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Single-Vector Co-expression of petase and mhetase in E. coli Enhances PET Depolymerization Compared to Co-culture
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DOI：10.1007/s10924-026-03933-0.png)
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Abstract
En 中文
Polyethylene terephthalate (PET) is a widely used synthetic polymer and a persistent environmental pollutant due to its resistance to natural degradation. Polyethylene terephthalate hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis offer a promising enzymatic route for PET hydrolysis. In this study, petase and mhetase genes were cloned separately and together into pETDuet-1 vectors, enabling co-expression of genes in a single host. Purified PETase and MHETase were characterized using soluble substrates and PET films. PETase showed the highest apparent activity toward p-nitrophenyl butyrate, with activity decreasing with increasing acyl chain length. The tested metal ions did not enhance PETase activity, and MgCl2 caused the strongest inhibition. Recombinant cell-based PET hydrolysis was evaluated at different temperatures (20, 25, and 37 °C) and pH values (6–9), with the highest soluble product formation observed at 37 °C and pH 8. HPLC analysis showed that co-culture and single-vector co-expression increased terephthalic acid (TPA) formation compared with single-enzyme systems. The BHET/TPA molar ratio decreased from 0.195 in co-culture to 0.156 under co-expression, indicating improved intermediate conversion when both genes were expressed in the same host. FTIR, DSC, and SEM analyses supported surface-level PET modification, including changes in ester-associated bands, crystallinity, and localized erosion. Overall, single-vector co-expression of petase and mhetase improved soluble product distribution and intermediate turnover compared with co-culture.
Keywords:
Co-culture
Co-expression
MHETase PETase
Polyethylene Terephthalate Depolymerization
Single Vector
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