane M is a protein marker, lane 1 is a BL21-BvEgl bacterial solution without IPTG induction, lane 2 is a total protein of the engineering strain BL21-BvEgl induced by IPTG, lane 3 is a crude enzyme solution of cellulase BvEgl, lane 4 is a column flow-through of the crude enzyme solution of cellulase BvEgl, lane 5 is an elution flow-through of NTA-0 buffer, lane 6 is an elution flow-through of NTA-10 buffer, and lane 7 is an elution flow-through of NTA-20 buffer;

[0023] Figure 2 The structural analysis results of cellulase BvEgl, wherein A indicates the GH5 catalytic domain and CBM3 binding domain of cellulase BvEgl, and B indicates the MGCGNL structural peptide of cellulase BvEgl;

[0024] Figure 3 The results of enzymatic property analysis of cellulase BvEgl, wherein A is the specific enzyme activity of cellulase BvEgl at different pH values, and B is the specific enzyme activity of cellulase BvEgl at different temperatures;

[0025] Figure 4 Figure 2 is the SDS-PAGE electrophoresis result of cellulase EglS and recombinant cellulase EglS-ML, where A is the SDS-PAGE electrophoresis result of cellulase EglS, M is protein marker, lane 1 is the BL21-EglS bacterial solution without IPTG induction, lane 2 is the crude enzyme solution of cellulase EglS, lane 3 is the NTA-0 buffer elution flow-through, lane 4 is the NTA-10 buffer elution flow-through, lane 5 is the NTA-20 elution flow-through, lane 6 is the NTA -40 buffer elution flow-through; B is the SDS-PAGE electrophoresis result of recombinant cellulase EglS-ML, M is protein marker, lane 1 is the BL21-EglS-ML bacterial solution without IPTG induction, lane 2 is the recombinant cellulase EglS-ML crude enzyme solution, lane 3 is the NTA-0 buffer elution flow-through, lane 4 is the NTA-10 buffer elution flow-through, lane 5 is the NTA-20 buffer elution flow-through, lane 6 is the NTA-40 buffer elution flow-through;

[0026] Figure 5These are the analysis results of the enzymatic properties of cellulase EglS and recombinant cellulase EglS-ML, wherein A is the analysis result of the pH stability of cellulase EglS and recombinant cellulase EglS-ML, and B is the analysis result of the temperature stability of cellulase EglS and recombinant cellulase EglS-ML. DETAILED DESCRIPTION

[0027] The present invention provides a structural peptide for improving the high temperature resistance and / or alkali resistance of cellulase. The amino acid sequence of the structural peptide is shown in SEQ ID NO.1.

[0028] The invention also provides the use of the structural peptide in improving the high temperature resistance and / or alkali resistance of cellulase.

[0029] The present invention also provides a nucleic acid molecule encoding the structural peptide, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0030] The present invention also provides a recombinant cellulase EglS-ML containing the structural peptide. The amino acid sequence of the recombinant cellulase EglS-ML is shown as SEQ ID NO.3.

[0031] The present invention also provides a gene encoding the recombinant cellulase EglS-ML, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.4.

[0032] The present invention also provides a method for preparing the recombinant cellulase EglS-ML, wherein the IGCGNV structural peptide in the amino acid sequence of the cellulase EglS is replaced with the MGCGNL structural peptide to obtain the recombinant cellulase EglS-ML;

[0033] The amino acid sequence of the cellulase EglS is shown in SEQ ID NO.5.

[0034] The present invention also provides a recombinant plasmid, which comprises the coding gene of the recombinant cellulase EglS-ML and an empty vector;

[0035] The empty vector is a pET28a vector.

[0036] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant plasmid and an empty vector bacterium;

[0037] The empty-load bacteria are Escherichia coli, preferably Escherichia coli BL21 (DE3) strain.

[0038] The present invention also provides the use of the recombinant cellulase EglS-ML prepared according to the preparation method in preparing products with high temperature resistance and / or alkali resistance.

[0039] The present invention also provides the use of the recombinant plasmid and the recombinant bacteria in preparing products with high temperature resistance and / or alkali resistance.

