Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6488097 | Enzyme and Microbial Technology | 2018 | 7 Pages |
Abstract
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 50â¯Â°C (t1/2, 50°C) increased from 1â¯min to 2.2â¯min, and the T5015 (temperature at which the activity of enzyme decreased by 50% in 15â¯min) increased from 44.8â¯Â°C to 47.2â¯Â°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 t1/2, 50°C and a 3.6â¯Â°C increase in T5015 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 t1/2, 50°C and T5015 increased to more than 768â¯min and 52.4â¯Â°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 t1/2,50°C increased to more than 2700â¯min while that of wild-type enzyme was only 1â¯min and T5015 was 9.8â¯Â°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.
Related Topics
Physical Sciences and Engineering
Chemical Engineering
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Authors
Ling Lin, Ying Wang, Mianbin Wu, Li Zhu, Lirong Yang, Jianping Lin,