title: Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.
abstract: Industrial application of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) for lactulose synthesis is limited by low enzyme activity and formation of epilactose as by-product. After four sequential rounds of random mutagenesis and screening, an optimal mutant G4-C5 was obtained. Compared with wild type (WT) enzyme, mutant G4-C5 demonstrated 2.8- and 3.0-fold increases in specific activity and kcat/Km for lactulose production, respectively, without compromising thermostability. DNA sequencing of mutant G4-C5 revealed five amino acid substitutions, namely, R5M, I52V, A12S, K328I and F231L, which were located on the protein surface, except for the mutation I52V. The yield of lactulose catalyzed by mutant G4-C5 increased to approximately 76% with no obvious epilactose detected, indicating that mutant G4-C5 was more suitable for lactulose production than the WT enzyme.
authors.affiliation: Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 94710, USA.
authors.forename: Qiuyun
authors.initials: Q
authors.lastname: Shen
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 94710, USA.
authors.forename: Yuzhu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: yrj@jiangnan.edu.cn.
authors.forename: Ruijin
authors.initials: R
authors.lastname: Yang
authors.affiliation: Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Siyi
authors.initials: S
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Yuting
authors.initials: Y
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Han
mesh_terms: D002475:Cellobiose / Q000737:chemistry*; D006094:Gram-Positive Bacteria; D007536:Isomerism; D007792:Lactulose / Q000737:chemistry*; D019998:Racemases and Epimerases / Q000737:chemistry*
chemical_list: D002475:Cellobiose; D007792:Lactulose; D019998:Racemases and Epimerases
keywords: Activity improvement; Cellobiose 2-epimerase; Lactulose; Random mutagenesis