Streptomyces Cs 528 균주로부터 Phospholipase D의 생산, 분리정제 및 특성분석

Abstract

Soil samples were screened for actinomyces strains that capable of producing phospholipase D, and Streptomyces Cs 528 showing a high phosholipase D activity was isolated. Streptomyces Cs 528 produced phospholipase D when grown in GCS medium containing glycerol 1.0%, soybean meal 1.0%, CaCO₃ 0.1% at 28℃. Analysis of the 16S rDNA showed that the strain Cs 528 was highly homologous to Streptomyces mirabilis DSM 40553T and Streptomyces olivochromogenes DSM 40451T. One form of extracellular phospholipase D was purified through ammonium sulfate fractionation, heat treatment, ultramembrane (YM10) filtration, sepharose CL-6B column chromatography, DEAE-sepharose CL-6B column chromatography, sephadex G-75 column chromatography, which produced a major band of 56KDa on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical properties of purified phospholipase D were examined. The optimal temperature for hydrolytic activity was 60℃. About 100% of enzyme activity kept at 60℃without any loss for 30min. The enzyme showed an optimal pH of 8.0 in hydrolytic reaction and was stable between pH 7.5-10. Triton X-100 increased enzyme activity, but activity of enzyme was inhibited by N-laurylsarcosine and SDS. The enzyme activity was enhanced in the presence of 2mM metal ions namely Ca^(2+) and activity also inhibited in the presence of 2mM metal ions, such as Co^(2+), Zn^(2+), EDTA.