Sun Xuting, Lin Xiaofan, Xian Yufan, Zhang Faying, Zhu Lingxuan, Geng Haitao, Wang Wenya, Zhang Guimin
College of Life Science and Technology, Beijing University of Chemical Technology, No. 15 Beisanhuan Road East, Chaoyang District, Beijing, 100029, China.
State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology, Beijing, China.
Appl Biochem Biotechnol. 2025 May 9. doi: 10.1007/s12010-025-05240-1.
Laccases (benzenediol:oxygen oxidoreductases) are important multi-copper oxidases with widespread applications in industry. Here, Bacillus subtilis laccase CotA that has been widely studied was engineered to improve catalytic activity and thermostability via rational design. After iterative mutation of beneficial mutation sites, a triple mutant of CotA laccase (DTA) was obtained, whose catalytic activity and thermostability were improved by 2.7-fold and 1.4-fold compared with the wild-type (WT) CotA, respectively. The enhanced activity of DTA is primarily due to strengthened intermolecular forces in the active site, while its improved thermostability is attributed to increased hydrophobic residues, augmented protein surface flexibility, collectively rendering DTA a more active and stable enzyme with potential industrial applications. Compared to WT, DTA can degrade mycotoxins aflatoxin B1 (55.09% vs 44.51%) and ZEN (zearalenone) (71.59% vs 41.09%) more efficiently, and DTA can also better pretreat lignocellulose, promoting the hydrolysis of cellulose by cellulase. All these indicate that DTA has the potential to be used in industry.
漆酶（苯二酚：氧氧化还原酶）是重要的多铜氧化酶，在工业上有广泛应用。在此，通过合理设计对已被广泛研究的枯草芽孢杆菌漆酶CotA进行改造，以提高其催化活性和热稳定性。经过对有益突变位点的迭代突变，获得了CotA漆酶的三重突变体（DTA），其催化活性和热稳定性分别比野生型（WT）CotA提高了2.7倍和1.4倍。DTA活性增强主要归因于活性位点分子间作用力的增强，而其热稳定性提高则归因于疏水残基增加、蛋白质表面柔韧性增强，这些共同使DTA成为一种更具活性和稳定性的酶，具有潜在的工业应用价值。与WT相比，DTA能更有效地降解霉菌毒素黄曲霉毒素B1（55.09%对44.51%）和玉米赤霉烯酮（ZEN）（71.59%对41.09%），并且DTA还能更好地预处理木质纤维素，促进纤维素酶对纤维素的水解。所有这些表明DTA具有工业应用潜力。
