Engineering Laccase from Bacillus pumilus for Improved Thermostability and Its Combination with Lytic Polysaccharide Monooxygenase for Lignin Depolymerization.

Abstract

Laccase from Bacillus is widely used to improve lignin depolymerization. However, wild-type laccase usually lacks heat resistance, and its catalytic process is usually accompanied by the repolymerization of lignin, limiting its application. Herein, we performed site-saturation mutation on Bacillus pumilus laccase, generating mutants A347H and N368L with activities increased by 2.37-fold and 2.46-fold, respectively. These mutants remain active at both 80 and 90 °C after 2 h. The above properties allowed them to efficiently catalyze native lignin depolymerization without mediators. Then, adding lytic polysaccharide monooxygenase (LPMO) and ascorbic acid further improved alkali lignin depolymerization, achieving a 40.87% depolymerization ratio. The insight into the product structure and reaction process suggested that the enhanced alkali lignin depolymerization was achieved by enhancing the cleavage of β-O-4 bond and C1-Cα bond, as well as inhibiting lignin repolymerization. This laccase-LPMO coupling system presents a new strategy for high-efficiency processing of lignin.