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Among the strains of the present invention, the Bacillus Velezii T23 strain was from the National Livestock and Poultry Aquatic Microbial Germplasm Resource Bank, and the Escherichia coli BL21 (DE3) strain was purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0042] Among the reagents of the present invention, C115-01 recombinase and E112-01 protein detection solution were purchased from Nanjing Novozyme Biotechnology Co., Ltd., pET28a vector was purchased from Merck, Germany, NTA series buffer (Cat. No.: C600304-0500) was purchased from Sangon Biotechnology (Shanghai) Co., Ltd., and M5 HiPerBacteria Genomic DNA Kit was purchased from Beijing Polymer Biotechnology Co., Ltd.;

[0043] In the instrument of the present invention, the 70666-3 nickel column was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0044] Example 1: Heterologous expression and purification of cellulase BvEgl

[0045] (1) Primer design: The entire genome of Bacillus velez T23 strain was extracted and sequenced using the M5 HiPerBacteria Genomic DNAKit bacterial genome extraction kit. The nucleotide sequence of the cellulase gene BvEgl in the genome was used as a DNA template to design primers Bv-28a-F / R for synthesizing the cellulase gene BvEgl. The primers were commissioned to Suzhou Genewise Biotechnology Co., Ltd. for synthesis. The nucleotide sequences of primers Bv-28a-F / R are shown in Table 1.

[0046] Table 1 Nucleotide sequence of primer Bv-28a-F / R

[0047]

[0048] (2) PCR amplification: The PCR amplification system was 20 μL, including 10 μL 2×PhantaMax MasterMix, 1 μL DNA template, 1 μL upstream primer Bv-28a-F with an initial concentration of 10 μmol / L, 1 μL downstream primer Bv-28a-R with an initial concentration of 10 μmol / L, and 7 μL ddH2O. The PCR amplification program was 95°C denaturation for 3 min → (95°C denaturation for 30 s → 60°C annealing for 30 s → 72°C extension for 1 min) × 35 cycles → 72°C extension for 5 min → 4°C storage to obtain the cellulase gene BvEgl, whose nucleotide sequence is shown in SEQ ID NO.8.

[0049] SEQ ID NO.8:

[0050]

[0051] The amino acid sequence of the cellulase BvEgl encoded by the cellulase gene BvEgl is shown in SEQ ID NO.7.

[0052] SEQ ID NO.7:

[0053] MAETKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISSHGLQWYGDFVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVIIDWHILNDGNPNQNKEKA KEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVTEWGTSDA SGNGGVFLDQSREWLNYLDSKNISWVNWNLSDKQESSSALKPGASKTGGWPLTDLTASGTFVRENIRGTKDLTKDGPETPAQDNPTQEKGVSVQYKAGDGRVNSNQIRPQLHIKNNGN ATVDLKDVTARYWYNAKNKGQNVDCDYAQMGCGNLTHKFVTLHKPKQGADTYLELGFKTGTLSPGASTGNIQLRLHNDDWSNYAQSGDYSFFQSNTFKTTKKITLYHQGKLIWGTEPN

[0054] (3) Vector construction and transformation: The cellulase gene BvEgl was connected to the pET-28a vector containing the T7 promoter obtained after double digestion with Nco I and Xho I using C115-01 recombinase to construct the expression vector pET28a-BvEgl. For specific steps, refer to the instruction manual of the pET-28a vector. Then, the Escherichia coli BL21 (DE3) strain was transformed. For specific steps, refer to the instruction manual of the Escherichia coli BL21 (DE3) strain to obtain the engineered strain BL21-BvEgl.

