Cutinases are special types of serine hydrolases that degrade the waxy repellent structure called cutin in plants, a polyester made up of fatty acids and glycerol that makes up the main component of plant cuticle. These enzymes have recently attracted biotechnological interest due to their ability to modify and depolymerize the ester linkages in polyethylene terephthalate (PET) and other synthetic polyesters. The goal of the project was to characterize cutinase catalyzed hydrolysis of PET and map the interaction surface of cutinase using NMR spectroscopy.
This thesis describes cutinase catalyzed hydrolysis of PET (PET film) and Bis (hydroxyethyl terephthalate (BHET, soluble PET substrate) using 1H NMR. The extent of hydrolysis monitored by time-resolved NMR displayed the relative amounts of monomers, where TPA (Terephthalic Acid) was twice more than MHET (Mono(2-hydroxyethyl) terephthalate), in PET hydrolysis and MHET was 5 times higher than TPA, in BHET hydrolysis. However, during BHET hydrolysis it was also observed that, at alkaline conditions, BHET is prone to autolysis at a slower rate. Based on the results it was observed that cutinase was hardly active on MHET, which could be caused by electrostatic repulsion between the carboxylate group at MHET and the overall electronegative surface at the catalytic site.
Two-dimensional 15N HSQC were recorded on pure concentrated 15N-labeled S120A cutinase sample for further characterization of its binding surface. Here 15N-labeled S120A cutinase was titrated with BHET for monitoring change in the chemical shift of the residues detected in the 15N-HSQC spectra. These results revealed the significantly perturbed amino acids A120, I183, A185, G41, D83, Y119, L176, L182, V184, H188, L189 that are located in and around the cutinase active site up on binding BHET. Among the 11 amino acids 7, were hydrophobic indicating that the interaction is mainly driven by hydrophobic interaction. The chemical shift changes observed for these amino acids were also used for determining the binding affinity of cutinase, which resulted in a Kd 150 +/- 50mM, being a weaker interaction.
In summary, the reported hydrolysis and interaction results of F. solani cutinase shows that the enzyme has the ability to hydrolyse PET, and that this hydrolysis can be observed by using NMR spectroscopy cutinase. Moreover, mapping the substrate-binding surface with BHET shows I183, A185, A120, seems to be crucial for binding. These insights provide essential information for further rational protein engineering of cutinase to make a potential candidate in large scale industrial applications for the eco-friendly treatment of plastic wastes