Abstract
Abstract
Laccases belong to the multicopper oxidase family (MCO). Compared with fungal lac-cases, 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 x10
5
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 °C. Both enzymes showed similar pH profiles, but the activity at 90 °C 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 °C, 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.