LC-Cutinase Variant F4
This is a LC-Cutinase mutated protein.
It has 4 amino acid mutations compared to the codon optimised protein.
The mutations are not restricted to distance from the active site of the protein.
pNP-Butyrate degradation assay
In order to evaluate the activity of the F4 mutant LC-Cutinase we used a pNP-Butyrate degradation assay (see Protocols *LINK*).
At first we used different concentrations of the substrate - 50/125/250μM and measured the absorbance at 405nm from the moment the enzyme was added.
After determining an optimal substrate concentration we measured the activity of each mutant variant with a substrate concentration of 125μM and different enzyme concentrations.
Protein concentrations were determined with spectrophotometric methods after purification with a cation exchange column.
After receiving O.D values above 1 we lowered the starting concentration of pNP-Butyrate to 75μM and measured the activity again. We received a linear trend from the start of the measurement, suggesting our variant is very fast at degrading the substrate.
PET degradation assay
The LC-Cutinase variants were grown on M9 minimal medium plates with shredded PET pellets as a sole carbon source to test their ability to degrade PET.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC