Purification and Properties of Thermophili Phospholipase A₂ Activity from Pyrococcus horikoshii

　

Yoshitsugu KOSUGI^a, Anisur Rahman KHAN^b, Yong-Goe JOH^c, Katsuhiko HIGUCHI^a, Katsuhiko ISHIKAWA^a, Eriko MATSUI^a, Hiroyasu ISHIDA^a, Yaeko MASUCHI^d, and Ikuo MATSUI^a

　

^a National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba 305, Japan

^b Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh

^c University of Dong-A, Pusan 604-714, Korea

^d RCAST, The University of Tokyo, 4-6-1 Komaba, Meguro-ku,Tokyo 192 Japan

　

Phospholipase A₂ (PLA₂) has attracted much attention because of its use in degumming process in the oil refinery. Nowadays enzymatic degumming is carried out at 60-65ーC for 3-6 hours using PLA₂ from porcine pancreas and the degummed oil contains less than 8 ppm phosphorous. Therefore, the enzymatic degumming process led to a simple physical refining and savings in the bleaching step.

PLA₂ activity showing high reaction temperature was found in a hyperthermophilic archaea Pyrococcus horikoshii which was grown in anaerobic condition at 95℃. PLA₂ was found in the supernatant at 105,000 g which is extract of the membrane fraction. The extracted PLA₂ was applied on TSK gel Phenyl-5PW column. The active fraction was analyzed by SDS PAGE. The molecular weight of the main protein band was 43,000. NBD-PC (i.e.l-hexadecanoyl-2-(N-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino hexanoyl)-sn-glycero-3-phosphocholine) was used as substrate for PLA₂ activity. The substrate could not be hydrolyzed non-enzymatically even at 95℃. The optimum temperature for the PLA₂ activity was around 95-105℃. The optimum pH for the activity was 6.7-7.2. Dithiothreitol (1.8-20 mM) hardly inhibit the PLA₂ activity. PLA₂ fraction was not active at 95ーC on the triacylglycerol such as triolein. The PLA₂ activity was higher with phosphatidyl ethanolamine than phosphatidyl inositol and was more active with the phosphatidyl inositol than with phosphatidyl choline (PC). The activity was higher in β-ara active on β-dioleoyl PC than β-palmitoyl PC. On the other hand the activity with the β-palmitoyl PC was more active than β-1inoleoyl PC. The results of substrate specificity are corresponding result with cytosolic PLA₂ (1). However, archaeal core membrane lipids are diether and tetraether containing isopranoid chains attached glycerol. On the contrary eubacteria and eukaryotes are ester linked phospholipids. There has been no report that Pyrococcus has ester lipid like phosphophatidyl choline. Biological meaning of PLA₂ and the true substrate of the PLA₂ in the archaea are interest targets for future studies.

1. Clark, J.D., Lin, L.-L., Kriz, R.W., Ramesha, C.S., Suitzman, L.A., Lin, A.Y., Milona N., and, Knopf, G.L. ( 1991 ) Cell., 65, 1043-1051.

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