Thermostabilization of firefly luciferase by a single amino acid substitution at position 217.

Random mutagenesis of the luciferase cDNA from "Genji" firefly, Luciola cruciata, was induced by hydroxylamine in an attempt to isolate thermostable mutants. Three mutants were isolated, and the cDNAs encoding these proteins were sequenced. All mutant cDNAs carried the same C to T transition mutation that conferred an amino acid substitution of Thr by Ile at position 217. The wild-type luciferase and the thermostable variant (Thr217Ile) were purified to homogeneity, and their enzymatic properties were determined. Thr217Ile was superior to wild-type in thermal and pH stability. Furthermore, the specific activity of the Thr217Ile mutant was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of luciferase, we replaced the Thr residue at position 217 with all of the rest of the possible amino acid residues by site-directed mutagenesis. The thermostability of these substitution mutants seemed to correlate with the hydrophobicity of the substituted amino acid residue.