Application of an endoglucanase editing gene and its encoding enzyme

Abstract

The present invention provides an endoglucanase editing gene and an application of its encoding enzyme, belonging to the field of gene technology. In order to solve the problems of low expression and poor thermal stability of endoglucanase in the prior art. The present invention provides an endoglucanase, the amino acid sequence of the endoglucanase is shown in SEQ ID NO.2. A new production method of endoglucanase is provided to improve the activity of endoglucanase.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to an endoglucanase for editing genes and the application of its encoded enzyme. Background Technology

[0002] To rationally utilize biomass resources and improve the utilization efficiency of agricultural waste, cellulase produced by filamentous fungi is used to degrade biomass cellulose and convert it into high-value-added chemicals and biofuels, effectively promoting the development of green agriculture. Cellulase primarily involves the combined action of endoglucanase, exoglucanase, and β-glucosidase. Endoglucanase acts on the amorphous regions of cellulose molecules, releasing oligosaccharides by randomly cleaving intermolecular β-1,4-glycosidic bonds. Exoglucanase acts on both ends of the long cellulose chain, hydrolyzing β-1,4-glycosidic bonds from the outside in to release cellulosic oligosaccharides, cellobiose, or glucose. β-glucosidase hydrolyzes cellobiose and cellooligosaccharides into glucose. However, the development of cellulose degradation technology is constrained by the unreasonable composition of cellulase systems in filamentous fungi, resulting in low enzyme activity and difficulty in controlling filamentous fungi during fermentation. Furthermore, different industrial applications require different proportions of cellulase components; for example, the textile industry requires highly active endoglucanase. Therefore, using yeast and other hosts for heterologous expression, clarifying the physicochemical properties of endoglucanase, improving specific enzyme activity, and reducing production costs are important ways to enhance the efficient utilization of cellulase. Summary of the Invention

[0003] The purpose of this invention is to solve the problems of low expression level and poor thermal stability of endoglucanase in the prior art.

[0004] The present invention provides an endoglucanase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0005] This invention provides a gene encoding an endoglucanase, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention provides a recombinant vector carrying the above-mentioned genes.

[0007] Further specifying, the originating vector of the recombinant vector is pPIC9K.

[0008] The present invention provides a recombinant microbial cell, wherein the recombinant microbial cell carries the above-mentioned gene or the above-mentioned recombinant vector.

[0009] The recombinant microbial cells provided by this invention are eukaryotic microbial cells or prokaryotic microbial cells.

[0010] The present invention provides the application of the above-mentioned endoglucanase, the above-mentioned encoding gene, the above-mentioned recombinant vector, and the above-mentioned recombinant microbial cells in the preparation of endoglucanase.

[0011] This invention provides a method for producing endoglucanase, wherein the method uses the above-mentioned recombinant microbial cell fermentation to obtain endoglucanase.

[0012] To further specify, the microbial cell is a Pichia pastoris cell.

[0013] The fermentation media used were MGY medium (g / L) (100 mL of 10% (v / v) glycerol, 800 mL of ddH2O, 100 mL of 10×YNB, and 2 mL of 500×B) and BMMY medium (g / L) (10 g of yeast extract, 20 g of peptone, 11.8 g of KH2PO4, and 5.03 g of K2HPO4·3H2O dissolved in 895 mL of water); fermentation conditions: 30℃, 250 rpm, cultured to OD. 600 Centrifuge at 2500×g for 8 min, retain the bacterial pellet, suspend the bacterial cells, and transfer to BMMY medium (BMMY). Incubate at 30℃ and 250rpm for 7 days, supplementing with methanol at a concentration of 0.5%-1% (v / v). After centrifugation, collect the supernatant.

[0014] The present invention provides the application of the above-mentioned endoglucanase in improving the tolerance of endoglucanase to the ionic liquid 1-ethyl-3-methylimidazolium acetate.