[0055] (4) Protein induction expression and purification: 5 mL of engineered strain BL21-BvEgl seed solution (OD 600 =5) was inoculated into 500 mL LB culture medium (50 μg / mL Kan) and cultured at 37°C until OD 600=0.8, to obtain BL21-BvEgl bacterial solution; add IPTG to a final concentration of 0.5mmol / L, induce expression at 16°C for 20h, collect the bacteria by centrifugation at 5000rpm for 10min, add NTA-0 buffer to resuspend, and then ultrasonically disrupt for 10min, set the ultrasonic power to 400W, and rest for 5s after each ultrasonication for 3s; after the ultrasonication, centrifuge at 5000rpm for 30min to collect the supernatant to obtain a crude enzyme solution; use the crude enzyme solution to wash the 70666-3 nickel column for a total of two washes, and wash twice. The flow rate was 1 mL / min, and then NTA-10 buffer, NTA-20 buffer, NTA-50 buffer, NTA-100 buffer, NTA-150 buffer, NTA-200 buffer, and NTA-300 buffer were used for gradient elution, and the elution flow rate was 1 mL / min. At the same time, E112-01 protein detection solution was used for detection and the elution flow-through was collected. After ultrafiltration and centrifugation to remove imidazole, a purified BvEgl enzyme solution was obtained; then SDS-PAGE electrophoresis was performed, and the results were as follows Figure 1 shown.

[0056] (5) Protein structure analysis: The Swiss-Model (https: / / swissmodel.expasy.org / ) was used to analyze the structure of cellulase BvEgl. Figure 2 shown.

[0057] PCR amplification results showed that the cellulase gene BvEgl was 1416 bp in length and the encoded cellulase BvEgl had 471 amino acids. Protein induction expression and purification results showed that the cellulase BvEgl with His-Tag was successfully purified using the E. coli expression system, and SDS-PAGE electrophoresis results showed that a single protein band appeared at about 53 kDa, which was consistent with the predicted results. The protein purity of the BvEgl enzyme solution was high and the expression level was large. The results of protein structure analysis showed that cellulase BvEgl was composed of a GH5 catalytic domain and a CBM3 binding domain. In the CBM3 binding domain, the H2 helical structure peptide MGCGNL located between the β4 and β5 folds played an important role in the structural stability of cellulase BvEgl. The amino acid sequence of the structural peptide MGCGNL was Met383-Gly384-Cys385-Gly386-Asn387-Leu388 (as shown in SEQ ID NO.1), and the nucleotide sequence encoding the amino acid residue sequence was shown in SEQ ID NO.2.

[0058] SEQ ID NO.1: MGCGNL;

[0059] SEQ ID NO. 2: ATGGGATGCGGCAATCTG.

[0060] Example 2: Optimal pH of Cellulase BvEgl

[0061] 0.1 mol / L citric acid-sodium citrate buffer solution (pH 3, 4, 5, 6, respectively), 0.2 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7, 8, respectively), 0.2 mol / L glycine-sodium hydroxide buffer solution (pH 9, 10, 11, 12, respectively) were prepared; 1% sodium carboxymethyl cellulose solution (CMC-Na) was used as a substrate, 100 μL 1% CMC-Na solution, 80 μL buffer solutions of different pH values ​​and 20 μL BvEgl enzyme solution with a final concentration of 400 mg / L were mixed evenly to obtain each reaction system, and the reaction was carried out in a water bath at 50°C for 30 min. The specific enzyme activity of cellulase BvEgl at different pH values ​​was determined by the DNS method, specifically: 300 μL DNS reagent was added to each reaction system to terminate the reaction, followed by a boiling water bath for 5 min for color development reaction, and after cooling to room temperature, the mixture was filled to 1 mL with distilled water, and 200 μL was taken in a 96-well ELISA plate, and the OD was determined by an ELISA instrument 540 The reducing sugar content in each reaction system was calculated according to the glucose standard curve formula, and the specific enzyme activity of cellulase BvEgl at different pH values ​​was calculated according to the specific enzyme activity formula. The optimal pH of cellulase BvEgl was determined with the highest enzyme activity as 100%. The results are shown in Table 2 and Figure 3 shown.

[0062] The formula of glucose standard curve is: y = 1.67x-0.0349 (R 2 =0.998);

[0063] The specific enzyme activity formula is:

[0064] Where: specific enzyme activity (U / mg is the amount of enzyme required for BvEgl enzyme solution to decompose substrate and release 1 μg of glucose in 1 min, G is the reducing sugar content (mg / mL), M is the dilution multiple of BvEgl enzyme solution, V is the volume of BvEgl enzyme solution (mL), T is the reaction time (min), and N is the cellulase concentration (mg / mL).

