在酿酒酵母中表达进化的尖孢镰刀菌漆酶。

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924, Lodz, Poland.

Institute of Physics, Lodz University of Technology, Wólczańska 219, 90-924, Lodz, Poland.

Sci Rep. 2020 Feb 24;10(1):3244. doi: 10.1038/s41598-020-60204-1.

Fusarium oxysporum laccase was functionally expressed in Saccharomyces cerevisiae and engineered towards higher expression levels and higher reactivity towards 2,6-dimethoxyphenol, that could be used as a mediator for lignin modification. A combination of classical culture optimization and protein engineering led to around 30 times higher activity in the culture supernatant. The winner mutant exhibited three times lower Km, four times higher kcat and ten times higher catalytic efficiency than the parental enzyme. The strategy for laccase engineering was composed of a combination of random methods with a rational approach based on QM/MM MD studies of the enzyme complex with 2,6-dimethoxyphenol. Laccase mediator system with 2,6-dimethoxyphenol caused fulvic acids release from biosolubilized coal.

尖孢镰刀菌漆酶在酿酒酵母中实现了功能表达，并通过工程改造提高了其对 2,6-二甲氧基苯酚的表达水平和反应活性，2,6-二甲氧基苯酚可作为木质素修饰的介体。经典的培养优化与蛋白质工程相结合，使培养上清液中的酶活提高了约 30 倍。与亲本酶相比，优胜突变体的 Km 值降低了三分之一，kcat 值提高了四倍，催化效率提高了十倍。漆酶工程化的策略由随机方法与基于QM/MM MD 研究酶与 2,6-二甲氧基苯酚复合物的合理方法相结合组成。使用 2,6-二甲氧基苯酚的漆酶介体系统可从生物溶解煤中释放出富里酸。