[0015] The present invention provides the application of the above-mentioned endoglucanase, the above-mentioned encoding gene, the above-mentioned recombinant vector, and the above-mentioned recombinant microbial cells in the degradation of cellulose.

[0016] This invention provides a method for producing reducing sugars. The method uses the aforementioned endoglucanase, with a reaction temperature of 60°C, a pH of 5.0, and a reaction time of 30 min.

[0017] Beneficial effects: (1) This invention provides an endoglucanase with a specific base sequence, and successfully uses the food-safe strain Pichia pastoris to heterologously express the endoglucanase with a high expression level. The method of this invention is safe and efficient and can be applied to the large-scale production of cellulase preparations.

[0018] (2) The endoglucanase pure enzyme provided by the present invention has a high specific activity, up to 94.17 U / mg, and the endoglucanase has good thermal stability.

[0019] (3) The endoglucanase of the present invention has a high conversion rate for sodium carboxymethyl cellulose as a substrate, and the yield of reducing sugar is calculated to be 104.3 mg.

[0020] (4) The endoglucanase provided by the present invention has good stability in ionic liquids and can be applied to the synergistic degradation of biomass by cellulase and ionic liquids. Attached Figure Description

[0021] Figure 1 This is for cloning the target gene; where M is the marker; 1 is the target band of eg5b;

[0022] Figure 2 Here is an electrophoresis image of the recombinant vector, where M is the marker; 1 is eg5b-pPIC9K; and 2 is pPIC9K.

[0023] Figure 3 This is a PCR diagram of the recombinant vector; M is the marker; 1-3 are positive clones; 4 is the negative control; and 5 is the blank control.

[0024] Figure 4 PCR validation of Pichia pastoris transformant (P. pastoris-eg5b); where M is the marker, 1-22 are the PCR validation results of recombinant Pichia pastoris transformant 1-22, and 4, 6, 7, 9, 11, 15, 17, and 19 are 8 HIS strains. + Mut + Phenotypic recombinant transformants;

[0025] Figure 5 The effect of induction time on the enzyme activity of recombinant strains; where the horizontal axis represents time and the vertical axis represents enzyme activity.

[0026] Figure 6 SDS-PAGE of supernatant of Pichia pastoris recombinant strains at different induction times; where M is the marker, lane 1 is induction for 168 h, lane 2 is induction for 144 h, lane 3 is induction for 120 h, lane 4 is induction for 96 h, lane 5 is induction for 72 h, lane 6 is induction for 48 h, and lane 7 is induction for 24 h.

[0027] Figure 7 Western blot validation of the recombinant protein; where M is the marker and 1-4 are EG5B.

[0028] Figure 8 The optimal temperature for the recombinant protein is represented by the x-axis, where temperature is the horizontal axis and relative enzyme activity is the vertical axis.

[0029] Figure 9 The optimal pH value for the recombinant protein; where the x-axis represents pH and the y-axis represents relative enzyme activity;

[0030] Figure 10The recombinant protein temperature stability is represented by the x-axis, where temperature is the horizontal axis and relative enzyme activity is the vertical axis. Detailed Implementation

[0031] 1-Ethyl-3-methylimidazolium acetate ([EMIM][CH3COO], product number: W131887) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0032] Example 1. Construction of a recombinant Pichia pastoris secretory expression system

[0033] 1. Cloning of the target fragment: Total RNA was extracted from *Talaromyces endophyticus* NEAU-6 and reverse transcribed into cDNA. Using the cDNA as a template, SignalP 5.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) was used to predict the signal peptide, and the first 18 amino acid sequences were obtained. MKPETLAAILSISAVAFG The signal peptide sequence was used. During the cloning of the target fragment, primers were designed by removing the signal peptide sequence region (eg5b-F: AGTCCCGCTCAAGAAGCGT, SEQ ID NO. 3; eg5b-R: TCAAGGGAAATAAGGCTTCAGAATC, SEQ ID NO. 4). High-fidelity polymerase was used for PCR to amplify the eg5b gene (the sequence shown in SEQ ID NO. 1 with the signal peptide removed), 945 bp (excluding the signal peptide). The amino acid sequence after translation of the gene sequence in SEQ ID NO. 1 is shown in SEQ ID NO. 2. Figure 1 ).

