Our team has chosen to improve the LC-Cutinase eznyme using a rational mutagenesis approach.
We have chosen to test our improved variants' activity using a pNP-Butyrate degradation assay with different substrate and enzyme concentrations.
Our experiments support that our designed proteins are significantly improved compared to the existing part BBa_K936000 due to the following reasons:
First, our experiments showed a significant improvement of pNP-B degradation activity in two of our LC-Cutinase variants:
Our conclusion is based on kinetic test results which showed faster rise in O.D levels compared to the W.T. protein in three different substrate concentration (Fig. 1,2,3):
These tests were conducted using a bacterial supernatant, without measurement of enzyme concentrations, which might suggest that the improvement in activity is accomplished by an increase of enzyme expression/concentration. Although it suggests an improvement in expression levels and not efficiency, this hypothesis is still considered an improvement over the W.T. as it increases the degradation rate of the substrate.
In order to minimize the effect of expression levels on activity, we isolated and cleaned the enzymes and performed the experiment again, with equal enzyme concentrations.
As seen from the results above, even in equal enzyme concentrations our LC-Cutinase variants display a significant improvement in pNP-B degradation in comparison to the W.T. LC-Cutinase.
Secondly, we have improved the characterization of the LC-Cutinase protein with regards to:
- pNP-Butyrate degradation - Previous data does not include kinetic tests, only final O.D values. We have also measured activity with different substrate concentrations at known enzyme concentrations, also, not performed previously.
PET degradation - We have shown evidence of LC-Cutinase PET degradation activity using two different experiments - Agar plates containing shredded PET and Electron microscopy of PET samples incubated with LC-Cutinase. All previous data in the registry does not include records of PET degradation activity, only pNP-B.