Toward reproducible PETase research: A standardized workflow for reliable enzyme production and comparison

Résumé fourni par la source

The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered Is PETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management. • Unified workflow standardizes PETase expression, purification, and QC steps. • Two-step Ni-NTA + SEC purification boosts enzyme purity and activity. • Activity of five Is PETase variants scales with their thermostability. • Protocol improves reproducibility and comparability across PETase studies.

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