Engineering of enhanced thermal stability of bis (2-hydroxyethyl) terephthalate (BHET) hydrolase from Stenotrophomonas pavanii for polyethylene terephthalate (PET) degradation

BHETase_B exhibited higher activity toward BHET than PET and MHET. • Half-lives of engineered BHETase_B (>5.0 h) was greater than WT at 50 °C. • Mutant Q7I/A106P achieves complete degradation of BHET within 2 h at 50 °C. • Mutant Q7I/A106P plus TurboPETase can synergistically degrade PET. Polyethylene terephthalate (PET) plastic waste poses significant environmental challenges. Enzymatic degradation of PET offers a sustainable recycling route, but intermediate products such as bis (2-hydroxyethyl) terephthalate (BHET) and mono (2-hydroxyethyl) terephthalate (MHET) can inhibit further hydrolysis. A bis (2-hydroxyethyl) terephthalate hydrolase (BHETase_B) from Stenotrophomonas pavanii exhibited hydrolytic activity toward both BHET and MHET, but its poor thermal stability limited industrial application. In this study, a combination of rational design and directed evolution strategies were employed to generate BHETase_B mutants, all of which exhibited significantly improved thermal stability with half-lives greater than 5.0 h at 50 °C, compared to the wild type (<1.5 h). Notably, mutant Q7I/A106P demonstrated a 7.6 °C increase in melting temperature (Tm = 52.5 °C for the wild type) and achieved complete degradation of BHET within 2 h at 50 °C. Mutant Q7I/A106P alone showed limited activity toward solid PET, but significantly enhanced product release when combined with PET hydrolases, especially TurboPETase, releasing 206.1 μM of products, a 1.4-fold increase compared with TurboPETase alone. These findings highlighted the potential of the engineered BHETase_B mutant as a downstream auxiliary enzyme for improving cascade PET degradation. Download: Download high-res image (155KB) Download: Download full-size image