Fumarases have been successfully applied in industry for the production of l-malate. However, the industrialization of fumarases is limited by their low thermostability. In this study, the thermostability of fumarase C from Corynebacterium glutamicum was enhanced through directed evolution, simulated mutagenesis, site-directed mutagenesis and saturated mutagenesis. Mutant 2G (A411V) was initially constructed through directed evolution. Its half-life at 50�°C (t 1/2, 50°C) increased from 1�min to 2.2�min, and the T 50 15 (temperature at which the activity of enzyme decreased by 50% in 15�min) increased from 44.8�°C to 47.2�°C. Besides, several different mutants were obtained by site-directed mutation. Among them, mutant 3G (A227V) showed significant improvement in thermostability with a 3.3-fold improvement of t 1/2, 50°C and a 3.6�°C increase in T 50 15 compared to the wild-type enzyme. Then, 2/3G (A227V, A411V) was obtained by combining the mutant 2G with the mutant 3G, for which the t 1/2, 50°C and T 50 15 increased to more than 768�min and 52.4�°C, respectively. Finally, site-saturated mutagenesis was employed on amino acid residues 175-Glu, 228-Gly, 297-Gly, 320-Lys and 464-Glu to maximize the thermostability of mutant 2/3G. The most thermostable mutant 175G with amino acid substitutions (A227V, A411V, E175K) was isolated. Its t 1/2,50°C increased to more than 2700�min while that of wild-type enzyme was only 1�min and T 50 15 was 9.8�°C higher than the wild-type enzyme. The thermostable mutated enzymes generated without affecting the activity in this study would be an attractive candidate for industrial applications.

» Author: Xiaoyi Li, Yan Xiao, Yingang Feng, Bin Li, Wenli Li, Qiu Cui

» Reference: Enzyme and Microbial Technology, Volume 114

» Publication Date: 01/08/2018

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