Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (ThcCut1) was expressed in P. pastoris. No significant differences between the expression of native ThcCut1 and of two glycosylation site knock out mutants (ThcCut1ₖoAsn and ThcCut1ₖoST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly (ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono (2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly (butylene succinate) (PBS) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) were hydrolyzed by ThcCut1 and ThcCut1ₖoST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with ThcCut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, ThcCut1ₖoST was found to be significantly more active on PBS than the native ThcCut1. Hydrolysis of PBS films by ThcCut1 and ThcCut1ₖoST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92% and 41% of weight loss were detected with ThcCut1ₖoST and ThcCut1, respectively. Furthermore SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.