Study On The Luciferase Stability In ATP-Bioluminescence

Currently, the main method for detecting microbial amount are plate count, ATP bioluminescent method, automated immunoassay, nucleic acid detection et al. ATP-bioluminescence has been paid much attention at home and aboard and widely applied in many fields, since it is simple, highly sensitive and rapid detection. ATP-bioluminescence is made of the bioluminescent reaction of luciferin and ATP, catalyzed by the luciferase. However, the luciferase performs poor stability and needs for long-term cryopreservation, which greatly limits its application. So it is necessary to carry out the study on improving the luciferase stability. In this paper, the main contents are as follows:(1) To build an optimal bioluminescent system, some important factors such as the suitable buffer and pH, reaction temperature, the background processing time, the concentration and the extracting time of ATP extractor were studied. The results showed that, the appropriate buffer and its pH were Glycylglycine and7.8; the luciferase activity was not significant reduction at room temperature (23℃) for2h; the new enzyme solution should be placed30min to reduce the background luminescence; the optimum concentration and extraction time of BAB were0.05%and3min.(2) The correlation between ATP bioluminescence and plate count method was investigated by choosing the laboratory stains(E.coli and S.aureus). The experimental results showed that there was significant relativity between two methods in microbial dection; the correlation coefficient R2reached above0.95. The detection limit of OD6oonm of E.coli and S.aureus were3.0×10-6and3.0×10-5; the corresponding colony number were2300cfu/mL and3200cfu/mL respectively. In addtion, the calculated ATP contents of E.coli and S.aureus were1.75x10-18mol/cfu and1.16x10-18mol/cfu.(3)The glassy state technique was adopted to conduct the research about improving the luciferase stability. The results showed that the best protectant formula for luciferase vitrification was0.4mol/mL trehalose,1000mg/L BSA and800mg/L DEAE-Dx and then the relative luminous intensity of glassy luciferase reached94.7%. The vacuum pump was choosed as the glassy apparatus of luciferase. The glassy enzyme was more stable under low temperature, However, the residual enzyme activities were65%and4%respectively when preserved under normal temperature and high temperature for30days, besides, the measurable limits of ATP concentration were decreased by1and3order of magnitude respectively. It was necessary to explore a better protectant formula to enhance the luciferase stability under normal and high temperature. The activity of glassy enzyme stored for18d at room temperature was superior to that of kit luciferase preserved at-20℃and both of the detection sensitivity were the same, illustrating that the glassy enzyme was much stable.