[0065] Table 2 Specific enzyme activity of cellulase BvEgl at different pH

[0066] pH Specific enzyme activity (U / mg) pH Specific enzyme activity (U / mg) 3 245.53±22.14 8 1594±44.28 4 1416.89±66.42 9 1217.64±40.74 5 2213.89±13.22 10 664.17±20.37 6 2036.78±22.14 11 199.25±15.94 7 1926.08±14.17 12 189.85±14.54

[0067] The results showed that the optimum pH for cellulase BvEgl to perform enzymatic function was 5; it had high specific enzyme activity at pH 4-8, all above 65%; it still had enzymatic activity at pH 9-10, with specific enzyme activities of 52.41% and 34.48%, respectively. This indicates that cellulase BvEg1 is a highly active cellulase with alkali resistance and can degrade cellulose under alkaline conditions.

[0068] Example 3: Optimum temperature of cellulase BvEgl

[0069] With 1% CMC-Na solution as substrate, 100 μL 1% CMC-Na solution, 80 μL 0.1 mol / L citric acid-sodium citrate buffer solution (pH=5) and 20 μL BvEgl enzyme solution with a final concentration of 400 mg / L were mixed evenly to obtain each reaction system, and the reaction was carried out in a water bath at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C for 30 min, respectively. The specific enzyme activity of cellulase BvEgl at different temperatures was determined and calculated according to the DNS method described in Example 2. The optimum temperature of cellulase BvEgl was determined with the highest enzyme activity as 100%. The results are shown in Tables 3 and 4. Figure 3 shown.

[0070] Table 3 Specific enzyme activity of cellulase BvEgl at different temperatures

[0071] Temperature(℃) Specific enzyme activity (U / mg) Temperature(℃) Specific enzyme activity (U / mg) 20 2014.64±88.56 70 1859.67±86.85 30 2125.33±42.28 80 1771.11±132.83 40 2213.89±44.28 90 1217.64±110.69 50 2147.47±40.29 100 442.78±90.05 60 2081.06±66.42

[0072] The results showed that the optimum temperature for cellulase BvEgl to perform enzymatic function was 40℃; its specific enzyme activity was higher than 80% at 20-80℃, and it still had about 70% specific enzyme activity above 80℃. This indicated that cellulase BvEgl was a high-activity cellulase with high temperature resistance, and could degrade cellulose under high temperature conditions.

[0073] Example 4: Substrate specificity of cellulase BvEgl

[0074] CMC-Na solution, crystalline cellulose solution (Avicel), beechwood xylan solution (Beechwood Xylan), barley glucan solution (Barley Glucan), β-mannan solution (β-mannan), sucrose solution (Sucrose), laminarin solution (Laminarin), and amylopectin solution (Amylopectin) were independently prepared with a final concentration of 1%, and 100 μL of each was taken as a substrate, and was mixed evenly with 80 μL of 0.1 mol / L citric acid-sodium citrate buffer solution (pH=5) and 20 μL of BvEgl enzyme solution with a final concentration of 400 mg / L to obtain each reaction system. The reaction was carried out in a water bath at 40°C for 30 min. The specific enzyme activity of cellulase BvEgl in each reaction system was determined and calculated according to the DNS method described in Example 2. The results are shown in Table 4.

[0075] Table 4 Specific enzyme activity of cellulase BvEgl on different substrates

[0076] Substrate Specific enzyme activity (U / mg) CMC-Na solution 2213.89±54.12 Microcrystalline cellulose solution 486.91±109.45 Beech Xylan Solution 130.32±7.77 Barley Glucan Solution —— β-Mannan solution —— Sucrose solution —— Laminarin Solution —— Pullulan solution ——

[0077] The results showed that cellulase BvEgl had a strong ability to degrade cellulose substrates containing β-1,4 glycosidic bonds, and the specific enzyme activity could reach 2213.89±54.12U / mg, indicating that cellulase BvEgl is a highly active cellulase that can specifically degrade cellulose substrates.

