Thermophilic phospholipase A2 in the cytosolic fraction from the archaeon Pyrococcus horikoshii |
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Authors: | Yan Feng Yong-Goe Joh Kazuhiko Ishikawa Hiroyasu Ishida Susumu Ando Tohru Yamagaki Hiroshi Nakanishi Shugui Cao Ikuo Matsui Yoshitsugu Kosugi |
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Affiliation: | (1) National Institute of Bioscience and Human-Technology, 305-8566 Tsukuba, Japan;(2) Key Laboratory of Molecular Enzymology and Engineering, Jilin University, Changchun, China;(3) Department of Food Science & Nutrition, University of Dong-A, 604-714 Pusan, Korea;(4) Department of Chemistry, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan |
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Abstract: | Hyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A2 in a cytosolic fraction. The enzyme displayed optimal activity at 90°C and pH 7 and preferentially hydrolyzed sn-2 fatty acids in the following order: linoleoyl> oleoyl>arachidoyl phosphatidylcholine. Phospholipase A2 had similar activities toward l-α-1-palmitoyl-2-arachidoyl derivatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A2 activity was unaffected by the addition of 0.0001–1 mM calcium, but was inhibited slightly by the addition of 2–10 mM calcium.
The activity was enhanced more than 5–18-fold in the presence of 3–20% (vol/vol) glycerol. The activity was unaffected by
the addition of 1–5 mM EDTA or 0.01–20 mM dithiothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544
disappeared upon incubation of the cytosolic fraction with total lipid. The phospholipase A2 was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using
10–15% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). No activity of phospholipase A2 activity was observed in the absence of proteins less than 19 kD in size, as fractionated by SDS-PAGE.
Portions of this article were presented at the Biocatalysis Symposium at the 91st Annual Meeting and Expo of the American
Oil Chemists’ Society in San Diego, CA, April, 2000. |
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Keywords: | Caldarchaetidic acid hyperthermophile phospholipase A2 Pyrococcus horikoshii thermostable enzyme |
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