[0034] 2. Construction of the recombinant vector: The eg5b fragment was inserted into the multiple cloning site downstream of the α-factor signal peptide sequence in pPIC9K, with EcoRI selected as the restriction enzyme site. To enable homologous recombination between the target fragment eg5b and plasmid pPIC9K, homologous recombination was designed again: primers EcoRI-eg5b-pPIC9K-F: GAGGCTGAAGCTTACGTAGAATTCAGTCCGCTCAAGAAGCGTTCTG (SEQ ID NO.5); EcoRI-eg5b-pPIC9K-R: TGCCGGCCGCCCTAGGGAATTCTCAATGATGATGATGATGATGAGGGAAATAAGGCTTCA (SEQ ID NO.6). The ligation product was transformed into E. coli DH5α for cloning and amplification. After plasmid extraction, plasmid PCR verification was performed, and sequencing was conducted. The alignment result was 100% (…). Figure 2 and Figure 3 ).

[0035] 3. Screening of recombinant Pichia pastoris

[0036] Linearization of the recombinant plasmid (using restriction endonuclease SacⅠ): The recombinant vector pPIC9K-eg5b obtained in step 2 was digested with restriction endonuclease SacⅠ to complete the linearization of the recombinant vector. Since the inserted recombinant vector fragment does not disrupt the AOX1 site present in the original yeast genome, HIS is generated. + Mut + Phenotypic positive clones.

[0037] Linearized plasmids were transformed into Pichia pastoris GS115, allowing the target gene to integrate into the yeast genome, yielding the recombinant transformant P. pastoris-eg5b. These recombinant transformants were screened on histidine-deficient MD plates; positive recombinant transformants were identified as His. + Phenotype. MD culture medium was purchased from Protech Biotechnology Co., Ltd., catalog number WS1020.

[0038] To further filter Mut + Phenotypic transformants were obtained by extracting genomic DNA from yeasts screened on histidine-deficient MD medium. Using this DNA as a template, the AOX1 region was amplified by PCR using primers 5'AOX1 (GACTGGTTCCAATTGACAAGC, SEQ ID NO. 7) and 3'AOX1 (GCAAATGGCATTCTGACATCC, SEQ ID NO. 8). If the Pichia pastoris recombinant transformant phenotype is Mut... + This will result in two bands: one representing the aox1 gene (2200 bp), and the other a slightly larger band representing the homologous recombination of the recombinant vector and yeast. Twenty-two recombinant strains of *P. pastoris-eg5b* were screened on MD plates. Genomic DNA was extracted from the recombinant transformants and used as a template for PCR verification of the AOX1 region. Figure 4 For PCR validation, lanes 4, 6, 7, 9, 11, 15, 17, and 19 showed a genome-specific band, aox1, at 2200 bp, with a band slightly larger than that of the eg5b gene. This indicates that eight HIS strains were successfully screened. + Mut + Recombinant transformants of the phenotype.

[0039] Example 2. Expression, isolation, and purification of endoglucanase

[0040] Extraction of recombinant protein: The recombinant transformant P. pastoris-eg5b ( Figure 4Transformants from lane 9 were streaked onto MD plates for isolation. Single bacteria were picked and inoculated into MGY medium (g / L) (100 mL 10% (v / v) glycerol, 800 mL ddH2O, 100 mL 10×YNB, 2 mL 500×B), and incubated at 30℃ and 250 rpm until OD500. 600 Centrifuge at 2500×g for 8 min, retain the bacterial precipitate, suspend the bacterial cells, and transfer to BMMY medium (g / L) (10g yeast extract, 20g peptone, 11.8g KH2PO4, 5.03g K2HPO4·3H2O dissolved in 895mL water), and incubate at 30℃ and 250rpm. Take samples at 24h, 48h, 72h, 96h, 120h, 144h, and 168h, and add a certain amount of methanol until the methanol concentration is between 0.5% and 1% (v / v). Finally, centrifuge the samples and collect the supernatant.

