Construction of fusion protein, method for degrading polymer by fusion protein and application
A fusion protein and polymer technology, applied in the field of enzyme engineering, can solve the problems of low degradation efficiency and limited binding ability, and achieve the effect of improving the degradation effect and improving the treatment effect
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Embodiment 1
[0036] Embodiment 1: the construction of genetically engineered bacteria
[0037] (1) According to the cutinase sequence derived from H.insolens in NCBI (as shown in SEQ ID NO.1), it was connected to the vector pET20b(+), and the plasmid pET20b(+)-Hic (recombinant plasmid) was constructed to obtain For the construction method, please refer to the literature: Sun Yirong, Wu Jing, Su Lingqia. Expression and fermentation optimization of Humicola insolens cutinase in Escherichia coli[J]. Food and Machinery, 2018(4).);
[0038] (2) According to the anchor peptide sequence (as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6) and spacer helical sequence (connecting peptide linker , as shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12), designed according to the pET20b(+)-Hic gene sequence Primers, and use PCR to amplify the gene anchor peptide and linker gene;
[0039] (3) Use the fragment recovered in step (2) as a Me...
Embodiment 2
[0041] Embodiment 2: Fermentation of genetically engineered bacteria produces enzyme
[0042] The genetically engineered bacteria constructed in Example 1 were inoculated in liquid LB medium (containing 100mg / L ampicillin) and grown for 8-10h, and the seeds were inserted into TB liquid fermentation medium (containing 100mg / L ampicillin) by 5mL / 100mL inoculum ampicillin); Escherichia coli was cultured and fermented on a shaker at 25°C for 48 hours, centrifuged at 4°C and 12,000 rpm for 15 minutes with a certain volume of fermentation broth, and the fermentation supernatant or broken wall supernatant was taken, which was the fermented crude enzyme liquid of the fusion protein.
[0043] Measure the enzyme activity of crude enzyme liquid, the result is as shown in table 1:
[0044] Table 1 Enzyme activity of recombinant bacteria
[0045]
Embodiment 3
[0046] Example 3: Degradation effect of fusion protein on PEA
[0047] Specific steps are as follows:
[0048] 1. Preparation of PEA solution: Take 1g of PEA into acetone, dilute to 100mL with acetone, shake well to obtain a solution with a concentration of 1%, and set aside.
[0049] 2. In a 25 mL Erlenmeyer flask with a stopper, add a fusion protein solution with a final concentration of 20 U / mL diluted with Tris-HCl buffer (50 mM, pH 8.0), and a PEA solution with a concentration of 0.1%, and the final volume is 10 mL. React at pH 5.0-9.0, 50°C. The turbidity change of the reaction system was measured by a spectrophotometer. The blank control group did not add enzymes, and the experimental groups were HiC treatment group and fusion protein treatment group.
[0050] 3. Draw the relative turbidity change diagram of the reaction system to judge the treatment of PEA.
[0051] Since PEA is insoluble in water, when the substrate solution is added to the reaction system, the so...
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