Purification and biochemical properties of phospholipase D (PLD57) produced by Streptomyces sp. CS-57.
ABSTRACT 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.
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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 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.Archives of Pharmacal Research 10/2009; 32(10):1461-7. · 1.54 Impact Factor
- Biotechnology and Bioprocess Engineering 04/2010; 15:595. · 1.28 Impact Factor
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ABSTRACT: A novel metal ion-independent phospholipase B (PLB684 ) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6,330 U/mg-protein). The enzyme functions as a monomer with a molecular weight of 38.9 kDa. Maximum activity was found at pH 8.4 and 50 °C. The substrate specificity was in the following order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent Km , Vmax and kcat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 mM, 15.8 mmol min(-1) mg-protein(-1) and 1.02 × 10(4) s(-1) , respectively. The positional specificity of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine hydrolysis was investigated using gas chromatography. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1:sn-2) was 45:55. The open reading frame of the gene is 1,239 bp in length and codes for a 30-amino-acid signal peptide and a 382-amino-acid mature enzyme. The deduced amino acid sequence of PLB684 shows 60% identity to a putative uncharacterized SGNH esterase of Streptomyces auratus AGR0001 (UniProt accession no. J1RQY0). The extracellular production of PLB684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB684 and the active site may include residues Ser330 and His332. This article is protected by copyright. All rights reserved.FEBS Journal 06/2013; · 4.25 Impact Factor