Biochemical and Functional Characterization of ANCUT4, an Oxidative Stress-Regulated Cutinase from Aspergillus nidulans with Activity on Synthetic Polyesters

Enzymes that efficiently degrade plastics are needed for waste management. Fungal cutinases are promising candidates. Among them, A. nidulans cutinases are differentially regulated, and two of them, ANCUT1 and ANCUT2, have demonstrated the ability to degrade synthetic polyesters. However, ANCUT4 is an oxidative stress-regulated enzyme that remained uncharacterized. This study aimed to produce, purify, and characterize this cutinase, evaluate its activity on synthetic polyesters, and understand its structural features. We identified ANCUT4 from an optimized medium containing cutin and H2O2 and heterologously expressed it for the first time in P. pastoris for characterization. The recombinant enzyme showed optimum activity at 30 °C and pH 7.0 and acted on both short-chain and long-chain synthetic nitrophenyl esters, and less on medium-chain ones. The low thermostability was associated with the lack of the third disulfide bond present in ANCUT1/2. Despite this, ANCUT4 degraded a variety of polyesters, achieving the highest degradation rate for PET and PCL (about 30% after four weeks of incubation). In summary, ANCUT4 is a novel catalyst for polyester hydrolysis, functionally distinct from the other three cutinases expressed by A. nidulans, and is expressed alongside other proteins that respond to oxidative stress.