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Enzyme Microb Technol. 2018 Aug;115:45-51. doi: 10.1016/j.enzmictec.2018.04.010. Epub 2018 Apr 25.

Enhancing the thermostability of fumarase C from Corynebacterium glutamicum via molecular modification.

Author information

1
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
2
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China. Electronic address: linjp@zju.edu.cn.

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 50 °C (t1/2, 50°C) increased from 1 min to 2.2 min, and the T5015 (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 t1/2, 50°C and a 3.6 °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 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 t1/2,50°C increased to more than 2700 min while that of wild-type enzyme was only 1 min and T5015 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.

KEYWORDS:

Directed evolution; Fumarase; Saturated mutagenesis; Site-directed mutagenesis; Thermostability

PMID:
29859602
DOI:
10.1016/j.enzmictec.2018.04.010
[Indexed for MEDLINE]

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