Zhang Ruohan, Wang Yuchen, Wang Xiaolu, Luo Huiying, Wang Yuan, Yao Bin, Huang Huoqing, Tian Jian, Guan Feifei
State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Bioresour Bioprocess. 2025 May 27;12(1):47. doi: 10.1186/s40643-025-00893-6.
Laccases with high catalytic efficiency and high thermostability can drive a broader application scope. However, the structural distribution of key amino acids capable of significantly influencing the performance of laccases has not been explored in depth. Thirty laccase 13B22 mutants with changes in amino acids at distances of 5 Å (first shell), 5-8 Å (second shell), and 8-12 Å (third shell) from the active center were validated experimentally with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate. Twelve of these mutants (first shell, 1; second shell, 4; third shell, 7) showed higher catalytic efficiency than the wild-type enzyme. Mutants D511E and I88L-D511E showed 5.36- and 10.58-fold increases in k/K, respectively, with increases in optimal temperature of 15 °C and optimal pH from 7.0 to 8.0. Furthermore, both mutants exhibited greater thermostability compared to the wild-type, with increases of 3.33 °C and 5.06 °C in T and decreases of 0.39 J and 0.59 J in total structure energy, respectively. The D511E mutation resides in the third shell, while I88L is in the second shell, and their performance enhancements were attributed to alterations in the rigidity or flexibility of specific protein structural domains. Both mutants showed enhanced degradation efficiency for benzo[a]pyrene and zearalenone. These findings highlight the importance of the residues located far from the active center in the function of laccase (second shell and third shell), suggesting broader implications for enzyme optimization and biotechnological applications.
具有高催化效率和高热稳定性的漆酶能够推动更广泛的应用范围。然而，能够显著影响漆酶性能的关键氨基酸的结构分布尚未得到深入研究。以2,2'-连氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)为底物，对30个活性中心距离为5 Å（第一壳层）、5-8 Å（第二壳层）和8-12 Å（第三壳层）处氨基酸发生变化的漆酶13B22突变体进行了实验验证。其中12个突变体（第一壳层1个、第二壳层4个、第三壳层7个）表现出比野生型酶更高的催化效率。突变体D511E和I88L-D511E的k/K分别增加了5.36倍和10.58倍，最佳温度升高了15 °C，最佳pH从7.0提高到8.0。此外，与野生型相比，这两个突变体均表现出更高的热稳定性，T分别增加了3.33 °C和5.06 °C，总结构能分别降低了0.39 J和0.59 J。D511E突变位于第三壳层，而I88L位于第二壳层，它们性能的增强归因于特定蛋白质结构域刚性或灵活性的改变。这两个突变体对苯并[a]芘和玉米赤霉烯酮的降解效率均有所提高。这些发现突出了远离活性中心的残基在漆酶功能（第二壳层和第三壳层）中的重要性，表明对酶的优化和生物技术应用具有更广泛的意义。
