In conclusion, our studies indicate that mutagenesis of some hydrophobic residue to Arg in the flexible loop fairly provides the basis for loop anchoring with increasing local interactions that finally increases thermostability and optimum temperature of luciferases. These findings show the special role of surface loops in the stability of the enzyme. Moreover, structural studies with CD and fluorescence (intrinsic, ANS and quenching) show that increase of exposed Arg in the surface flexible loop and probability loop anchoring is responsible for more protein rigidity. The results show that surface loop anchoring with mutation of hydrophobic residue to Arg can increase stability of the firefly luciferase that can be confirmed the previous studies [55, 64]. The results show that loops have important roles in low thermal features of firefly luciferases. Red The red light transmitted through live tissue more efficiently than other wavelengths of visible light, thus by red-shifting emission of bioluminescent reporters, we may expand their utility for in vivo monitoring of biological processes. These findings are important as the red emitter thermostable mutant luciferases that described here may be useful for various biological applications.