Enhanced Enzyme Activity of Thermoalkaliphilic Laccase (CtLac) of Caldalkalibacillus thermarum Strain TA2.A1 by Random and Site-directed Mutagenesis and Its Application to Lignin Degradation

Abstract Background: Thermoalkaliphilic laccase, CtLac, has advantageous properties such as high enzyme activity and stability at 70℃ and pH 8.0 in the presence of organic solvents and surfactants, which confers a potential industrial application. Moreover, CtLac depolymerized lignin and produced high-value benzaldehyde chemicals such as vanillin. In the present study, the directed evolution approach using the combination of random and site-directed mutagenesis was performed to enhance the laccase activity of CtLac. Two methods of spectrophotometric assay and real-time oxygen measurement were employed for comparing and evaluating laccase activity among mutants. Results: Random mutagenesis was carried out by error-prone PCR to introduce 6-8 mutations per 1000 bps of CtLac coding gene. Among the 1,300 tested transformants, RM484 with higher laccase activity was selected, and V243 was targeted for site-directed mutagenesis. V243D showed 35% and 25% higher laccase activity than wild-type CtLac from the spectrophotometric assay and the oxygen consumption measurement, respectively. Besides, V243D showed higher catalytic efficiency with the decreased Km and the increased kcat value than wild-type CtLac. Furthermore, V243D showed the enhanced oxidative degradation of lignin under the optimal enzymatic conditions, 70℃ and pH 8.0, with 10% higher GGGE oxidation and 50-95% increased high-value aldehyde production than wild-type CtLac.Conclusions: CtLac was engineered for improved laccase activity through the directed evolution approach involving random and site-directed mutagenesis in the lack of protein structural information. Considering the limitation of the spectrophotometric assay, the real-time measurement of oxygen consumption could be a more accurate method to evaluate oxidation of laccase activity in mutagenesis research.