Abstract

Cellulases are promising catalysts for the depolymerization of cellulose under mild conditions. Reengineered cellulases are required to match application demands in biorefineries and to avoid cost-intensive downstream processing. This manuscript provides a novel fluorescence-based high throughput screening method for directed evolution of cellulases, based on 4-methylumbelliferyl-beta-D-cellobioside (4-MUC). The 4-MUC high throughput screening system was successfully employed to identify CelA2 variants with enhanced stability and activity in mixtures of water with deep eutectic solvents like choline chloride : glycerol (ChCl : Gly), and seawater. The cellulase variant 4D1 (L21P; L184Q; H288R; K299I; D330G; N442D) was isolated and showed, compared to wild type, an increase in specific activity in 30% (v/v) ChCl : Gly (7.5-fold; 0.4 to 3.0 U mg(-1)) and in concentrated seawater (1.6-fold; 5.5 to 9.3 U mg(-1)). In addition, the residual activity of 4D1 in the presence of 3-fold concentrated seawater is unaffected whereas CelA2 wild type loses >50% of its activity. Furthermore, the position H288 was identified as a key position for activity and resistance in 4D1.