Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET. P oly(ethylene terephthalate) (PET) is a thermoplastic polyester with excellent tensile and impact strength, transparency, and appropriate thermal stability (1). Because of its high production (36 million tons per year [2]), PET constitutes a significant waste material, which, although not representing a direct hazard to the environment, is not readily decomposed in nature.Enzymatic recycling of polymers and particularly of PET basically would break down the polymer into its building blocks ethylene glycol (EG) and terephthalic acid (TA). These have a high value and can be reused in chemical synthesis, including the production of PET. This would avoid current limitations in PET recycling, which, e.g., requires pure PET fractions or has to fight with enrichment of contaminants (3). In addition, the surface modification of PET to increase its hydrophilicity is an essential step in processing for many applications ranging from textiles to medical and electronics. Synthetic polymers and particularly PET show excellent chemical resistance, but this feature makes them very difficult to be functionalized and is a great drawback for the processing steps. Current methods, including the use of harsh chemicals, concentrated acid or alkali, or different types of plasma approaches pursue to insert reactive moieties. Therefore, enzymatic strategies have recently received considerable attention (for a review, see reference 2) due to their environmentally friendly profile compared to currently used techniques such as those based on concentrated alkali (4) or the limitation of plasma methods (5) to planar surfaces.Compared to traditional approaches, enzymatic partial hydrol...