A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity.
An extracellular phospholipase D (PLD(St)) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD(St) was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca(2+), and the maximum activity was at 2 mM CaCl(2). The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 micromol min(-1) mg(-1), respectively. More importantly, PLD(St) could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD( St ) does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD(St) may therefore be a novel type of PLD enzyme.
Mander
Poonam
P
Department of Pharmacy, College of Pharmacy, Chosun University, Gwangju, Korea.
Simkhada
Jaya Ram
JR
Cho
Seung Sik
SS
Park
Sung Ju
SJ
Choi
Hong Seok
HS
Lee
Hei Chan
HC
Sohng
Jae Kyung
JK
Yoo
Jin Cheol
JC
D002122:Calcium Chloride / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000737:chemistry; D010739:Phospholipase D / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D010713:Phosphatidylcholines; D010739:Phospholipase D; D002122:Calcium Chloride