High-yield production and stability enhancement of recombinant BhrPETase in Escherichia coli

Polyethylene terephthalate (PET) pollution poses a global environmental challenge, and enzymatic degradation represents a promising eco-friendly solution. The BhrPETase mutant Bhr6M demonstrated high degradation efficiency, showing considerable potential for PET biodegradation. To facilitate its practical application, we scaled up recombinant Bhr6M production in Escherichia coli via a 3-L bioreactor and identified optimal induction conditions of 30 °C and lactose feeding at 0.3 g·L^-1·h^-1. However, the enzyme solution exhibited limited stability, with a half-life (t_1/2) of approximately four days at 30 °C and four hours at 70 °C, hindering its broader application. To address this issue, we rationally designed via AlphaFold3, PROSS and combinatorial mutagenesis, obtaining the double mutant Bhr-S184H/M57I with synergistic performance improvements. This mutant achieved a yield of 3.17 g·L^-1 in approximately 31 h of fermentation, representing the highest productivity reported for PET hydrolases. Its stability was enhanced with a 66-fold longer t_1/2 at 70 °C than Bhr6M, and over 90% residual activity after 30 days at 30 °C. In addition, the mutant degraded 97% of amorphous PET at 70 °C within 48 h, a process that required 96 h for Bhr6M at its optimal temperature of 60 °C. After that, we further validated its practical utility by achieving over 95% degradation of pretreated PET bottles. Molecular dynamics (MD) simulations revealed the enhanced stability originates from new hydrogen-bonding networks and reduced conformational flexibility in key regions. This study provides a scalable strategy to simultaneously improve enzyme yield and stability, laying a foundation for industrial PET biodegradation.