Background: In the bioluminescence phenomenon which has been observed in many organisms, a luciferase catalyzes the oxidation of luciferin to oxyluciferin and a visible light is produced. Light production of luciferase is one of the most sensitive tools in the detection of ATP for measuring microbial contamination, study of protein-protein interactions in cells and construction of biosensors and genetic bioreporters. In spite of these applications, the weakness of firefly luciferase is its rapid inactivation. Many studies have been done to develop thermostable luciferases by site directed mutagenesis. One of these modifications was LRR mutant in which the Leu300 was substituted with Arg in the E354RR356 Lampyris turkestanicus luciferase as template. LRR was more thermostable than the wild-type but with only 0.02% activity. Materials and Methods: In this research by using the method of site-directed mutagenesis, glutamate 270 was substituted with alanine in LRR mutant as template. The kinetics parameters of the mutated enzyme were determined and its structural characteristics were also investigated with fluorescence and CD spectroscopy. Results: The kinetics results revealed that the mutant 270 was more active than LRR. Furthermore the structural studies showed the significant changes in secondary and tertiary structures in the mutated enzyme in comparison with LRR and wild-type samples. Conclusion: The 270 mutant was more active than the LRR and it is recommended that this mutant be used in medical assays kits production.