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Fireflies fluoresce at night, its basic biochemical reactions can catalyze fluorescence luciferase factors oxide fluorescein and emit fluorescence following reaction equation, said: the luciferase ATP O 2 (?) oxidation fluorescein AMP PPi CO 2 light. This reaction must have the participation of ATP and its luminescence intensity and the concentration of ATP is a positive correlation within a certain concentration range. ATP widely exists in which the living cells and the content thereof is quite stable in normal cell life. ATP content can indicate the food as well as the number of micro-organisms and food processing environment pollution, so this method not only can be used for the rapid detection of food security, health conditions can also detect food-contact surfaces in order to strictly control the food the safety of the industrial production. In addition, the luciferase gene has been widely used as reporter gene, can be detected by this reaction in the gene expression level. Firefly luciferase system has been gradually applied to the cell's metabolic research, biosensors, environmental monitoring, drug screening, and other fields. This study selected extracted from China yellow firefly luciferin - luciferase system luminescent organs for the experimental material, first is the use of SDS-PAGE and HPLC analysis to calculate the difference of the molecular weight of the enzyme and several other fluorescein and purity. Suitable reaction conditions in order to establish the luciferase, and explore the kinetics of the enzymatic reaction curve characteristics, appropriate buffer species, ionic concentration, temperature, and the optimum pH value. Determine a reasonable buffer system. Several metal ions and EDTA (ethylene diamine tetra-acetic acid) activity also discussed. While the stability of luciferase in the low temperature and room temperature, the light affect its stability discussed, and select one of several methods to maintain the activity of the stabilizer and dark. After the establishment of the above-described basic reaction conditions, the present study also DTT (dithiothreitol), GSH (glutathione), BSA (bovine serum albumin), DEAE-Dx (diethylaminoethyl dextran) CoA (coenzyme A) several activator activity enhancement system. Some surfactants and inhibitors of luciferase to do a preliminary study. The results of this study show that the luciferase is more suitable for the reaction system is HEPES buffer solution, the optimum pH is 5.5, the majority of luciferase optimum pH value of 7.6 to 7.8, results are inconsistent with previous reports, show this The specificity of the enzyme. The optimum temperature is 20 ° C, the optimum ionic strength in the 10 to 100 mmol / L, molecular weight of about 64KD, is an atypical Michaelis-Menten's enzyme, ATP kinetic curves for the S-type on one of the substrates. The purity of the enzyme extract at 80%, with the decrease in pH of the buffer, to the luciferase reaction no red shift occurs, the wavelength of the emission spectra at the peak emission wavelength peak wavelength is always near at 570nm, this point and the nature of the North American firefly different. Room temperature the nature of the enzyme is unstable and easier to inactivation, light sensitive, and is easily oxidized in air. BSA, DTT and GSH can significantly protect and enhance the activity of the enzyme, metal ion Mg 2 of Mn 2 and the of Ca 2 enhance enzyme activity. DEAE-DX and CoA of the luminous intensity of the reaction can be improved. Most of the high concentrations of metal ions and detergents will reduce the activity of the enzyme, and even to make it completely inactivated. Through this research, the establishment of a more ideal firefly luciferase - the luciferase reaction system and method of operation. Due to the nature of the different types of luciferase certain differences, so the results of this study for better understanding and use of the firefly luciferase provides some useful information.
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