A Study On The Properties And Applications Of Firefly Luciferase

Firefly can give rise to luminescence during the night, the basic reaction involved the firefly luciferase(FL) and luciferin and ATP in the body of firefly.The reaction function can be explained like this:The essential substrate of the reaction was ATP, and the intenstiy of the luminescence and the concentration of ATP possessed the positive relativity. Since the amount of ATP was relatively stable duing the whole life of the cell, so the existence of ATP can indicate the hygience and germ amount of the food and drinking during the curing process. This detection method was so fast that can be applied into controlling the safety during the food and drinking industry. Besides that, firefly luciferase gene have been a reporter gene to detect the level of the certain gene. Recently,firefly luciferase-luciferin bioluminescence system have been widely applied into the study on the metabolism, biosensor, environmental detection and drugs screening.The firefly luciferase-luciferin system in this study was extracted from Lucoila Chinesis L. first of all, the MW of the luciferase and purification of this luciferase was detected by the SDS-PAGE and HPLC analysing. In order to explore the optimum for the ATP detection, the dynamic property,the best buffer solution system ,intensity of ions ,temperature and pH value were explored. The effect from several metal ions and EDTA on the activity of the enzyme were studied. Besides, the stability of the firefly luciferase duing the low and normal temperature and the effect of light on it were also studied in order to select a good method to keep the stability of this enzyme.Base on the reaction environment, several enhancer and inhibitor were studied on the firefly luciferase,such as DTT, GSH,BSA,DEAE-Dx and CoA.The results showed that ,the optimum buffer solution system was HEPES buffer solution system (pH value 5.5), which was different from any other firefly luciferase system.(normally the optimum pH value is 7.6～7.8).The optimum temperature was 20 ℃. The MW of the enzyme protein was 64KD,which was a non characteristic Michaelis-Menten enzyme. The dynamic curve of ATP substrate was S shape. Unlike other firefly luciferase, there was no "red shift" phenomena with the decreasing of the pH value of the buffer solution and peak emission wavelenght is 570nm stablely.In normal temperature, the enzyme was easy to lose its activity and sensitive to light. The stability and activity can be enhanced by BSA,DTT and GSH. Some metal ions such as Ca²⁺,Mg²⁺ and Mn²⁺ also did the same thing. In a addition, DEAE-Dx and CoA did have positive effect on this enzyme.In this study, a sound reaction condition and standard operation process were formed and the property of this enzyme was dugged in. Since the different kind and different function and character, what have been done in this paper would shed a light on the study of the firefly luciferase.