Method for enhancing activity of endoglucanase on basis of error-prone PCR (Sequential Error-Prone) technology
An endoglucanase and technology technology, which can be applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., and can solve problems such as laborious and time-consuming
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Embodiment 1
[0041] Embodiment 1: Obtaining of mutant endoglucanase gene;
[0042] (1) Experimental method
[0043] 1. Preparation of mutation template
[0044] Inoculate a loop of Escherichia coli DH5α containing the pMD19-T-Cen plasmid and culture in 10 mL LB (Amp) liquid medium at 37°C overnight, and use the plasmid extraction kit to extract the pMD19-T-Cen plasmid according to the instructions. The extracted plasmid was detected by double enzyme digestion with BglII and Sph I, and it was used as a template for error-prone PCR after the detection was correct.
[0045] 2. Error-prone PCR method
[0046] Mg in error-prone PCR system 2+ The concentration is 7mmol / L, Mn 2+ The concentration is 0.1 mmol / L; the ratio of dCTP, dTTP and dATP, dGTP is 5:1. Mix well after adding the sample, and then enter the PCR cycle.
[0047] 3. For error-prone PCR mutations, use the pMD19-T-Cen plasmid as a template to
[0048] P1: 5'TAATCCAACCCGGAATTCGCAGAGACAAAAACGCCAGTAGC-3' and
[0049] P2: 5'-FAG...
Embodiment 2
[0054] Example 2: Construction of endoglucanase E. coli mutant library
[0055] (1) Experimental method
[0056] 1. Construction of mutant endoglucanase gene pET-32a(+) expression vector
[0057] 1) Preparation of plasmid vector pET-32a(+)
[0058] pET-32a(+) carries T7lac promoter and ampicillin resistance gene, cultivate Escherichia coli DH5α containing pET-32a(+) plasmid in 10mL LB(Amp) liquid medium at 37°C overnight, use plasmid extraction reagent The pET-32a(+) plasmid was extracted according to the instruction manual.
[0059] 2) Double digestion treatment of error-prone PCR product and pET-32a(+) plasmid
[0060] Take the above-mentioned error-prone PCR product (endoglucanase gene) and pET-32a(+) plasmid for EcoR I and Not I double enzyme digestion respectively. The enzyme digestion method is as follows:
[0061]
[0062]
[0063] Digest overnight at 37°C. Use the gel recovery kit to recover and purify. After recovering the 1.4Kb fragment of the endoglucanas...
Embodiment 3
[0085] The screening of embodiment 3 high enzyme activity bacterial strains
[0086] (1) Experimental method
[0087] 1. Transfer the colonies grown on the LB (Amp) plate (the constructed mutant library, each colony on the plate is a clone) to the LB (Amp) plate and LB (Amp, IPTG, CMC- Na) plate, after transferring the plate, place the plate in a constant temperature incubator at 37°C for culture, take out the LB (Amp) plate and store it in a 4°C refrigerator after culturing for 8 hours, and continue culturing the LB (Amp, IPTG, CMC-Na) plate for 36 hours. Then Congo red staining was used to observe the size of the hydrolysis circle, and at the same time, the original bacteria and the empty vector without endoglucanase gene were used as positive and negative controls on each plate. Screen the target strain according to the size of the hydrolysis circle, and then find out the target clone strain from the LB (Amp) plate.
[0088]2. Inoculate 10mL LB (Amp) liquid culture medium...
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