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

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.

Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.

A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.

Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca.

Several bacterial polyester hydrolases are able to hydrolyze the synthetic polyester polyethylene terephthalate (PET). For an efficient enzymatic degradation of PET, reaction temperatures close to the glass transition temperature of the polymer need to be applied. The esterases TfH, BTA2, Tfu_0882, TfCut1, and TfCut2 produced by the thermophilic actinomycete Thermobifida fusca exhibit PET-hydrolyzing activity. However, these enzymes are not sufficiently stable in this temperature range for an efficient degradation of post-consumer PET materials. The addition of Ca2+ or Mg2+ cations to the enzymes resulted in an increase of their melting points between 10.8 and 14.1°C determined by circular dichroism spectroscopy. The thermostability of the polyester hydrolases was sufficient to degrade semi-crystalline PET films at 65°C in the presence of 10 mM Ca2+ and 10 mM Mg2+ resulting in weight losses of up to 12.9% after a reaction time of 48 h. The residues Asp174, Asp204, and Glu253 were identified by molecular dynamics simulations as potential binding residues for the two cations in TfCut2. This was confirmed by their substitution with arginine, resulting in a higher thermal stability of the corresponding enzyme variants. The generated variants of TfCut2 represent stabilized catalysts suitable for PET hydrolysis reactions performed in the absence of Ca2+ or Mg2+.