The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus alpha/beta-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic alpha/beta-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.
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 we report 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). The carboxylesterase TfCa from Thermobifida fusca KW3 was used for validating the method. High correlation and regression coefficients between the experimental and fitted data confirmed 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 indicated that the enzyme preferentially hydrolyzed the less bulky molecule BHET. The high-throughput assay will facilitate the detection of novel enzymes for the biocatalytic modification or degradation of PET.
The mol-ecule of the title compound, C(12)H(16)N(2)O(4), is centrosymmetric and the amide group is twisted relative to the benzene ring by 14.40 (13) degrees . The mol-ecules are hydrogen bonded into a three-dimensional framework, with the hydr-oxy O atoms acting as acceptors in N-Hcdots, three dots, centeredO hydrogen bonds and as donors in O-Hcdots, three dots, centeredO=C inter-actions.
