Improving enzymatic delignification of lignocellulosic biomass through enzyme engineering

Introduction

Laccase and laccase-like multicopper oxidases can degrade lignin through oxidative cleavage. In addition to producing various phenolic compounds from lignin, this enzymatic reaction could potentially be used in lignocellulosic biomass pretreatment for isolating cellulosic materials essential for the biomass valorization. Although these enzymes play a major role in ecological lignin degradation, low activity of native enzymes is considered as a major bottleneck towards enzyme-assisted lignin/biomass valorization at industrial scale. Improving catalytic activity of these enzymes through enzyme engineering could potentially address this problem.

Methods & Results

Two previously reported bacterial laccases were recombinantly produced in E. coli, and their activity was confirmed on a commercial lignin. Potential engineering sites were identified by analysing their substrate binding pockets through docking a lignin model compound. Site saturation libraries were generated by mutating the selected residues in the binding pockets. Multiple single mutants were identified with higher enzymatic activity compared to the wild type enzymes. Various single mutants with higher activity were combined to generate double and triple mutants which showed improved enzyme activity. The enzyme activity was further improved through hotspot mutation of the residues away from the substrate binding pocket. Activity of selected enzymes was confirmed on lignin model compounds, commercial lignin, and lignocellulosic materials. The enzymes were characterized for their pH and temperature profiles.

Conclusions

Catalytic activity of a laccase and a laccase-like multicopper oxidase was improved through semi-rational enzyme engineering approach. The engineered enzymes showed improved performance for degrading lignin model compounds and commercial lignin and could potentially be used in enzyme assisted lignin valorization and enzymatic delignification of lignocellulosic agrifood side-streams.