Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate films by polyester hydrolases

Abstract

The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 m Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 m, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 m MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 m, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the K i values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.

Keywords: 3‐(N‐morpholino)propanesulfonic acid; Tris; biocatalysis; inhibition; polyester hydrolase; polyethylene terephthalate.

Figure 1

Initial hydrolysis rates of PET films (9 cm²) as a function of the concentration of TfCut2 (dashed line) and LCC (solid line) in (A) Tris; (B) MOPS; (C) sodium phosphate (0.2 m, pH 8.0). Error bars indicate the standard deviation of triplicate determinations.

Figure 2

Initial hydrolysis rates of PET films (9 cm²) of LCC (light bars) and TfCut2 (dark bars) as a function of buffer concentration of (A) Tris, (B) MOPS, and (C) sodium phosphate (pH 8.0). In each buffer, LCC and TfCut2 were employed in concentrations corresponding to their maximum initial hydrolysis rates (see Fig. 1). Error bars indicate the standard deviation of triplicate determinations.

Figure 3

Double reciprocal plots of initial hydrolysis rates of PET films versus substrate concentration for LCC (A) and TfCut2 (B) at different concentrations of Tris: ● 0 m, ▲ 0.2 m, and ♦ 0.4 m.

Figure 4

Double reciprocal plots of initial hydrolysis rates of PET films versus substrate concentration for LCC (A) and TfCut2 (B) at different concentrations of MOPS: (A) ● 0 m, ▲ 0.2 m, and ♦ 0.4 m and (B) ● 0 m, ▲ 0.05 m, and ♦ 0.075 m.

Figure 5

Docking of Tris and MOPS to LCC and TfCut2. An overlay of multiple binding modes of the inhibitors is presented. The structures of the enzymes are shown in red with the catalytic triad highlighted as blue sticks and Tris and MOPS as yellow sticks. (A) docking of Tris (neutral) to LCC; (B) docking of Tris (neutral) to TfCut2; (C) docking of MOPS to LCC; (D) docking of MOPS to TfCut2. The docking was performed with AutoDock Vina 41.