Functional characterizations of polyethylene terephthalate-degrading cutinase-like enzyme Cut190 mutants using bis(2-hydroxyethyl) terephthalate as the model substrate

Abstract

A mutant of cutinase-like enzyme from <em>Saccharomonospora viridis</em> AHK190, Cut190_S226P/R228S, designated as Cut190*, possesses high-thermal stability and has high polyethylene terephthalate (PET)-degrading activity. The functional characterizations of PET-degrading enzymes are generally conducted using accessible substrates such as poly(butylene succinate-<em>co</em>-adipate) (PBSA) and <em>p</em>-nitrophenyl butyrate (pNPB), even though their structures are different from that of PET. Bis(2-hydroxyethyl) terephthalate (BHET) is a component of PET, and the structure is similar to that of PET compared to PBSA and pNPB. Therefore, the analysis using BHET as the substrate is important to evaluate effective PET degradation. In this study, we analyzed the enzymatic activity of Cut190* using BHET under various conditions including Ca²⁺ concentration and pressure. Although terephthalate was supposed to be the final product, the intermediate product, mono(2-hydroxyethyl) terephthalate (MHET), was the only product generated, possibly due to the low binding affinity of MHET. The Cut190* activity towards BHET was observed even in the absence of Ca²⁺ and increased with increasing Ca²⁺ concentration, which is similar to its activity towards pNPB, but different from its activity towards PBSA. The difference can be attributed to the size of substrates. We also analyzed the activities of Cut190* and another high-activity mutant, Cut190*Q138A/D250C-E296C, under high pressures up to 400 MPa. BHET was non-enzymatically hydrolyzed under high pressure at 37 ℃. Enzyme activities were maintained under high pressures, and degradation of BHET in the presence of enzyme was higher than that in the absence of enzyme. Furthermore, the structural analysis of Cut190* under high pressure using Fourier transform infrared spectroscopy showed that most of the enzyme molecules were populated in the native structure below 400 MPa. These results indicate that BHET can contribute to the effective functional analysis of PET-degrading enzyme, and the combination of enzyme and pressure can lead to eco-friendly PET degradation.