Construction method of recombinant escherichia coli for expressing endoglucanase
A technology of recombinant Escherichia coli and endoglucanase, which can be applied in the field of genetic engineering and can solve problems such as damage to the main structure of fibers
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Embodiment 1
[0019] Example 1 Acquisition and transformation of endo-β-1,4-glucanase gene ph1171-sgc-mut-sd
[0020] Pyrococcus kuyerii P. horikoshii (American Culture Collection Center No. ATCC700860) extracts genomic DNA as a template for PCR amplification, primers:
[0021] phEG-SG-C-F: 5ˊ-CAGCAGAT CATATG GAAAATACAACATATCAACACCG-3ˊ
[0022] phEG-SG-C-R: 5ˊ-CTCTAA GCGGCCGC TCAAGAACTTTTGGAACAACTATC-3ˊ
[0023] Mut-SD-F: 5ˊ-CGCTTGGTGGGGTGGTAATCTAATG-3ˊ
[0024] Mut-SD-R: 5ˊ-CATTAGATTACCACCCCACCAAGCG-3ˊ
[0025] PCR amplification system: Add the following reagents in sequence in a 0.5ml PCR tube: 5 μL of 10× PCR buffer; 1 μL of dNTPs mixture (2.5 mM each); 1 μL of 10 μM phEG-SG-C-F; 10 μM phEG-SG-C-R 1 μL; Genomic DNA 1 μL; Vent DNA polymerase 0.5 μL; Sterile water 40.5 μL;
[0026] PCR amplification conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 72 s, 30 cycles; final extension at 72°C for 5 min; i...
Embodiment 2
[0030] Expression of endo-β-1,4-glucanase gene ph1171-sgc-mut-sd
[0031] The expression conditions were 37°C, 200 r / min, cultured in LB medium, adding ampicillin with a final concentration of 100 μg / mL at the time of inoculation, and cultured until OD 600 = 0.6, then add IPTG to a final concentration of 0.1 mM to induce expression for 3 h. In the middle, samples were taken at 1.5 h and 3 h after induction, respectively. Bacteria were collected by centrifugation at 4167 r / min, ultrasonically disrupted (conditions: total time 2 min, 2 s on, 2 s off, power 50%, ice bath), 80 μL was sampled as a whole protein sample, and the remaining sample was placed at 12000 r / min. min, centrifuged at 4°C for 10 min, and then 80 μL of the supernatant was taken as the bacterial supernatant, and 20 μl of 5×SDS PAGE loading buffer was added respectively, and then heat-treated, and the results were analyzed by 12% SDS-PAGE electrophoresis.
[0032]
Embodiment 3
[0034] Purification and optimum temperature determination of endo-β-1,4-glucanase gene ph1171-sgc-mut-sd
[0035]After the above bacterial cells were ultrasonically disrupted, they were directly heated at 75°C for 30 min, and centrifuged at 12,000 r / min at 4°C for 30 min, then the supernatant and precipitate were collected to prepare electrophoresis samples, and 12% SDS-PAGE electrophoresis samples were taken. analyze. Then, the supernatant after heating and centrifuging was used as the crude enzyme solution, the protein content was determined and standardized by the Bradford method, and 0.5% carboxymethylcellulose sodium (CMC-Na) was used as the hydrolysis substrate, pH5.6, 100 mM Acetic acid buffer solution was reacted at 75°C, 80°C, 85°C, 90°C, 95°C, and 100°C for 30 min, and the DNS method was used to detect the reaction products, and the optimum reaction temperature was determined according to the detection results.
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