Streptomyces Cs 684 균주로부터 Phospholipase D의 생산, 분리정제 및 특성분석

Abstract

Soil samples were screened for several actinomyces strains, that capable of producing phospholipase D, and it was revealed that a finally selected Streptomyces Cs 684 has a high phosholipase D activity. The production of phospholipase D from Streptomyces Cs 684 was carried out in GYT medium that contains glucose 2.0%, yeast extract 1.5%, tryotone 0.5%, calcium carbonate 0.1% at 28℃. 16S rDNA analysis showed that Streptomyces Cs 684 was highly homologous to Streptomyces jumonjinensis JCM 4947 and Streptomyces nanningensis YIM 33098. A extracellular phospholipase D was purified through several purification procedures such as ammonium sulfate fractionation, heat treatment, ultramembrane (YM10) filtration, sepharose CL-6B column chromatography and DEAE-sepharose CL-6B column chromatography. The purified phospholipase D was identified with a major band of 48KDa on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature for hydrolytic activity was 55℃. Most of enzyme activity was retained for 30min incubation at 55℃ without a significant loss in enzyme activity. The enzyme showed an optimal pH of 7.0 in hydrolytic reaction and was stable between pH 6.0-9.0. Tween 20 increased enzyme activity, but was inhibited by polyoxyethylene-4-laurylether, N-laurylsarcosine and SDS. The enzyme activity was a little affected by Ca+2 metal ion. .