[0078] Example 5: Optimization of cellulase by MGCGNL peptide

[0079] (1) Construction and transformation of recombinant cellulase vector: Using the nucleotide sequence of the cellulase gene EglS of Bacillus subtilis 168 strain disclosed by Wolf et al. (Wolf M, Geczi A, Simon O, et al. Genes encoding xylan and β-glucan hydrolysing enzymes in Bacillus subtilis: characterization, mapping and construction of strains deficient inlichenase, cellulase and xylanase [J]. Microbiology (Reading), 1995, 141 (Pt 2): 281-290) (shown in SEQ ID NO. 6) as a template, the IGCGNV structural peptide encoding fragment ATTGGATGCGGCAATGTG in the nucleotide sequence of the cellulase gene EglS was artificially designed to replace the MGCGNL structural peptide encoding fragment ATGGGATGCGGCAATCTG, thereby obtaining the recombinant cellulase gene EglS-ML (shown in SEQ ID NO. 6). NO.4), thereby replacing the IGCGNV structural peptide (sequence: ILe383-Gly384-Cys385-Gly386-Asn387-Val388) in the amino acid sequence of cellulase EglS with the MGCGNL structural peptide (sequence: Met383-Gly384-Cys385-Gly386-Asn387-Leu388) to obtain the recombinant cellulase EglS-ML (as shown in SEQ ID NO.3); constructing the expression vector pET28a-EglS-ML of the recombinant cellulase according to the method described in Example 1, and transforming to obtain the engineered strain BL21-EglS-ML.

[0080] The nucleotide sequence of the cellulase gene EglS is shown in SEQ ID NO.6.

[0081] SEQ ID NO.6:

[0082]

[0083] The amino acid sequence of the cellulase EglS encoded by the cellulase gene EglS is shown in SEQ ID NO.5.

[0084] SEQ ID NO.5:

[0085] MAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISSHGLQWYGEYVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVIIDWHILNDGNPNQNKEKA KEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVTEWGTSDA SGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGASKTGGWRLSDLSSASGTFVRENILGTKDSTKDIPETPSKDKPTQENGISVQYRAGDGSMNSNQIRPQLQIKNNGN TTVDLKDVTARYWYKAKNKGQNFDCDYAQIGCGNVTHKFVTLHKPKQGADTYLELGFKNGTLAPGASTGNIQLRLHNDDWSNYAQSGDYSFFKSNTFKTTKKITLYDQGKLIWGTEPN

[0086] The nucleotide sequence of the recombinant cellulase gene EglS-ML is shown in SEQ ID NO.4.

[0087] SEQ ID NO.4:

[0088]

[0089] The amino acid sequence of the recombinant cellulase EglS-ML encoded by the recombinant cellulase gene EglS-ML is shown in SEQ ID NO.3.

[0090] SEQ ID NO.3:

[0091] MAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISSHGLQWYGEYVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVIIDWHILNDGNPNQNKEKA KEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVTEWGTSDA SGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGASKTGGWRLSDLSSASGTFVRENILGTKDSTKDIPETPSKDKPTQENGISVQYRAGDGSMNSNQIRPQLQIKNNGN TTVDLKDVTARYWYKAKNKGQNFDCDYAQMGCGNLTHKFVTLHKPKQGADTYLELGFKNGTLAPGASTGNIQLRLHNDDWSNYAQSGDYSFFKSNTFKTTKKITLYDQGKLIWGTEPN

[0092] (2) Protein induction expression and purification of recombinant cellulase: Protein induction expression and purification were performed according to the method described in Example 1 to obtain purified EglS enzyme solution and EglS-ML enzyme solution; SDS-PAGE electrophoresis was then performed. The results were as follows: Figure 4 shown.

[0093] (3) pH stability analysis: EglS enzyme solution and EglS-ML enzyme solution were diluted to 400 mg / L, and then 0.2 mol / L glycine-sodium hydroxide buffer solution (pH 9 and 10, respectively) was added to each of them and treated at 50°C. During this period, the specific enzyme activities of cellulase EglS and recombinant cellulase EglS-ML were measured and calculated according to the method described in Example 2, with the highest enzyme activity measured in the reaction without treatment being 100%. The results are shown in Tables 5 and Figure 5 shown.

