Karmacharya Jayram, Shrestha Prasansah, Han So-Ra, Lee Jun Hyuck, Oh Tae-Jin
Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University, Asan, 31460, Republic of Korea.
Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University, Asan, 31460, Republic of Korea; Genome-based Bio-IT Convergence Institute, Asan, 31460, Republic of Korea.
Chemosphere. 2025 Mar;372:144137. doi: 10.1016/j.chemosphere.2025.144137. Epub 2025 Jan 22.
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.
漆酶在应对环境挑战方面具有特别的意义，例如降解三苯甲烷（TPM）染料，包括结晶紫（CV）和考马斯亮蓝（CBB），这些染料常用于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中蛋白质的可视化。然而，这些染料由于难以降解而带来了重大的环境问题，这使得从工业废水中去除它们成为一项重大挑战。为了解决这个问题，当前的研究调查了一种源自南极细菌分离株芽孢杆菌属PAMC28748的新型CotA漆酶对这些顽固染料进行脱色的潜力。CotA基因已成功克隆并表达，该酶在pH 3和50°C时表现出最佳活性，这有利于其最大催化性能。重组芽孢杆菌属PAMC28748 rBCLac在没有额外介体的情况下能有效使CBB脱色，而CV的降解则需要使用氧化还原介体ABTS。使用ABTS时，在0.35%浓度的CV孵育240分钟后，脱色率超过90%。通过分子对接研究的进一步调查表明，酶与染料分子之间的氢键和疏水相互作用对于有效降解至关重要，突出了该酶的特定相互作用机制。除了其催化效果外，该研究还通过超速离心和丙酮沉淀回收并重复使用ABTS和rBCLac，展示了rBCLac系统的实际潜力。尽管酶活性略有下降，但该过程在三个循环中保持了超过75%的效率，从而展示了该系统的可持续性和可重复使用性。这些发现共同表明，从极端南极环境中分离出的rBCLac作为去除含有持久性染料的工业废水的候选物具有很大的潜力，通过酶及其介体再利用还具有经济高效且可持续的水处理潜力。
