Engineering the degradation of polyethylene terephthalate (PET) through synergistic action of diverse thermostable enzymes

Abstract

Abstract: The synthetic thermoplastic polymer, polyethylene terephthalate (PET), is a human creation. It is formed by the building blocks, terephthalate and ethylene groups linked through ester linkages. With a half-life of several decades, there is a dire need to disintegrate PET into its monomers through green methods which are enzyme-assisted or microbial consortia-assisted. PET hydrolyzing enzymes (PHEs) degrade the water-insoluble plastic, PET, into (a) water-soluble degradation intermediates (DIs), such as OET (oligoethylene terephthalate), BHET (bis-hydroxyethyl terephthalate), and MHET (mono-hydroxyethyl terephthalate), and (b) the water-soluble terminal product of degradation, TPA (terephthalic acid). Generally, high surface hydrophobicity of PHEs leads to high binding affinity and efficient degradation of PET. Most catalytically efficient enzymes are expected to invade PET, releasing and accumulating TPA and DIs in solution. High surface hydrophobicity causes the enzymes to remain bound to the PET surface such that the solution containing released DIs is devoid of any enzyme. However, pure TPA is required for any condensation of TPA to PET during the recycling process. For this, the contaminating DIs are required to be further degraded to TPA. Therefore, wepropose that complete degradation of PET into TPA can be achieved by deploying two different enzymes at two different locations, in a synergistic ‘dual’ division of labor (where one enzyme works mainly upon PET, at its surface, to generate TPA and DIs and the other enzyme works mainly upon DIs in the solution surrounding PET). In this study, we report the engineering of PET degrading systems through the use of thermophilic hydrolases: TTCE (carboxylesterase from Thermus thermophilus), LCC (leaf branch compost cutinase) and TfCut2 (cutinase from Thermobifida fusca). Owing to differences in their PET-binding abilities, we demonstrate that synergy in action, amongst these enzymes, leads to improved yields of TPA that is also pure. Through structural analyses of the above enzymes, and comparison with a PETase from Ideonella sakaiensis, we created three PET-binding- compromised variants of LCC with improved catalysis and five variants of TfCut2 (out of a total of twenty-two) demonstrating 1.3 to 7-fold higher activity than wild-type TfCut2. Moreover, a serendipitously formed truncated version of a chimera designed from wild-type LCC andTfCut2 serves as an efficient PET binding module (similar to carbohydrate-binding modules present in many cellulases) that can have applications in immobilizing hydrolases onto PET surface.