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

作　　者：冯蔚宗[1] 林俊涵[1] 菜少丽[1] 邹有土[1] 陈国仁[1] 黄平[1] 林雅静[1] 王冰冰[1] 林琳[1] 

机构地区：[1]福建师范大学生命科学学院,福州350108

出　　处：《生物工程学报》2011年第4期584-591,共8页Chinese Journal of Biotechnology

基　　金：工业微生物高通量选育与保藏技术研发平台建设(No.2006H0085);福建省自然科学基金(Nos.C0410009;B0120001);国家自然科学基金(No.30270033)资助~~

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 nm,发射波长465 nm下测定荧光值。通过GraphPad Prism软件分析得出脂肪酶的Tm。利用该方法测定了野生型扩展青霉脂肪酶及其突变体的Tm值,结果与传统的NaOH滴定和平板透明圈法的测定结果相似。同时,利用该方法可在96孔板上快速完成数十个样品的Tm测定,从而成为一种快速、可靠、可用于高通量的酶稳定性的测定方法。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.

关 键 词：脂肪酶 热稳定性 8-苯胺基-1-萘磺酸(ANS) 

分 类 号：Q814[生物学—生物工程]