Purificiation Of Cryptococcus Laurentii Lipase And Rapid Measurement Of Lipase Thermostability

As one of the most widely use industry enzyme, lipase (EC 3.1.1.3) was used in medicine, food, daily chemical industry and other industrial fields. The lipase from Cryptococcus laurentii B40, which was reported as a lipase, was purified by DEAE Sepharose fast flow ion exchange column. The purified protein shows a single band on SDS-PAGE with the molecular about 44.3KDa estimated by SDS-PAGE electrophoresis. The N-terminal sequence of it is D-F-G-P-I-T-I-Y-T-P-P-A. The optimum pH and temperature are 10.2 and 25℃respectively. The enzyme could catalyse triglyceride below 12 carbon and could activated by some reagents like Sodium Dehydrocholate, but inhibited by many heavy metal such as Hg2+Lipase is a widely used industry enzyme but usually with poor thermo-stability. One of the methods to improve the thermo-stability is generating mutations by mutagenesis or DNA Shuffling and selecting the useful mutants from a mutant library. It is necessary to develop a rapid method to measure the thermo-stability of lipases since the traditional reaction activity analysis. Here we develop a rapid and high throughput ANS Fluorescence signal measurement to evaluate the thermo-stability of the lipases:incubate lipase at 25-65℃for 30min, then combine 0.20mg/mL lipase,0.05mM 1,8-ANS (8-Anilino-l-naphthalenesulfonic acid, ANS) in the buffer of 20mM Tris-HCl, 100mM-500mM NaCl, pH7.2. Read fluorescence signal at EX 378nm, EM 465nm with fluorescence photometer or plate reader. Then calculate the Tm with GraphPad Prism5.0. We tested PEL (Penicillium expansum lipase, mutant PEL-ep8-K115R and PEL-ep8-K202A with above method and got the similar Tm value that measured by traditional method.