Improvement of thermoalkaliphilic laccase (CtLac) by a directed evolution and application to lignin degradation

ABSTRACT: Thermoalkaliphilic laccase (CtLac) from the Caldalkalibacillus thermarum strain TA2.A1 has advantageous properties with potential industrial applications, such as high enzyme activity and stability at 70 °C and pH 8.0. In the present study, a directed evolution approach using a combination of random and site-directed mutagenesis was adopted to enhance the laccase activity of CtLac. Spectrophotometric assay and real-time oxygen measurement techniques were employed to compare and evaluate the enzyme activity among mutants. V243 was targeted for site-directed mutagenesis based on library screening. V243D showed a 25–35% higher laccase activity than wild-type CtLac in the spectrophotometric assay and oxygen consumption measurement results. V243D also showed higher catalytic efficiency than wild-type CtLac with decreased K(m) and increased k(cat) values. In addition, V243D enhanced oxidative degradation of the lignin model compound, guaiacylglycerol-β-guaiacyl ether (GGGE), by 10% and produced a 5–30% increase in high-value aldehydes than wild-type CtLac under optimal enzymatic conditions (i.e., 70 °C and pH 8.0). Considering the lack of protein structural information, we used the directed evolution approach to predict Val at the 243 position of CtLac as one of the critical amino acids contributing to the catalytic efficiency of the enzyme. Moreover, it found that the real-time oxygen measurement technique could overcome the limitations of the spectrophotometric assay, and apply to evaluate oxidase activity in mutagenesis research. KEY POINTS: • CtLac was engineered for enhanced laccase activity through directed evolution approach • V243D showed higher catalytic efficiency (k(cat)/K(m)) than wild-type CtLac • V243D produced higher amounts of high-value aldehydes from rice straw than wild-type CtLac SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-022-12311-4.