[0041] like Figure 5 As shown, the enzyme activity of the recombinant increased with increasing induction time. At 120 h of induction, the highest endoglucanase activity (35.88 U / mL) was observed in the recombinant *P. pastoris-eg5b*. The endoglucanase activity was determined as follows: 1.5 mL of 1% sodium carboxymethyl cellulose (CMC-Na) solution was added to a test tube, and 0.5 mL of the test solution was added to the experimental group. The control group received the same volume of inactivated test solution. The tubes were incubated at 50°C for 30 min. After incubation, 1.5 mL of DNS reagent was added, and the tubes were immediately boiled for 5 min. After cooling, the volume was adjusted to 10 mL with distilled water. The mixture was thoroughly mixed on a vortex mixer, and the absorbance was measured. Under the above experimental conditions, the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar from 1% CMC-Na solution per minute was defined as one unit of enzyme activity (U).

[0042] SDS-PAGE analysis of recombinant proteins: Fermentation supernatants were collected at different induction times and analyzed by SDS-PAGE electrophoresis. Figure 6 As shown, a protein of approximately 45 kDa was obtained, which is larger than the predicted target protein, possibly due to varying degrees of glycosylation during post-translational modification.

[0043] Western blot verification: The target protein was transferred to a PVDF membrane under a constant current of 200 mA. The membrane was washed with 1×TBST for 8 min. It was then blocked with 5% (w / v) skim milk at 37°C for 1 h. After washing again for 8 min, primary antibody diluted with 5% (w / v) skim milk was added, and the membrane was incubated overnight at 4°C. After washing four times, diluted secondary antibody was added and incubated at room temperature for 1 h. After washing four more times, the PVDF membrane was exposed according to the instructions of the ECL chemiluminescence kit from Shanghai Beyotime Biotechnology Co., Ltd. The Pichia pastoris recombinant successfully expressed the endoglucanase EG5B (… Figure 7 ).

[0044] The recombinant transformant P. pastoris-eg5b successfully expressed the endoglucanase EG5B. The target protein was purified by nickel column chromatography to near electrophoretic purity, and the enzymatic properties of the purified recombinant enzyme were analyzed.

[0045] Example 3. Optimal temperature and pH of endoglucanase

[0046] Relative enzyme activity = enzyme activity at different temperatures or pH values ​​÷ maximum enzyme activity × 100%.

[0047] 1. Optimal temperature

[0048] The effect of temperature on the activity of recombinant endoglucanase was investigated within the temperature range of 30℃ to 90℃. Figure 8 As shown, the optimal temperature is 60℃.

[0049] 2. Optimal pH value

[0050] The effect of buffer solutions with different initial pH values ​​on the activity of recombinant endoglucanase was investigated at 60℃. Figure 9 As shown, the optimal pH for recombinant enzyme EG5B is 5.0.

[0051] Example 4. Temperature stability of endoglucanase and the effect of metal ions

[0052] Relative enzyme activity = Remaining enzyme activity ÷ Original enzyme activity × 100%.

[0053] 1. Temperature stability

[0054] like Figure 10 As shown, after incubation at 50℃ for 1 hour, the enzyme activity was still 100%; after incubation at 60℃ for 1 hour, the enzyme activity was still 81.6%; and after incubation at 70℃ for 1 hour, the enzyme activity was still 54.6%.

[0055] 2. The Influence of Metal Ions

[0056] At 60℃ and pH 5.0, 1mM, 5mM, and 10mM of metal ions (Cu) were added. 2+ Ca 2+ K + Zn 2+ Mg 2+ Mn 2+ Co 2+ ), where Mn 2+ and Cu 2+ The promoting effect is quite obvious.

