Abstract
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.
Highlights
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
The substrate would play the role of a mediator in the Laccase Mediator System (LMS) for brown coal biosolubilization
P-coumaric acid, sinapic acid (SA), fulvic acid and DMP were selected according to the best MOE docking score (Supplemental Table S1), economic and feasibility analysis of the use of such a compound in LMS (Fig. 1)
Summary
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. Biosolubilization of brown coal is a clean coal technology that aims at the conversion of the lignite to its cleaner form or to change its structure to gain new features[4,5]. Such solubilized material could be used as a source of value-added products[4]. S. cerevisiae strain enables easy genetic manipulations and convenient protocol for directed evolution of expressed proteins These features were used in laccase mutagenesis towards higher activity towards 2,6-dimethoxyphenol (DMP) which can act as laccase mediator in brown coal degradation. The impact of the laccase variants on solubilized coal was assessed
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