高通量脂肪酶稳定性测定法-ANS法的建立

Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay

摘要:

根据蛋白质变性后结构变化的特点和荧光探针8-苯胺基-1-萘磺酸 (8-Anilino-1-naphthalenesulfonic acid，ANS) 的特性，建立了一种快捷而准确的脂肪酶热稳定性 (Tm) 测定的新方法——ANS脂肪酶稳定性测定法，即将脂肪酶在25 ℃~65 ℃的不同温度下保温30 min后，加入到ANS反应体系 (0.2 mg/mL酶蛋白，0.05 mmol/L ANS，20 mmol/L Tris-HCl (pH 7.2)，100~500 mmol/L NaCl) 中。在激发波长378

Abstract:

We have developed a rapid and high throughput lipase-ANS (8-Anilino-1-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal’s increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 °C to 65 °C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement.