Engineering White-Rot Fungal Lignin Peroxidase for Industrial Application through Optimization of in vitro Refolding and Enhanced Heme–Protein Interaction

Abstract

Lignin degradation has garnered significant attention for its potential in biotechnological applications, particularly in the valorization of lignocellulosic biomass for achieving carbon neutrality. Lignin peroxidase from Phanerochaete chrysosporium (PcLiP), with its remarkable oxidative capabilities, is recognized as a key enzyme in lignin degradation. However, challenges such as low expression efficiency and poor stability have limited its industrial applications. This study focused on optimizing recombinant production and enhancing the stability of PcLiP to address these challenges. Refolding and following production processes from a recombinant insoluble expression of PcLiP01 (PcLiP isozyme 1) were refined to improve production yield. By refining refolding and dialysis processes, with a focus on minimizing inclusion bodies’ aggregation chance and optimizing further activation through controlled dialysis pH, an activity yield of 94.0 U l−1 was achieved, representing a 6.27-fold increase from the initial condition. From bioreactor cultivation, activity yields were improved up to 700 U l−1 with a specific activity of 28–30 U mg−1. Next, protein engineering was conducted to improve thermal stability by enhancing interactions between the enzyme and its heme cofactor. By ‘keystone cofactor heme-interaction approach’ to enhance ligand binding and stabilize lignin peroxidase, the melting temperature of PcLiP01 E40S/V181A was increased by 8.66℃, and its half-life at 60℃ was extended 29-fold compared to the wild type. Molecular dynamics simulations revealed the detailed mechanisms for stability improvement caused by enhanced heme-protein interactions. Additionally, a stability index derived from these simulations accurately predicted stabilizing mutations in other PcLiP isozymes. The constructed PcLiP01 mutant exhibited improved efficiency in milled wood lignin degradation at 40℃. Furthermore, to explore the industrial potential of a thermally stable PcLiP variant, the kinetics and kinetic thermostability of PcLiP isozymes were assessed under pH 5.5 and 37℃, mimicking skin conditions. As a result, PcLiP04 (PcLiP isozyme 4) was selected as a suitable candidate for melanin decolorization under skin-mimicking environments. In vitro melanin decolorization assays demonstrated that PcLiP04 exhibited 2.9-fold higher melanin decolorization efficiency than PcLiP01, supporting the potential of PcLiP04 and thermal stable PcLiP variants for skin-whitening and other industrial applications. These results suggest the feasibility of engineering lignin peroxidase for industrial and environmental applications, contributing to lignin bioconversion and enzyme-based technologies. Additionally, the molecular dynamics simulation results suggest the possibility of stabilization approaches for other cofactor-containing proteins using similar strategies.