Design of thermostable luciferases through arginine saturation in solvent-exposed loops

In most bioluminescence systems the oxidation of luciferin and production of light is catalyzed by luciferases. Protein engineering studies have shown that thermostable proteins from thermophilic organisms have a higher frequency of Arg, especially in exposed states. To further clarify the arginine saturation effect on thermostability of firefly luciferase, some of hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase are changed to arginine. All of these residues are located at the surface loops of L. turkestanicus luciferase. Starting with a luciferase mutant (E(354)Q/Arg(356)), single mutation (-Q(35)R, -(IR)-R-182, -(IR)-R-232 and -(LR)-R-300), double mutation (-Q(35)R/(IR)-R-232) and triple mutation (-Q(35)R/(IR)-R-232/(IR)-R-182) are added. Bioluminescence emission spectra indicate that substitution of Arg by these residues, do not effect on the maximum wavelength of emission spectrum. It should be noted, introduction of double mutation (-Q(35)R/(IR)-R-232) and triple mutation (-Q(35)R/(IR)-R-232/I-182 R) were kept specific activity of firefly luciferase. By addition of positively charged residue, some specific mutations (-(IR)-R-232, -Q(35)R/(IR)-R-232 and -Q(35)R/(IR)-R-232/(IR)-R-182) showed that optimum temperature of activity was increased to 40 degrees C which are 12 and 15 degrees C higher than E(354)Q/Arg(356) and wild-type luciferases, respectively. Also, after 40 min incubation of enzymes at 40 degrees C, the relative remaining activity of wild type was only 5%, whereas for -(IR)-R-232, -Q(35)R/(IR)-R-232 and -Q(35)R/(IR)-R-232/(IR)-R-182 was 60, 80 and 80% of original activity, respectively.