Part:BBa_K1921004
PETase MutateD
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
The PET-ase is an enzyme, which can hydrolyze PET. And this mutation protein is changed on the basis of the PETase. This protein is changed from R to T at 64 position. This mutation protein can hydrolyze PET in low temperature, which is a big problem in the world. It also can increase in the bacteria, which is easy to get.
Biology
Compared with the sequence of PETase in NCBI, the homology of PETase and Enzyme TfCut2 was 52%. The paper says that scientists Ren Wei found that change TfCut2 enzyme amino acid 63 T into other amino acids, the degradation products of the reaction will inhibit the reaction, and the vicinity amino acid sequence at the site of TfCut2 enzyme has a high homology with PETase. Based on the literatures, we decided to mutate the amino acid R at this position to T for lower inhibition from product.
Protein Expression
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ 5h. After taking samples, we used 1mM IPTG 5μl induced in 16℃ for 4-5h. Then we make the expression .
Figure 1.The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.
Figure 2.The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.
HPLC Result
Figure 3.The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.
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