Abstract
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°C, and the enzyme was stable at or below 60°C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca2+, and the maximum activity was at 2 mM CaCl2. The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 µmol 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.
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References
Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254 (1976).
Carrea, G., D’arrigo, P., Piergianni, V., Roncaglio, S., Secundo, F., and Servi, S., Purification and properties of two phospholipases D from Streptomyces sp. Biochim. Biophys. Acta., 1255, 273–279 (1995).
Imamura, S. and Horiuti, Y., Enzymatic determination of phospholipase D activity with choline oxidase. J. Biochem., 83, 677–680 (1978).
Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685 (1970).
Leiros, I., Secundo, F., Zambonelli, C., Servi, S., and Hough, E., The first crystal structure of a phospholipase D. Structure, 8, 655–667 (2000).
Lerchner, A., Mansfeld, J., Schaffner, I., Schops, R., Beer, H. K., and Ulbrich-Hofmann, R., Two highly homologous phospholipase D isoenzymes from Papaver somniferum L. with different transphosphatidylation potential. Biochim. Biophys. Acta., 1737, 94–101 (2005).
Lineweaver, M. H. and Burk, D., The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56, 658–666 (1934).
Morris, A. J., Frohman, M. A., and Engebrecht, J., Measurement of phospholipase D activity. Anal. Biochem., 252, 1–9 (1997).
Ogino, C., Negi, Y., Daido, H., Kanemasu, M., Kondo, A., Kuroda, S., Tanizawa, K., Shimizu, N., and Fukuda, H., Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity. Biochim. Biophys. Acta., 1530, 23–31 (2001).
Qin, W., Pappan, K., and Wang, X., Molecular heterogeneity of phospholipase D (PLD): cloning of PLDγ and regulation of plant PLDγ, −β, and -α by polypohopsphoinositides and calcium. J. Biol. Chem., 272, 28267–28273 (1997).
Seong, C. N., Jo, J. S., Choi, S. K., Kim, S. W., Kim, S. J., Lee, O. H., Han, J. M., and Yoo, J. C., Production, purification, and characterization of a novel thermostable serine protease from soil isolate, Streptomyces tendae. Biotechnol. Lett., 26, 907–909 (2004).
Shimbo, K., Yano, H., and Miyamoto, Y., Purification and properties of phospholipase D from Streptomyces lydicus. Agric. Biol. Chem., 54, 1189–1193 (1990).
Simkhada, J. R., Cho, S. S., Lee, H. J., and Yoo, J. C., Purification and biochemical properties of phospholipase D (PLD57) produced by Streptomyces sp. CS-57. Arch. Pharm. Res., 30, 1302–1308 (2007).
Simkhada, J. R., Lee, H. J., Jang, S. Y., Kim, J. H., Lee, H. C., Sohng, J. K., and Yoo, J. C., A novel low molecular weight phospholipase D from Streptomyces sp. CS684. Bioresour. Technol., 100, 1388–1393 (2009a).
Simkhada, J. R., Lee, H. J., Jang, S. Y., Cho, S. S., Park, E. J., Sohng, J. K., and Yoo, J. C., A novel alkalo- and thermostable phospholipase D from Streptomyces olivochromogenes. Biotechnol. Lett., 31, 429–435 (2009b).
Ulbrich-Hofmann, R., Lerchner, A., Oblozinsky, M., and Bezakova, L., Phospholipase D and its application in biocatalysis. Biotechnol. Lett., 27, 535–544 (2005).
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Mander, P., Simkhada, J.R., Cho, S.S. et al. A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity. Arch. Pharm. Res. 32, 1461–1467 (2009). https://doi.org/10.1007/s12272-009-2017-0
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DOI: https://doi.org/10.1007/s12272-009-2017-0