The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.
Enzyme Microb Technol. 2022 Apr;155:109977. doi: 10.1016/j.enzmictec.2021.109977. Epub 2021 Dec 23.
Congo Red (CR) is a typical azo dye with highly toxic and carcinogenic properties. This study aimed to improve the decolorization activity of Bacillus pumilus W3 CotA-laccase for azo dye CR. This work analyzed the interaction between CotA-laccase and CR based on homology modeling and molecular docking. The three amino acids (Gly323, Thr377, Thr418) in the substrate-binding pocket were rationally modified through saturation mutation. Finally, the obtained multi-site mutants T377I/T418G and G323S/T377I/T418G decolorized 76.59% and 59.37% of CR within 24 h at pH 8.0 without a mediator, which were 3.15- and 2.44-fold higher than the wild-type CotA. The catalytic efficiency of the multi-site mutants T377I/T418G and G323S/T377I/T418G to CR were increased by 2.21- and 2.01-fold compared with the wild-type CotA, respectively. The mechanism of activity enhancement of mutants was proposed by structural analysis. This evidence suggests that the mutants T377I/T418G and G323S/T377I/T418G could be used as novel bioremediation tools.
刚果红（CR）是一种典型的偶氮染料，具有高毒性和致癌性。本研究旨在提高解偶氮染料刚果红（CR）的解聚活性。这项工作基于同源建模和分子对接分析了 CotA-漆酶与 CR 的相互作用。通过饱和突变合理修饰了底物结合口袋中的三个氨基酸（Gly323、Thr377、Thr418）。最终，获得的三突变体 T377I/T418G 和 G323S/T377I/T418G 在 24 h 内无需介体即可将 CR 脱色 76.59%和 59.37%，分别比野生型 CotA 提高了 3.15 倍和 2.44 倍。与野生型 CotA 相比，三突变体 T377I/T418G 和 G323S/T377I/T418G 对 CR 的催化效率分别提高了 2.21 倍和 2.01 倍。通过结构分析提出了提高突变体活性的机制。这一证据表明，突变体 T377I/T418G 和 G323S/T377I/T418G 可以用作新型生物修复工具。
