Engineering FAST-PETase with enhanced catalytic efficiency and acidic adaptability for sustainable PET waste recycling

This study presented a comprehensive protein engineering strategy to enhance the catalytic efficiency and environmental adaptability of FAST-PETase for PET plastic degradation. Through systematic site-directed mutagenesis of key basic residues (Arg84, Arg117, and Lys227) surrounding the active site, we identified R84D as the optimal single mutant, exhibiting 2.1-fold higher activity than FAST-PETase. Iterative saturation mutagenesis at 19 targeted positions yielded the quadruple mutant R84D/A203T/S230K/E198Q, demonstrating superior kinetic parameters: 3.3-fold higher catalytic efficiency (k_cat/K_m = 10.8 L·mmol^-1·min^-1) and significantly improved substrate affinity (K_m = 72.6 mM) at acidic conditions. Structural characterization revealed this mutant's optimized active site geometry, featuring enhanced hydrogen bonding and hydrophobic interactions. It exhibited broad pH adaptability (maintaining >80% activity at pH 7.59.0) with a 0.5-unit acidic shift in optimal pH (7.5 vs wild-type's 8.0), while retaining thermal stability up to 50°C. Molecular dynamics simulations confirmed the mutant's enhanced structural rigidity, with 3050% lower RMSD values compared to FAST-PETase. When tested in a two-module reactor system, the quintuple mutant R84D/A203T/S230K/E198Q/S239V enabled efficient PET conversion to TPA, achieving a space-time conversion rate of 3.51 g·(L·h)^-1. These results demonstrated its practical potential for industrial application. It established an effective framework for engineering plastic-degrading enzymes with enhanced performance under industrially relevant conditions.