Location: Healthy Processed Foods ResearchTitle: Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis Author
|Shen, Qiuyun - Jiangnan University|
|Yang, Ruijin - Jiangnan University|
|Hua, Xio - Jiangnan University|
|Zhang, Wenbin - Jiangnan University|
|Zhao, Wei - Jiangnan University|
Submitted to: Journal of Molecular Catalysis B: Enzymatic
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2015
Publication Date: 7/22/2015
Citation: Shen, Q., Zhang, Y., Yang, R., Hua, X., Zhang, W., Zhao, W. 2015. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis. Journal of Molecular Catalysis B: Enzymatic. 120:158-164.
Interpretive Summary: Lactulose is a non-digestible disaccharide widely used as a food additive to improve taste and promote intestinal health. It is also used as a pharmaceutical product for the treatment of chronic constipation and hepatic encephalopathy. It is desirable to produce lactulose with a cleaner, enzymatic method rather than using the current chemical method for the isomerization of lactose under alkaline conditions. Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) is a good candidate enzyme but its activity and stability need to be improved for industrial applications. The best CsCE mutant obtained in this study have a half-life more than three times that of the wild type enzyme. These results can facilitate the development of highly stable CsCE for potential industrial applications.
Technical Abstract: Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (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 °C. Combinatorial mutations were subsequently introduced, and the superior mutant was double mutant E161D/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 °C to 87.5 °C, and catalytic efficiency (kcat/Km) for lactulose production was increased 29%. Moreover, this mutant E161D/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.