Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolydcus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE. A total of eight single-site mutants were designed, and five of them showed prolonged half-life of inactivation at 80 degrees C. Combinatorial mutations were subsequently introduced, and the superior mutant was double mutant El 61D/N365P. The half-life was approximately 4-fold higher than that of the wild type enzyme. In addition, the reaction temperature for maximum activity increased from 80 degrees C to 87.5 degrees C, and catalytic efficiency (k(cat)/K-m) for lactulose production was increased 29%. Moreover, this mutant El 61D/N365P was more stable against chemical denaturation and showed also a broader pH profile. The second most stable variant were mutant E161D/S180P/S351G with a 3.3-fold increase in half-life. These results provided new insights into the thermostability of CsCE and suggested further potential industrial applications. (C) 2015 Elsevier B.V. All rights reserved.