Details

Autor(en) / Beteiligte

• Shen, Qiuyun
• Zhang, Yuzhu
• Yang, Ruijin
• Hua, Xiao
• Zhang, Wenbin
• Zhao, Wei

Titel

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

Ist Teil von

• Journal of molecular catalysis. B, Enzymatic, 2015-10, Vol.120, p.158-164

Ort / Verlag

Elsevier B.V

Erscheinungsjahr

2015

Link zum Volltext

Quelle

Elsevier ScienceDirect Journals Complete

Beschreibungen/Notizen

• [Display omitted] •Semi-rational design was employed to enhance thermostability of CsCE.•Five beneficial mutations lead to increased thermostability of CsCE.•The t1/2 of mutant E161D/N365P increased to 4-fold of that of wild type enzyme.•Mutant E161D/N365P showed increased activity.•Mutant E161D/N365P was more stable against chemical denaturation. 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.