Evaluation of enzymatic degradation of petase against PBT and PEN nanoplastics using dye release assay

Plastic pollution poses a significant environmental challenge in recent years, driving interest in enzymatic degradation of plastic as one of the sustainable solutions. This study focuses on the expression, purification, and activity testing of different variants (WT, V3 and FAST) of PETase, an enzyme known to degrade polyethylene terephthalate (PET), using Escherichia coli (E. coli) SHuffle cells as the expression host. Following purification, the enzymatic activity of PETase was assessed on plastic-based nanoparticle substrates, specifically polyethylene naphthalate (PEN) and polybutylene terephthalate (PBT). Substrate degradation was monitored by a dye release assay using plastic nanoparticle- encapsulated dyes, Acridine Orange (AO) and Toluidine Blue O (TBO), providing an effective method for quantifying degradation. Fluorescence measurements obtained via spectrophotometry were found correlated with high-performance liquid chromatography (HPLC) data, which served as a reference standard for validation. This work contributes to the growing field of biocatalytic plastic degradation and highlights the potential for fluorescence-based assays in rapid enzyme screening.