[0094] (4) Temperature stability analysis: EglS enzyme solution and EglS-ML enzyme solution were diluted to 400 mg / L, respectively, and then treated at 60°C and 80°C, respectively. During the enzymatic hydrolysis, the specific enzyme activities of cellulase EglS and recombinant cellulase EglS-ML were measured and calculated according to the method described in Example 2 at an enzymatic hydrolysis pH of 5 and an enzymatic hydrolysis temperature of 40°C. The highest enzyme activity measured in the reaction without treatment was taken as 100%. The results are shown in Tables 6 and 7. Figure 5 shown.

[0095] Table 5 pH stability results of cellulase EglS and recombinant cellulase EglS-ML

[0096]

[0097] Table 6 Temperature stability results of cellulase EglS and recombinant cellulase EglS-ML

[0098]

[0099] The results showed that both cellulase EglS and recombinant cellulase EglS-ML could be expressed soluble in E. coli. In terms of pH stability, compared with the highest enzyme activity, when pH was 9, the specific enzyme activity of cellulase EglS dropped to 48% after 240 min of treatment, while the specific enzyme activity of recombinant cellulase EglS-ML was still more than 90% within 360 min of treatment; when pH was 10, the specific enzyme activity of recombinant cellulase EglS-ML after 60 min of treatment was more than 1.5 times that of cellulase EglS. In terms of temperature stability, compared with the highest enzyme activity, after treatment at 60°C for 120 min, the specific enzyme activity of recombinant cellulase EglS-ML was 3.25 times higher than that of cellulase EglS; after treatment at 80°C for 30 min, the specific enzyme activity of recombinant cellulase EglS-ML was 3.1 times higher than that of cellulase EglS. This indicates that the MGCGNL structural peptide can significantly improve the enzyme activity stability of cellulase under high temperature conditions and alkaline environment, and can be used for the functional optimization of commercial cellulase.

[0100] As can be seen from the above examples, the present invention provides a structural peptide for improving the high temperature and / or alkali resistance of cellulase and a preparation method and application of recombinant cellulase EglS-ML. The MGCGNL structural peptide of the present invention is used to optimize the existing cellulase using the method of synthetic biology, which can significantly improve the enzyme activity stability of cellulase under high temperature conditions and alkaline environment.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A structural peptide for improving the high temperature resistance and / or alkali resistance of cellulase, characterized in that: The amino acid sequence of the structural peptide is shown in SEQ ID NO.

1.

2. Use of the structural peptide according to claim 1 in improving the high temperature resistance and / or alkali resistance of cellulase.

3. A nucleic acid molecule encoding the structural peptide according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A recombinant cellulase EglS-ML containing the structural peptide according to claim 1, characterized in that: The amino acid sequence of the recombinant cellulase EglS-ML is shown in SEQ ID NO.

3.

5. The gene encoding the recombinant cellulase EglS-ML according to claim 4, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

6. The method for preparing the recombinant cellulase EglS-ML according to claim 4, characterized in that: The IGCGNV structural peptide in the amino acid sequence of the cellulase EglS is replaced with the MGCGNL structural peptide described in claim 1 to obtain the recombinant cellulase EglS-ML; The amino acid sequence of the cellulase EglS is shown in SEQ ID NO.

5.

7. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the coding gene of the recombinant cellulase EglS-ML according to claim 5 and an empty vector; The empty vector is a pET28a vector.

8. A recombinant bacterium, characterized in that: The recombinant bacteria include the recombinant plasmid according to claim 7 and empty bacteria; The empty-load bacteria are Escherichia coli.

9. Use of the recombinant cellulase EglS-ML prepared according to the preparation method of claim 6 in preparing products with high temperature resistance and / or alkali resistance.

10. Use of the recombinant plasmid according to claim 7 and the recombinant bacteria according to claim 8 in preparing products with high temperature resistance and / or alkali resistance.

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