Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride

Abstract

ObjectivesFire y luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of re y luciferase in the presence of sodium chloride. ResultsWe rst obtained two mutant luciferase enzymes whose inhibition were alleviated and identi ed these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

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“…Disinfectants (sodium hypochlorite, ethanol, and benzalkonium chloride) [16] Up to 93% inhibition of luciferase Salts in seawater [34] Inhibition of the luciferase reaction NaCl [60] Up to 79% inhibition of luciferase activity at 140 mM NaCl Extractants (Triton X-100 and dodecyltrimethylammonium bromide) [36] Strong inhibition of the luciferin-luciferase reaction…”

“…Mutant forms of luciferase resistant to inhibition by sodium chloride were specially obtained in other work [60]. These enzymes at 140 mM NaCl maintained their activity at 67 and 79% of the initial level, respectively, whereas the enzyme without mutations had only 44% of its initial activity.…”