Purification and biochemical properties of phospholipase D (PLD57) produced by Streptomyces sp. CS-57.
Streptomyces sp. CS-57, which was isolated from Korean soil, was found to produce phospholipase D (PLD57) as an extracellular enzyme when cultured in medium containing 2% glucose, 1.5% yeast extract, 0.5% trypton, and 0.1% calcium carbonate at 28 degrees C, and 160-rpm. PLD57 was purified using Sepharose CL-6B column chromatography, and DEAE-Sepharose CL-6B ion exchange column chromatography. The specific activity of the purified enzyme increased 6.7 fold with 3% recovery. The purified enzyme was then analyzed using 12% SDS-PAGE, which revealed that the molecular mass of the purified enzyme was 55 kDa. PLD57 showed both hydrolytic (H) and transphosphatidylation (T) activity, and the optimum temperatures of these activities were found to be 45 degrees C and 35 degrees C, respectively. Similarly, both of these activities were found to be optimal at a pH of 7.5. In addition, even after being heat treated at 45 degrees C for up to 2 h, the enzyme activity remained at 100%, and the H-activity was found to be stable at a pH of 6 to 8. Further, enzyme activity occurred in the presence of EDTA, indicating that metal ions are not required for their activity, although some metal ions did marginally increase the activity. Enzyme activity also increased by 75% in the presence of Triton-X 100 at a concentration of 0.375 %; however, none of the other detergents evaluated in this study were found to enhance enzyme activity.
Department of Pharmacy, College of Pharmacy, Chosun University, Gwangju 501-759, Korea.
Jaya Ram
JR
Simkhada
Seung Sik
SS
Cho
Hyo Jung
HJ
Lee
Jin Cheol
JC
Yoo
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D002614:Chelating Agents / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D003902:Detergents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007723:Korea; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D017830:Octoxynol / Q000737:chemistry; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D012988:Soil Microbiology; D013302:Streptomyces / Q000145:classification / Q000201:enzymology*; D013696:Temperature
D001426:Bacterial Proteins; D002614:Chelating Agents; D003902:Detergents; D008670:Metals; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C047264:sepharose CL 6B; C043982:DEAE-Sepharose CL-6B; D017830:Octoxynol; D012685:Sepharose; D004492:Edetic Acid; D010739:Phospholipase D