Part:BBa_K1921003
PETase MutateM
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 PETase is an enzyme, which can hydrolyze PET. And this mutation protein is changed on the basis of the PETase. This protein is changed from S to F at 181 position. On the one hand, this mutation protein can hydrolyze PET in low temperature, which is a big problem in the world. On the other hand, it can increase the activity in the high concentration to the native protein. This mutation can continue high activity more times than native. 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 Est119 was 50%. The paper says that scientists Kengo Kitadokoro found that the phenylalanine at position 248 is the key site for the flexible binding of this enzyme to carbon chains of different lengths, and the vicinity amino acid sequence at the site of Enzyme Est119 has a high homology with PETase. Based on the literatures, we decided to mutate the amino acid S at this position to F To expand the hydrophobic response pocket (polar amino acids to non-polar amino acids).
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.
Improvement
Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control.
| None |