Exploiting CotA laccase from Antarctic Bacillus sp. PAMC28748 for efficient mediator-assisted dye decolorization and ABTS regeneration

Abstract

Laccases are of particular interest in addressing environmental challenges, such as the degradation of triphenylmethane (TPM) dyes, including crystal violet (CV) and Coomassie Brilliant Blue (CBB), which are commonly used in SDS-PAGE for protein visualization. However, these dyes present significant environmental concerns due to their resistance to degradation, which makes their removal from industrial wastewater a major challenge. To address this, the current study investigates the potential of a novel CotA laccase derived from Bacillus sp. PAMC28748, an Antarctic bacterial isolate, for decolorizing these stubborn dyes. The CotA gene was successfully cloned and expressed, and the enzyme demonstrated optimal activity at pH 3 and 50 °C, which favors its maximum catalytic performance. The recombinant Bacillus sp. PAMC28748 rBCLac effectively decolorized CBB without additional mediators, whereas the degradation of CV required the use of the redox mediator ABTS. With ABTS, over 90 % decolorization was achieved at a 0.35 % concentration of CV after 240 min of incubation. Further investigation through molecular docking studies revealed that hydrogen bonding and hydrophobic interactions between the enzyme and the dye molecules are critical for effective degradation, highlighting the enzyme's specific interaction mechanisms. In addition to its catalytic effectiveness, the study also demonstrated the practical potential of the rBCLac system by recovering and reusing both ABTS and rBCLac through ultracentrifugation and acetone precipitation. The process maintained over 75 % efficiency across three cycles, despite a slight decline in enzyme activity, thus showcasing the system's sustainability and reusability. These findings collectively suggest that rBCLac, isolated from an extreme Antarctic environment, holds considerable promise as a candidate for the removal of industrial wastewater containing persistent dyes, with the added potential for cost-effective and sustainable water treatment through the reuse of both the enzyme and its mediator.