Purification and Characterization of Phospholipase D from Streptomyces tendae

Abstract

초 록 Streptomyces tendae 에서 생산되는 Phospholipase D 의 정제 및 특성분석

Poonam Mander Advisor: Prof. Jin Cheol Yoo College of Pharmacy Graduate School of Chosun University

Streptomyces tendae는 한국 토양에서 분리한 방선균의 일종으로, extracellular phospholipase D 를 생산하는 균주이다. PLD를 생산을 위한 발효과정은 정제 및 특성분석과 함께 수행하였다.

최적배지 조건을 알아보기 위해 탄소원, 질소원, 금속이온을 다양한 농도에서 실험한 결과 2% sucrose, 2% soybean, 0.05 % CaCl2 , 0.1% CaCO3 배지 조성에서 최대의 PLD 활성을 나타내었다. 28°C 에서 5일간 배양 시 배양액 속에 hydrolytic activity가 3.7 U/ml 존재하였다. Sepharose CL-6B column chromatography 와 DEAE Sepharose CL-6B 이온 교환 column chromatography를 사용하여 정제된 효소는 10% SDS-PAGE에서 42.8kDa 하나의 밴드를 나타내었다. 효소의 최적 온도는 60°C 이고, 최적 pH는 pH8 이었다. 효소의 온도 안정성을 알아보기 위해 60°C에서 90분간 방치하였을 때 100% 안정함을 나타내었다. pH 8~10에서 안정한 것으로 보아 알칼리성 PLD임을 알 수 있었다. 계면활성제와 금속이온에 대한 영향을 살펴본 결과Triton X-100 와 Ca 2+.에 의해 효소의 활성이 증가하였다.|ABSTRACT Purification and Characterization of Phospholipase D from Streptomyces tendae

Poonam Mander Advisor: Prof. Jin Cheol Yoo College of Pharmacy Graduate School of Chosun University

Streptomyces tendae, a member of actinomycetes isolated from Korean soil, produced an extracellular phospholipase D. The fermentation process was carried out along with its purification and characterization. Various concentrations of carbon, nitrogen and mineral sources were supplied to the culture medium for media optimization. It was able to produce maximum PLD activity when cultured in a medium containing 2% sucrose, 2% soybean, 0.05 % CaCl2 and 0.1% CaCO3. During fermentation process most of the PLD was secreted during early growth phase. The culture broth exhibited 3.7 U/ml of hydrolytic activity when cultivated at 28°C for 5 days. The enzyme was purified using Sepharose CL-6B column chromatography followed by DEAE Sepharose CL-6B ion exchange column chromatography. The enzyme was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 42.8 kDa. Optimal activity was found at 60°C and pH 8. Additionally, the enzyme activity remained virtually unaltered when treated at 60°C for 90 min and was stable at pH range of 8-10, suggesting that the enzyme is thermostable and slightly pH stable in alkaline range. Further, the enzyme activity was found to be enhanced by 1.5% Triton X-100 and 2 mM Ca 2+. This work presents the purification and characterization of an enzyme from Streptomyces tendae.