Surface Display and Engineering of Laccase CotA for Increased Growth of Pseudomonas putida on Lignin

Lignin is an underutilized raw material. Existing processes in which bacteria degrade lignin and produce added value products are not economically feasible yet due to limited lignin degradation. The aim of our study was to enhance microbial lignin degradation by surface-display of the bacterial laccase CotA from the thermophilic Bacillus coagulans. Enzyme engineering of surface-displayed CotA was used to increase its activity at 30 degrees C for the application at moderate temperatures. Libraries of CotA were created using error-prone PCR and site saturation mutagenesis. Two different substrates with distinct binding properties in CotA were utilized for activity screening to detect variants with enhanced activity regardless of substrate specificity. Combination of favorable mutations resulted in the variant CotA T260S/L385K/F416R which showed 1.95-fold and 13.4-fold activity of CotA with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP) as substrate, respectively. All mutations were located within the substrate binding site. The K-M-value of CotA T260S/L385K/F416R was increased with ABTS (2.08 mM versus 0.44 mM of ancestor CotA) and decreased with 2,6-DMP (0.31 mM versus 3.79 mM of ancestor CotA). Surface display of the identified variant CotA T260S/L385K/F416R on Pseudomonas putida enhanced its growth on lignin.