Efficiency and Stability Enhancement of Cis-epoxysuccinic Acid Hydrolase by Fusion with a Carbohydrate Binding Module and Immobilization onto Cellulose
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Cis-epoxysuccinic acid hydrolase (CESH) is an enzyme that catalyzes cis-epoxysuccinic acid to produce enantiomeric L(+)-tartaric acid. The production of tartaric acid by using CESH would be valuable in the chemical industry because of its high yield and selectivity, but the low stability of CESH hampers its application. To improve the stability of CESH, we fused five different carbohydrate-binding modules (CBMs) to CESH and immobilized the chimeric enzymes on cellulose. The effects of the fusion and immobilization on the activity, kinetics, and stability of CESH were compared. Activity measurements demonstrated that the fusion with CBMs and the immobilization on cellulose increased the pH and temperature adaptability of CESH. The chimeric enzymes showed significantly different enzyme kinetics parameters, among which the immobilized CBM30-CESH exhibited twofold catalytic efficiency compared with the native CESH. The half-life measurements indicated that the stability of the enzyme in its free form was slightly increased by the fusion with CBMs, whereas the immobilization on cellulose significantly increased the stability of the enzyme. The immobilized CBM30-CESH showed the longest half-life, which is more than five times the free native CESH half-life at 30 °C. Therefore, most CBMs can improve enzymatic properties, and CBM30 is the best fusion partner for CESH to improve both its enzymatic efficiency and its stability.
KeywordsCis-epoxysuccinic acid hydrolase Carbohydrate binding module Fusion enzyme Enzyme stability Affinity immobilization
We thank Zai-Kun Xu for help with the molecular cloning and Dr. Yifei Li for critical reading of the manuscript. This work is supported by the National Basic Research Program of China (973-project, grant no. 2011CB707404), the One Hundred Talented People Program (KSCX2-YW-G-066) of the Chinese Academy of Sciences, and the National Natural Science Foundation of China (grant no. 30970050).
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