Production, purification and characterization of phospholipase D from Streptomyces sp. VN-3

Abstract

베트남 해안에서 분리된 VN-3는 Streptomyces cheonanensis (99.43%)과 Streptomyces spinoverrucosus (97.12%)에 가까운 계통발생을 보여졌다. 이에 16S rDNA 데이터를 통해 이 종은 Streptomyces sp. (Streptomyces cheonanensis)라 생각된다. 본 연구는 Streptomyces sp. VN-3로부터 Phospholipase D의 생산,정제 및 특성분석이 이루어졌다. 최적 배양 배지는 28oC 하에서 Glycerol 1.0%, Soybean 1.0%, CaCO3 0.1%라 보여진다. 배양액으로부터 생산된 Phospholipase D는 phosphotidylcholine의 hydrolytic activity뿐만 아니라 phosphatidycholine에 대해 transphosphotidlation activity까지 지니고 있다. 그 중에서도, 배양액의 hydrolytic activity는 1.97 units/mg의 활성이 나타났다. Phospholipase D 는 5-45%의 ammonium sulfate fractionation, ultra membrane (YM10) filtration, Sepharose CL-6B column chromatography와 Poros 20 HQ strong anion exchange column chromatography를 통해 정제 되었고 10% SDS-PAGE에서 58kDa의 밴드를 나타냈다. Hydrolytic activity의 경우 이 효소는 pH 8.0, 45 oC에서 최적의 반응을 나타내었다. 이 효소는 비교적 높은 온도와 알칼리 pH에서 안정함을 보였다. 효소의 활성을 증가시키기 위해 유화제와 금속이온은 각각 1.5% Triton X-100 과 Mn2+ (2 mM)을 사용하였다.|VN-3 strain, isolated from Vietnam sea showed a close phylogeny to Streptomyces cheonanensis (99.43%) and Streptomyces spinoverrucosus (97.12%). The 16S rDNA data suggested that this species might be Streptomyces cheonanensis. This study is an attempt to produce, purify and characterize phospholipase D from Streptomyces sp. VN3. The optimum culture medium was determined as glycerol 1.0%, soybean 1.0%, CaCO3 0.1% at 28°C. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphophatidylation activity on phosphotidylcholine. In particular, the culture broth showed 1.97-units/mg of hydrolytic activity. The phospholipase D was purified through ammonium sulfate fractionation (5-45%), ultra membrane (YM10) filtration, Sepharose CL-6B column chromatography and Poros 20 HQ strong anion exchange column chromatography, which produced a major band of 58 kDa on 10% SDS-PAGE. The enzyme showed an optimum pH of 8.0 and temperature of 45°C for hydrolytic activity. The enzyme was relatively stable at high temperature and alkaline pH. The detergent 1.5% Triton X-100 and the metal ion Mn2+ (2 mM) were found to be enhanced the enzyme activity.