Induction of actinobacterial enzymes for enhanced biological depolymerization of polyethylene terephthalate plastics

Abstract

Polyethylene terephthalate (PET), a widely used plastic, has been accumulating in the environment at an alarming rate, posing significant ecological challenges. This research addresses PET pollution through a biological depolymerization-based approach, involving three integrated strategies: (1) physicochemical modification of PET to enhance degradability, (2) optimization of carbon sources and inducer sources for cutinase enzyme production by Thermobifida fusca YX, and (3) an in silico investigation to identify and characterize potential PET-hydrolyzing enzymes (PHEs) in the bacterium. The first phase of the study focused on improving PET biodegradability through pretreatment techniques, including ultraviolet (UV) photooxidation, thermal oxidation, and size reduction of PET films. These treated samples were subjected to enzymatic depolymerization using both commercial immobilized enzymes and free (non-immobilized) enzymes derived from T. fusca YX. Analytical techniques such as Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) revealed that higher temperatures (above 80 °C) favored whole-cell biological depolymerization, while lower temperatures were more suitable for free enzyme-mediated depolymerization. Reduced particle size was also found to significantly improve biological depolymerization efficiency. Following these findings, cutinase production by T. fusca YX was optimized to increase enzyme yield. Various carbon sources (including sodium acetate, sodium butyrate, and sodium lactate) and natural cutin inducers (extracted from Roma tomatoes, Royal Gala apples, and watermelon peels) were tested. Using Response Surface Methodology (RSM), enzyme production was enhanced by 42%, demonstrating a viable strategy for scalable and cost-effective cutinase generation for industrial applications. In the final phase, an in silico analysis was conducted to explore the genomic potential of T. fusca YX for PET depolymerization. Six candidate cutinase enzymes were identified: three extracellular, two membrane-associated, and one intracellular. Molecular docking studies were performed to analyze their binding affinities with PET and cutin ligands, representing the first structural investigation of such interactions in T. fusca YX. Overall, this study offers a comprehensive approach to improving PET biological depolymerization under environmentally benign conditions, while reducing reliance on harsh chemical depolymerization processes. The use of a naturally occurring microbial strain highlights the potential for further biotechnological advancements in sustainable plastic waste management. Keywords Polyethylene terephthalate, physicochemical pretreatments, chemical hydrolysis, biological depolymerization, cutinase, Thermobifida fusca YX, response surface methodology, central composite rotatable design, bioinformatics, PET-hydrolyzing enzymes