Expression, Purification And Application Of Firefly Luciferase And Saccharomyces Cerevislae Atp Sulfurylase In Escherichia Coli

Luciferase, a high-efficiency biocatalyst, is composed by a polypeptide chain. Under condition of existing of ATP, Mg2+, O2, the luciferase can catalysis luciferin to release green light. In this article, Photinus pyralis firefly luciferase gene is cloned into E. coli and mutated randomly but luciferase mutants with high tolerance to pH or temperature have not been got. By analysis of the structure of luciferase, we found that there are too many negatively charged amino acids around isoleucine232. After site-directed mutagenesis of isoleucine232into lysine, the luciferase gene was connected to the expression vector pET30a(+), and then the combination plasmid was transduced into the expression strain BL21(DE3). Finally, recombinant strain Q2/pET30a-luc and strain Q3/pET30a-luc-I232K which can highly express firefly luciferase were obtained. Luc and Luc(I232K) were purified by Ni-affinity chromatography and the specific activity is1.94×1012RLU/mg,4.68×1012RLU/mg respectively.In this article, the reaction kinetics curves of substrate luciferin and ATP to two luciferases are s shape. Mg2+and CoA can both activate the activity of two enzymes, and CoA can also extend the light-emitting time, the optimal concentration is10mM,20μM respectively. This experiment explored the effect of a couple of enzyme preservation solution for two firefly luciferases activity. The most appropriate protection condition of these two luciferases is2.0mM EDTA,1.0mM DTT,30μg/mL BSA. The concentration of (NH4)2SO4and TritonX-100for Luc is1mM,0.1%respectively. But Luc(I232K) can suffer higher concerntration of (NH4)2SO4or TritonX-100, which is2mM、0.15%respectively. When continued to increase the concerntration of these two materials, the activity of two enzymes would be decreased. The activity of Luc(I232K) is higher than Luc in pH5. The optimal temperature of Luc(I232K) is22-28℃, which is broader than Luc(22℃～25℃). When stored in-20℃,4℃,22℃, the enzyme survival rate is higher than Luc. When placed in40℃for5minutes, the enzyme survival rate of Luc (I232K) and Luc is 77%,8.6%respectively. And when placed in40℃for1hour, Luc will show no activity, while the enzyme survival rate of Luc (I232K) is10%.The purified Luc(I232K) was used in measuring ATP content of microbial cells. This article used quaternary ammonium surfactants benzalkonium chloride(BAC) to extract cellular ATP. When the concentration of BAC is less than0.03%, there is little interference in luciferase activity. The optimal BAC concentration for Vibrio cholerae, E. coli, Pseudomonas putida and Bacillus subtilis is0.02%,0.03%,0.02%,0.01%respectively. The optimal extracting ATP time for Vibrio cholerae, E.coli, Pseudomonas putida and Bacillus subrilis is2min,3min,3min,2min respectively. The experiment found that there is a good correlation between ATP concentration and luminous intensity. Also bacterial number and luminous intensity have a good correlation within a certain range. By measuring luminous intensity can calculate ATP content of one cell. The results are as follows:the ATP content of Vibrio cholerae is about0.24-0.45×10-18mol/cell; E.coli is about0.65-1.27×10-17mol/cell; Pseudomonas putida is about0.14-0.2×10-16mol/cell; Bacillus subtilis is about0.08-0.25×10-16mol/cell. At different growth stages the ATP content of these four tested bacteria are essentially the same. Also mutant enzyme Luc(I232K) was used in testing the effect of nano-material ZnO on Pseudomonas putida cell activity. The result is that though nano-material ZnO can promote biofilm formation, but it has no effect on cell activity. Maybe the detection limit of the instrument is not low enough and the ATP extracting agent benzalkonium chloride has inhibitory of the activity of luciferase Luc (I232K), resulting in sensitivity is not high enough. Another possible reason is that this approach can not be used in determination of trace change of ATP concerntration.The ATP sulfurylase gene met3was connected to the expression vector pET30a(+), and then the combination plasmid was transduced into the strain BL21(DE3). At last, recombinant strain Q4/pET30a-met which can highly express ATP sulfurylase was obtained. ATP sulfurylase was purified by Ni-affinity chromatography, and coupled with Luc (I232K) to detect the activity of ATP sulfurylase, and the specific activity is2.79×103U/mg. Another application of these two coupled enzymes is detecting the content of PPi which released in PCR reaction to evaluate the activity of DNA polymerase. Experiments show that DNA polymerase activity was decreased when placed in4℃and28℃for a month.