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

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-C_α bond, as well as inhibiting lignin repolymerization. This laccase-LPMO coupling system presents a new strategy for high-efficiency processing of lignin.