Application of supercritical carbon dioxide for preparation of starfish phospholipase A 2

Abstract

Pyloric ceca of starfish ( Asterina pectinifera) were treated by supercritical carbon dioxide (SCO 2) to remove the lipids. Then, phospholipase A 2 (SC-PLA 2) 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-PLA 2 was nearly homogeneous in SDS–PAGE and native-PAGE. The molecular weight of the SC-PLA 2 was estimated as approximately 20,000. N-terminal amino acid sequence of the SC-PLA 2 was SVYQF. Temperature and pH optimums of the SC-PLA 2 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 Ca 2+. The SC-PLA 2 was stimulated most by adding Ca 2+ followed by Mg 2+ and Co 2+, while it was strongly inhibited by adding Zn 2+ and EDTA. The SC-PLA 2 hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine. These characteristics of the SC-PLA 2 were the same as those of the starfish PLA 2 (CM-PLA 2) purified from the pyloric ceca defatted by chloroform–methanol (2:1, v/v) solution. Therefore, we concluded the SCO 2 defatting process is useful as a substitute for organic solvent defatting process.