A detailed study and comparison was made on the catalytic activities of cutinases from Humilica insolens (HiC), Pseudomonas mendocina (PmC), and Fusarium solani (FsC) using low-crystallinity (lc) and biaxially oriented (bo) poly(ethylene terephthalate) (PET) films as model substrates. Cutinase activity for PET hydrolysis was assayed using a pH-stat to measure NaOH consumption versus time, where initial activity was expressed as units of micromoles of NaOH added per hour and per milliliter of reaction volume. HiC was found to have good thermostability with maximum initial activity from 70 to 80 degrees C, whereas PmC and FsC performed best at 50 degrees C. Assays by pH-stat showed that the cutinases had about 10-fold higher activity for the lcPET (7% crystallinity) than for the boPET (35% crystallinity). Under optimal reaction conditions, initial activities of cutinases were successfully fit by a heterogeneous kinetic model. The hydrolysis rate constant k(2) was 7-fold higher for HiC at 70 degrees C (0.62 mu mol/cm(2)/h) relative to PmC and FsC at 50 and 40 degrees C, respectively. With respect to PET affinity, PmC had the highest affinity, while FsC had the lowest value. In a 96 h degradation study using lCPET films, incubation with PmC and FsC both resulted in a 5% film weight loss at 50 and 40 degrees C, respectively. In contrast, HiC-catalyzed lcPET film hydrolysis at 70 degrees C resulted in a 97 +/- 3% weight loss in 96 h, corresponding to a loss in film thickness of 30 mu m per day. As degradation of lCPET progressed, crystallinity of the remaining film increased to 27% due to preferential degradation of amorphous regions. Furthermore, for all three cutinases, analysis of aqueous soluble degradation products showed that they consist exclusively of terephthalic acid and ethylene glycol.