Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site‐directed mutagenesis

Abstract

Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.

Halomonas elongata: a microbial source of highly stable enzymes for applied biotechnology

“…Waste effluents contain many salts that hamper bioremediation processes with mesophilic microorganisms. In contrast, the azoreductase from H. elongata retained more than 50% of activity in presence of NaCl concentration as high as 50 g/L (Nakhaee et al 2018). Furthermore, the thermostability of the enzyme was enhanced by introducing a simple disulfide bond.…”

Editorial: Extremozymes: characteristics, structure, protein engineering and applications

“…Furthermore, advances in X-ray crystallography and cryo-electron microscopy are key for detailing extremozyme structures at atomic levels. Techniques like directed evolution and rational design have improved extremozymes' stability and efficiency (Nakhaee et al, 2018 ; Rigoldi et al, 2018 ; Liu et al, 2019 ). An interdisciplinary approach incorporating bioinformatics, synthetic biology, and materials science will further enhance the application of extremozymes in addressing global challenges.…”