Multilevel approach to the improvement of the enzymes involved in biodegredation of poly(ethylene terephthalate) plastic

Abstract

The employment of enzymatic systems to provide a green alternative to waste management is a quickly developing field that is currently limited by the intrinsic catalytic and structural limitations of select enzymes. Detailed characterization of the functional and structural limitations of biotechnologically interesting systems is needed to address the actualized and perceived shortcomings of these potentially lucrative enzyme systems. Here we have described unique methods for characterizing enzymes found in the biodegradation of xenobiotic aromatic pollutants that can be used to further our understanding of these systems and their limitations and guide strategic improvements. We have developed a substrate, pH, and temperature adaptable method able to quantify minute esterase activity discrepancies. We demonstrated the proficiency of this technique by characterizing substrate activity variability among a suite of esterases using substrate BHET, a diester subunit of PET. Results classified the enzymes with no activity, activity on only BHET, and activity on BHET and the resulting monoester MHET. We also looked to advance the understanding of the functional and structural limitations of a Rieske oxygenase, terephthalate dioxygenase, which is involved in the bioconversion of terephthalic acid. This work presented the first in-depth study of the structural limitations of this family of multimeric metalloproteins. Results from spectroscopic measurements including small angle X- ray scattering and differential scanning calorimetry detail a two-phase, non-concerted structural dissociation occurring between 40 and 50°C. Temperature dependent kinetic assays support these findings with corresponding activity loss. The lability of the catalytically necessary mononuclear iron cofactor as outlined by X-ray absorption spectroscopy and the extremely limited turnover number have also defined the operational boundaries of this protein. The results presented in this work advance the understanding of enzymes critical to the bioremediation of plastic waste. Detailed knowledge of their limitations both in structure and in function can be used to guide development of these enzymes to make stronger, more effective systems.