[0057] Table 1

[0058]

[0059] 5. Specific activity of endoglucanase

[0060] The concentration of purified protein was determined using the BSA method, and the concentration of purified protein EG5B was 0.54 mg / mL. When the substrate was CMC-Na, the specific activity of the recombinant enzyme EG5B was 94.17 U / mg.

[0061] Example 5. Application of Endocellulase

[0062] 1. Add 0.5 mL of the above-obtained endoglucanase with a protein concentration of 0.41 mg / mL, using 15 mg of sodium carboxymethyl cellulose as a substrate. The enzymatic hydrolysis reaction was carried out at 60 °C and pH 5.0 for 30 min. The amount of reducing sugar produced was determined using the DNS method, and the yield of reducing sugar was 104.3 mg.

[0063] 2. Tolerance of recombinant proteins to ionic liquids

[0064] The enzyme activity of recombinant protein EG5B was determined at different concentrations (0 g / L-250 g / L) of [EMIM]CH3COO under conditions of 60℃ and pH 5.0. Relative enzyme activity = residual enzyme activity ÷ original enzyme activity × 100%. Recombinant protein EG5B exhibited good tolerance; at an IL concentration of 50 g / L, it retained 76.4% of its enzyme activity; at an IL concentration of 100 g / L, it retained 66.4% of its enzyme activity. Therefore, this endoglucanase can be used in the synergistic degradation of cellulose by cellulase and ionic liquids.

[0065] Example 6.

[0066] Bioinformatics analysis: The obtained endoglucanase has an isoelectric point of 4.59 and a molecular weight of approximately 34.29 kDa.

[0067] 1. Signal peptide prediction

[0068] The recombinant protein signal peptide was predicted to be a secretory protein. The first 18 amino acids (…) MKPETLAAILSISAVAFG It may be a signal peptide sequence.

[0069] Gene sequence (full length 999 bp), SEQ ID NO.1:

[0070]

[0071]

[0072] Amino acid sequence, SEQ ID NO.2:

[0073] MKPETLAAILSISAVAFG SPLKKRSGLQWFGSNEAGAEFGTAIPGTLGKDYTFADTSTIQTLIDSGMNIFRVPFLMERMVPNSLTGSIDSAYYQGYSEVINYITSKGAWAIVDPHNYGRYNGAVISSTNDFGAFWKTVASKFSNNTKVIFDTNNEFHDMDESLVVSLNQAAIDAI RSTGATSQYIFVEGNSWTGAWTWTTVNDDMKALTDSSAASDDLLVYEMHQYLDSDGSGTSVDCVNSTIGADRVKAATEWLKTNKKRAFLGEFAGGATSTCETAVTGLLDYLSSNSDVWLGATWWAAGPWWADYIYSMEPTTGTAYKAYLSILKPYFP.

Claims

1. An endoglucanase, characterized in that The amino acid sequence of the endoglucanase is shown in SEQ ID NO.

2.

2. A gene encoding endoglucanase, characterized in that The nucleotide sequence encoding endoglucanase is shown in SEQ ID NO.

1.

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant microbial cell, characterized in that The recombinant microbial cell is a recombinant microbial cell carrying the gene according to claim 2 or the recombinant vector according to claim 3.

5. The recombinant microbial cell according to claim 4, characterized in that The recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

6. The recombinant microbial cell according to claim 5, characterized in that The microbial cells are Pichia pastoris cells.

7. Use of the coding gene according to claim 2, the recombinant vector according to claim 3 or the recombinant microbial cell according to any one of claims 4 to 6 in the production of endoglucanase.

8. Use of the endoglucanase according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3 or the recombinant microbial cell according to any one of claims 4 to 6 in degrading cellulose.

9. A method for producing reducing sugar, characterized in that: The method comprises using the endoglucanase according to claim 1, with a reaction temperature of 60°C, a pH value of 5.0, and a reaction time of 30 minutes.

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