Fast Turbidimetric Assay for Analyzing the Enzymatic Hydrolysis of Polyethylene Terephthalate Model Substrates.

Abstract

Synthetic plastics such as polyethylene terephthalate (PET) can be cooperatively degraded by microbial polyester hydrolases and carboxylesterases, with the latter hydrolyzing the low-molecular-weight degradation intermediates. For the identification of PET-degrading enzymes, efficient and rapid screening assays are required. Here a novel turbidimetric method in a microplate format for the fast screening of enzyme activities against the PET model substrates with two ester bonds bis-(2-hydroxyethyl) terephthalate (BHET) and ethylene glycol bis-(p-methylbenzoate) (2PET) is reported. The carboxylesterase TfCa from Thermobifida fusca KW3 is used for validating the method. High correlation and regression coefficients between the experimental and fitted data confirm the accuracy and reproducibility of the method and its feasibility for analyzing the kinetics of the enzymatic hydrolysis of the PET model substrates. A comparison of the hydrolysis of BHET and 2PET by TfCa using a kinetic model for heterogeneous catalysis indicates that the enzyme preferentially hydrolyzes the less bulky molecule BHET. The high-throughput assay will facilitate the detection of novel enzymes for the biocatalytic modification or degradation of PET.