Directed evolution of laccase from Thermus thermophilus HB27 reveals a variant with enhanced high temperature catalytic activity and electrochemical properties
Laccases belong to the multicopper oxidase family (MCO). Compared with fungal laccases, bacterial laccases have high stability and activity under extreme pH and temperature conditions. Because of these characteristics, bacterial laccases have become highly impactful in industrial processes. In the present work, we engineered the Thermus thermophilus HB27 laccase (Tth-Lac) by directed evolution. The library had a size of 1.2 x105 colonies, and we screened mutants by malachite green decolorization on solid medium. The results showed that Q36H mutant exhibited higher catalytic activity than Tth-laccase (WT) at 90 degrees Celsius. Both enzymes showed similar pH profiles, but the activity at 90 degrees Celsius of Q36H mutant was X-fold higher than that of the wild-type enzyme. In contrast, the kinetic constants were similar for both enzymes. In the voltammetric responses, at 61.5 degrees Celsius, the Q36H mutant showed increased current, and the T1 copper potential shifted by 100 mV relative to the tth-Lac. These results suggest that the Q36H mutation has high potential for industrial applications because replacing a glutamine (Q) with a histidine (H) at position 36 enhances the enzyme's structural stability, allowing it to function efficiently under extreme temperature conditions.