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Polybutylene adipate terephthalate (PBAT) is a biodegradable alternative to polyethylene and can be broadly used in various applications. These polymers can be degraded by hydrolases of terrestrial and aquatic origin. In a previous study, we identified tandem PETase-like hydrolases (Ples) from the marine microbial consortium I1 that were highly expressed when a PBAT blend was supplied as the only carbon source. In this study, the tandem Ples, Ple628 and Ple629, were recombinantly expressed and characterized. Both enzymes are mesophilic and active on a wide range of oligomers. The activities of the Ples differed greatly when model substrates, PBAT-modified polymers or PET nanoparticles were supplied. Ple629 was always more active than Ple628. Crystal structures of Ple628 and Ple629 revealed a structural similarity to other PETases and can be classified as member of the PETases IIa subclass, α/β hydrolase superfamily. Our results show that the predicted functions of Ple628 and Ple629 agree with the bioinformatic predictions, and these enzymes play a significant role in the plastic degradation by the consortium.
Abstract
Background
Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparin‐induced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6‐, 8‐, 10‐, and 12‐mer size and a hypersulfated 12‐mer (S12‐mer).
Methods
We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and anti‐PF4/heparin antibodies.
Results
The synthetic heparin‐like compounds display stronger binding characteristics to PF4 than animal‐derived heparins of corresponding lengths. Upon complexation with PF4, 6‐mer and S12‐mer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8‐, 10‐, and 12‐mer heparins. Anti‐PF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins ≤ 8‐mer than with complexes formed between PF4 and heparins ≥ 10‐mer. Addition of one sulfate group to the 12‐mer resulted in a S12‐mer, which showed substantial changes in its binding characteristics to PF4.
Conclusions
We provide a template for characterizing interactions of newly developed heparin‐based anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.
The large amount of waste synthetic polyester plastics has complicated waste management and also endangering the environment due to improper littering. In this study, a novel carboxylesterase from Thermobacillus composti KWC4 (Tcca) was identified, heterologously expressed in Escherichia coli, purified and characterized with various plastic substrates. Irregular grooves were detected on polybutylene adipate terephthalate (PBAT) film by scanning electron microscopy (SEM) after Tcca treatment, and Tcca can also hydrolyze short–chain diester bis(hydroxyethyl) terephthalate (BHET). The optimal pH and temperature for Tcca were 7.0 and 40 °C, respectively. In order to explore its catalytic mechanism and improve its potential for plastic hydrolysis, we modeled the protein structure of Tcca and compared it with its homologous structures, and we identified positions that might be crucial for the binding of substrates. We generated a variety of Tcca variants by mutating these key positions; the variant F325A exhibited a more than 1.4–fold improvement in PBAT hydrolytic activity, and E80A exhibited a more than 4.1–fold increase in BHET activity when compared to the wild type. Tcca and its variants demonstrated future applicability for the recycling of bioplastic waste containing a PBAT fraction.