Substrate Report for: HEB

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structurefunction relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS analysis. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40 degrees C and pH 7.0 and stable for several days at pH 7.0 and 37 degrees C while the half-life times decreased to 3 days at 40 degrees C and only 6 h at 45 degrees C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), respectively, to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2 degrees +/-1.7 degrees to 62.6 degrees +/-1.1 degrees due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.

The functionalization of synthetic polymers such as poly(ethylene terephthalate) to improve their hydrophilicity can be achieved biocatalytically using hydrolytic enzymes. A number of cutinases, lipases, and esterases active on polyethylene terephthalate have been identified and characterized. Enzymes from Fusarium solani, Thermomyces insolens, T. lanuginosus, Aspergillus oryzae, Pseudomonas mendocina, and Thermobifida fusca have been studied in detail. Thermostable biocatalysts hydrolyzing poly(ethylene terephthalate) are promising candidates for the further optimization of suitable biofunctionalization processes for textile finishing, technical, and biomedical applications.