Results

Overview

In the course of our research term, we managed to purify all six PETase variants (i.e. the mutant generated by Austin et al (2018): W159H S238F, PETase variant “Tianyu”, PETase variant “Dimi 1”, PETase variant “Dimi 2”, PETase variant “Dimi 3”, and PETase variant “Dimi 4”) using their C terminal hexahistidine tag using affinity purification. The catalytic activity of these variants was subsequently compared to wildtype PETase using a p-nitrophenol butyrate (pNPB) assay, which mimics the ester bond cleaved by PETase. It is frequently used in the literature as a substrate for assessing hydrolase activity, as the product of the reaction (p-nitrophenolate) conveniently fluoresces at 405 nm. A variety of substrate concentrations were used from 30 uM to 2500 uM, with controls for autohydrolysis of pNPB in solution. Prior to executing this assay, we endeavoured to improve the characterization of the wildtype PETase. We discovered that its cleavage of pNPB was optimal at pH 8.0-9.0, as opposed to "physiological pH" of 7.0. The comparison of all PETase variants was thus executed at pH 9.0. The assay temperature was also kept constant at 30 C, due to this temperature for PETase characterization used by Yoshida et al.

Subsequently, a time-course (with a one minute kinetic interval for the first 10 minutes, followed by a five minute kinetic interval up to 30 minutes) was executed for all proteins. Due to the high catalytic activity of all of our variants in comparison to wildtype, the absorbance of the spectrophotometer was maxed out at ~15 minutes at substrate concentrations above 625 uM. We have thus chosen to show the linear portion of the graph (from 0-10 minutes) that was used in the experimental estimation of the Km constants for each protein.

From this graph, it is clear that all of our variants seem to present an improvement on the wildtype PETase, but do not match the remarkable activity of the 2018 mutant engineered by Austin et al: W159H S238F. Importantly, alternative assays will be required to follow up on this result. Running HPLC assays of the supernatant of purified PETases upon overnight incubation on PET film would result in the precise detection of the products of PET degradation (MHET, BHET, and TPA). This would rule out the possibility that we had pulled down nonspecific esterases in the E. coli genome capable of degrading pNPB. Another curiosity is the PETase variant "Tianyu", which was optimized for hydrophobicity and catalytic activity, using a machine learning algorithm. This resulted in a PETase that displayed very amino acid sequence conservation with the wildtype and an Instability Index of 50.08 (unstable) by Benchling's estimator. We were surprised to see the protein fold and apparently have activity comparable to one of our FuncLib variants, "Dimi 1", which had a much larger dataset for estimating thermostability.

Team:Toronto/Results

Results

Overview