Influence of natural deep eutectic solvents on stability and structure of cellulase

To address the issue of low stability and short half-life of cellulase, thermal stability and conformation change of Cellic® CTec2 were studied in various natural deep eutectic solvents (NADESs), with the aim of finding green and suitable solvents to enhance its stability, and thus facilitating utilization of lignocellulosic biomass. Fourteen NADESs (20%, v/v), with betaine or choline chloride (ChCl) as hydrogen bond acceptor (HBA), different polyols, amides or organic acids as hydrogen bond donor (HBD) were examined. When compared with the cellulase incubated in buffer at 50 °C, seven NADESs (20% in buffer, v/v) prolonged half-life time of the enzyme, among which betaine/1,4-butanediol and ChCl/1,4-butanediol (molar ratio 1:2) were the most prominent, with half-life time increased by about 11 and 9 folds, respectively, and >80% residual activity remained after 72 h. The influence of HBDs was more evident than HBAs in the aspect of cellulase stability, particularly HBDs of polyols (esp. diols) were better than amides, whereas HBDs of organic acids seemed to be destructive for cellulase. Associated conformational changes of cellulase during incubation in different solvents were also characterized with fluorescence spectrophotometer and circular dichroism spectrometer. The result demonstrated that cellulase in those NADESs enhancing its stability exhibited a higher fluorescence intensity, as well as minor spectrum intensity decreased over time, when comparing with that in buffer. Further investigation of NADESs concentration suggested that 10–20% (v/v) was optimal for stability enhancement, among which 15% and 20% (v/v) betaine/1,4-butanediol exhibited the best performance, evidenced by relative activity of about 450% after 48 h incubation at 50 °C, showing great promise in enhancing celluase stability.