Purification and characterization of lipase from Spirulina platensis

Abstract

A lipase from photosynthetic cyanobacterium Spirulina platensis ( Arthrospira) was purified by sequential operation of ammonium sulphate precipitation, dialysis, DEAE-Sepharose anion exchange chromatography, and Sepharose-6B gel filtration chromatography for the first time. This purification procedure resulted in 375-fold purification of lipase with 29.35% final yield. The purified lipase showed a prominent single band on SDS-PAGE. It is a monomeric protein of 45 kDa molecular weight and its isoelectric point is 5.9. The purified lipase exhibited maximal hydrolytic activity at a temperature of 45 °C and pH of 6.5. The values of K m and V max calculated from the Lineweaver–Burk plot using p-nitrophenyl palmitate ( p-NPP) as hydrolysis substrate were 0.02 mM and 38.9 μmol min −1 mg −1, respectively. The catalytic efficiency ( k cat/ K m) of purified lipase was determined as 1.5 × 10 6 M −1 s −1. The remaining activity of the lipase was about 95% of its original activity at 25 °C for 24 h of preincubation. However, the remaining activity was about 26% of the original activity at 45 °C for 24 h. The purified lipase appears to be unique since it cleaved triolein at only 3-position releasing 1,2-diolein. Lipase activity was stimulated by Ca 2+, Mg 2+, Zn 2+, Triton X-100 and SDS, and inhibited by Li +, Fe 2+, Mn 2+, EDTA and PMSF.