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 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 degrees 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-PLA(2) was stimulated most by adding Ca2+ followed by Mg2+ and Co2+, while it was strongly inhibited by adding Zn2+ 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 SCO2 defatting process is useful as a substitute for organic solvent defatting process. (C) 2010 Elsevier Ltd. All rights reserved.