Application of supercritical carbon dioxide for preparation of starfish phospholipase A2

Pyloric ceca of starfish (Asterina pectinifera) were treated by supercritical carbon dioxide (SCO2) to remove the lipids. Then, phospholipase A2 (SC-PLA2) was extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200, DEAE-cellulose, and Sephadex G-50. The purified SC-PLA2 was nearly homogeneous in SDS–PAGE and native-PAGE. The molecular weight of the SC-PLA2 was estimated as approximately 20,000. N-terminal amino acid sequence of the SC-PLA2 was SVYQF. Temperature and pH optimums of the SC-PLA2 were at around 50 °C and pH 9.0, respectively, and the enzyme activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca2+. The SC-PLA2 was stimulated most by adding Ca2+ followed by Mg2+ and Co2+, while it was strongly inhibited by adding Zn2+ and EDTA. The SC-PLA2 hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine. These characteristics of the SC-PLA2 were the same as those of the starfish PLA2 (CM-PLA2) purified from the pyloric ceca defatted by chloroform–methanol (2:1, v/v) solution. Therefore, we concluded the SCO2 defatting process is useful as a substitute for organic solvent defatting process.