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Issue 10, 2012

Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater

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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-β-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.

Graphical abstract: Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater

Supplementary files

Article information


Submitted
22 May 2012
Accepted
19 Jul 2012
First published
24 Jul 2012

Green Chem., 2012,14, 2719-2726
Article type
Paper

Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater

C. Lehmann, F. Sibilla, Z. Maugeri, W. R. Streit, P. Domínguez de María, R. Martinez and U. Schwaneberg, Green Chem., 2012, 14, 2719 DOI: 10.1039/C2GC35790A

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