Cloning, expression and determination of pet degradation potential of a fungal cutinase enzyme

Environmental plastic pollution has reached alarming levels globally. As current mechanical and chemical recycling methods are inadequate, the development of innovative and sustainable solutions is vital. Polyethylene terephthalate (PET) pollution is a significant environmental problem due to the polymer's widespread use, crystalline structure, and natural resistance to biodegradation. While some natural microorganisms demonstrate the ability to hydrolyze PET, their industrial applicability is limited. Enzymes such as polyethylene terephthalate hydrolase (PETase), esterase, lipase, and cutinase have been identified that can degrade PET polymer. However, cutinase and PETase enzymes with high PET degradation potential are more preferred in industrial applications. In this thesis, the PET degradation potential of the biocontrol agent Trichoderma harzianum CBS 226.95 cutinase was investigated. Following enzyme characterization studies in an E. coli expression system, optimum enzymatic conditions were determined: In the presence of 10 mM pNPA, pH was 6.0, temperature was 50 °C, and incubation time was 5 minutes. The Km, Vmax, and specific activity parameters of the pure enzyme were calculated as 0.85 mM, 11.52 (µM/min), and 11.52 µmol/min/mg, respectively. The present findings demonstrate that Trichoderma species used for pathogen control in agricultural applications have an effective enzymatic profile for degrading environmental PET waste.