Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay.
- Author:
Weizong FENG
1
;
Junhan LIN
;
Shaoli CAI
;
Youtu ZOU
;
Guoren CHEN
;
Ping HUANG
;
Yajing LIN
;
Bingbing WANG
;
Lin LIN
Author Information
1. College of Life Science, Fujian Normal University, Fuzhou 350108, China.
- Publication Type:Journal Article
- MeSH:
Anilino Naphthalenesulfonates;
chemistry;
Enzyme Stability;
High-Throughput Screening Assays;
methods;
Hot Temperature;
Lipase;
metabolism;
Spectrometry, Fluorescence
- From:
Chinese Journal of Biotechnology
2011;27(4):584-591
- CountryChina
- Language:Chinese
-
Abstract:
We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-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 degrees C to 65 degrees 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.
