Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance.
Luciola lateralis luciferase (LlL) was the most tolerant, followed by
Luciola cruciata luciferase (LcL) and
Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu
490
Lys mutation contributes to improved resistance to BAC. The corresponding Glu
490
Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L
490
K mutant, having both an Ala217Leu and a Glu
490
Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu
490
Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L
490
K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L
490
K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.
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