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

Abstract

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+.

A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

“…The T m and the values of the disulfide bridge variants were indeed increased by up to 24.9 and 17.3 °C, respectively. Compared to a previously reported increase of T m by 14 °C effected by a substitution of the calcium binding site with a salt bridge, a further gain of more than 10 °C in the T m could thus be obtained . The most stable variant has the D204C‐E253C disulfide bridge.…”

“…When the probability density of Ca 2+ within a radius of 3.7 Å to D204C‐E253C was calculated, E26 with a probability density of 42.5 ± 21.0% (SD), D174 with 27.8 ± 20.0%, E202 with 22.4 ± 18.4% and E64 with 21.4 ± 10.4% were observed in close vicinity to the dication. The occupancy map of Ca 2+ in D204C‐E253C revealed that the binding occurred close to the original calcium binding site D174‐D204‐E253 (Fig. ).…”

“…The crystal structure of TfCut2 (PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4CG1) was used as a template . MD simulations were performed using the gromacs 5 software (Uppsala University, Uppsala, Sweden) adopting AMBER99SB force field parameters as described previously . Equilibrations and MD simulations were carried out at 373 K. The probability densities of amino acid residues were calculated using GROMACS 5 excluding densities below 5% and those not present in all three rounds of simulation.…”

“…Est119 shares a sequence identity of 82% and a highly structural similarity with TfCut2 . In our previous work, the stabilization effect of calcium in several T. fusca polyester hydrolases has been analyzed . The T m of TfCut2 was found to be increased by more than 12 °C in the presence of 10 m m CaCl 2 .…”

Renewable building blocks for sustainable polyesters: new biotechnological routes for greener plastics

“…The resulting monomers are potentially reusable as substrates for polymerization after separation of dyes and contaminants. Protein engineering approaches were used to enable the practical applicability of enzymes in PET degradation and, more specifically, to increase the thermal stability, the activity and the absorption of the enzyme on PET substrates.…”