Efficient Cloning and Expression of IsPETase in the E. coli SHuffle Cells; Activity Analysis using HR-LC/MS

Polyethylene terephthalate (PET), a highly persistent and challenging-to-recycle material, poses a very concerning threat to the environment due to is over accumulation. To combat this issue, we focus on PETase, a biomolecule with immense potential for PET recycling due to its exceptional activity and substrate specificity compared to other PET-degrading enzymes. In this study, the overexpression of IsPETase from Ideonella sakaiensis, was investigated by optimizing its codon usage, cloning it into the pET plasmid, and successfully expressing it in the cytoplasm of Escherichia coli using SHuffle cells, which facilitate the expression of proteins requiring disulfide bonds for proper folding and functionality. Under the optimized conditions, the Shuffle expression system yielded an average of 69.3 mg of IsPETase, with an uncertainty of ±16.8 mg (n = 3), in 1-L culture. This represents an impressive 11.5-fold increase compared to its conventional expression in E. coli. After production, we confirmed the identity of the PETase enzyme using a bottom-up proteomics approach, achieving an impressive 97% coverage and identifying 21 unique peptides. Our research findings were strengthened through additional investigations utilizing High-Resolution Liquid Chromatography-Mass Spectrometry (HR-LC/MS) and Scanning Electron Microscopy (SEM). These analyses confirmed the active production of the PETase in the E. coli SHuffle cells, and its effective PET depolymerization. The application of HR-LC/MS allowed us to quantitatively monitor PETase activity, providing a distinctive approach for future PET recycling and enzyme engineering studies. Overall, our research presents a promising solution to the efficient production of active IsPETase enzyme.