Enhanced Catalytic Turnover and Robustness of Laccase from Trametes pubescens through Carrier-Free Aggregation

Abstract

Green biocatalysis has revolutionized industrial processing by replacing whole-cell systems with robust immobilized biocatalysts, significantly streamlining downstream purification. This study reports on the fabrication and biocatalytic characterization of carrier-free Crosslinked Enzyme Aggregates (CLEAs) using laccase produced by the white rot fungus Trametes pubescens through solid state fermentation. The resulting L-CLEAs exhibited superior thermal robustness and catalytic performance compared to the soluble laccase (SL). Kinetic analysis revealed that while the apparent substrate binding affinity (Km) increased 5.4 fold due to mass transfer limitations, this was also largely compensated for a 4.1 fold enhancement in the catalytic rate constant (kcat). Furthermore, L-CLEAs demonstrated a significant 2.2-fold reduction in activation energy (Ea) and an increased inactivation energy (Ed), indicating a fortified structural conformation. Thermal stability assays confirmed that L-CLEAs nearly doubled the enzyme's half-life (t1/2) at 60°C, extending it to 406 minutes. Moreover L-CLEAs also retained >80% activity after 20 consecutive cycles of use, demonstrating excellent operational durability and ease of recovery through simple centrifugation. These findings, coupled with exceptional reusability, position L-CLEAs as a highly efficient and stable hybrid system for the industrial degradation of phenolic compounds and other green chemistry applications.