Characterization and rational engineering of a novel laccase from Geobacillus thermocatenulatus M17 for improved lignin degradation activity.

Lignin, with its complex, high-molecular-weight aromatic polymer structure and stable ether or ester bonds, greatly impedes the efficient degradation of lignocellulosic waste. Bacterial laccases have gained attention for their potential in lignocellulosic waste degradation due to their resilience in extreme conditions and ability to be produced in large quantities. In this study, a novel laccase from Geobacillus thermocatenulatus M17 was identified and expressed in E. coli BL21 (DE3). The enzymatic properties of this M17 laccase, including its tolerance to pH, temperature, metal ions, inhibitors, and organic solvents, were thoroughly investigated. The M17 laccase demonstrated optimal activity at pH 3-6 and at temperatures of 50-60 °C, with Co enhancing its activity over Cu, and exhibited strong resistance to organic solvents. Further optimization through mutagenesis led to the engineered D217K variant. The efficiency of the engineered laccase was validated with alkali lignin and various sources of plant biomass. The degradation rate of D217K variant for alkali lignin increased significantly, rising from 66.33 % to 83.27 %. Additionally, for high-lignin-content biomass, the degradation rates improved as follows: wheat stover increased from 7.63 % to 10.29 %, switchgrass from 6.02 % to 7.00 %, and corn stalk from 4.51 % to 6.59 %. In conclusion, this study identified a new bacterial laccase and further enhanced its activity through rational engineering, suggesting its promising application in plant biomass degradation.