Effects of 1,2-diacylglycerol and cholesterol on the hydrolysis activity of phospholipase D in egg-yolk phosphatidylcholine bilayers

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging toStreptomyces(73%) andMicromonospora(10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of theStreptomycesgenus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

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Detection of material-derived differences in the stiffness of egg yolk phosphatidylcholine-containing liposomes using atomic force microscopy

Chemistry and Physics of Lipids ◽

10.1016/j.chemphyslip.2020.104992 ◽

2020 ◽

Vol 233 ◽

pp. 104992

Author(s):

Yuki Takechi-Haraya ◽

Masaki Matsuoka ◽

Hirotaka Imai ◽

Kenichi Izutsu ◽

Kumiko Sakai-Kato

Keyword(s):

Atomic Force Microscopy ◽

Egg Yolk ◽

Force Microscopy ◽

Egg Yolk Phosphatidylcholine ◽

Atomic Force

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Hypochlorite-Induced Peroxidation of Egg Yolk Phosphatidylcholine is Mediated by Hydroperoxides

Free Radical Research ◽

10.3109/10715769709097832 ◽

1997 ◽

Vol 27 (1) ◽

pp. 1-12 ◽

Cited By ~ 17

Author(s):

O. M. Panasenko ◽

J. Arnhold ◽

Yu. A. Viadimirov ◽

K. Arnhold ◽

V. I. Sergienko

Keyword(s):

Egg Yolk ◽

Egg Yolk Phosphatidylcholine

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Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers

Biochemical Journal ◽

10.1042/bj3270133 ◽

1997 ◽

Vol 327 (1) ◽

pp. 133-138 ◽

Cited By ~ 33

Author(s):

Antonio CRUZ ◽

Cristina CASALS ◽

Kevin M. W. KEOUGH ◽

Jesús PÉREZ-GIL

Keyword(s):

Aqueous Phase ◽

Protein Interactions ◽

Pulmonary Surfactant ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Egg Yolk ◽

Surfactant Protein ◽

Scanning Calorimetry ◽

Adsorption Activity ◽

Egg Yolk Phosphatidylcholine

Pulmonary surfactant-associated protein B (SP-B) has been incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) by two different procedures to characterize the dependence of lipid–protein interactions on the method of reconstitution. In method A the protein was dissolved in a small volume of either methanol or 60% (v/v) acetonitrile and injected into an aqueous phase containing phospholipid vesicles. In method B the vesicles were prepared by injection of a mixture of phospholipid and SP-B dissolved in methanol or aqueous acetonitrile. Both methods of reconstitution led to the extensive interaction of SP-B with PC bilayers as demonstrated by co-migration during centrifugation, marked protection against proteolysis, change in the fluorescence emission intensity of SP-B, and protection of SP-B tryptophan fluorescence from quenching by acrylamide. SP-B promoted the rapid adsorption of DPPC on an air/liquid interface irrespective of the method of protein reconstitution. However, the interfacial adsorption activity of SP-B reconstituted by method B remained stable for hours, but that of SP-B prepared by method A decreased with time. Electron microscopy showed that the injection of SP-B into an aqueous phase containing PC or DPPC vesicles (method A) induced a rapid aggregation of vesicles. By contrast, a much longer time was required for detecting vesicle aggregation when the protein was reconstituted by co-injection of SP-B and phospholipids (method B). The presence of 5% (w/w) SP-B in DPPC bilayers prepared by method B broadened the differential scanning calorimetry thermogram and decreased the enthalpy of the transition. In contrast, the injection of SP-B into preformed DPPC vesicles (method A) did not influence the gel-to-liquid phase transition of DPPC bilayers. Taken together, these results indicate that the mode and extent of interaction of SP-B with surfactant phospholipids depends on the conditions of preparation of lipid/protein samples, and that care should be taken in the interpretation of findings from reconstituted systems on the role of these surfactant proteins in the alveolar space.

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Lecithin hydrophobicity modulates the process of cholesterol crystal nucleation and growth in supersaturated model bile systems

Biochemical Journal ◽

10.1042/bj3180139 ◽

1996 ◽

Vol 318 (1) ◽

pp. 139-144 ◽

Cited By ~ 18

Author(s):

Hidenori OCHI ◽

Susumu TAZUMA ◽

Goro KAJIYAMA

Keyword(s):

Acyl Chain ◽

Egg Yolk ◽

Nucleation And Growth ◽

Particle Size Analysis ◽

Crystal Nucleation ◽

Size Analysis ◽

Cholesterol Crystal ◽

Egg Yolk Phosphatidylcholine ◽

Bile Cholesterol ◽

Model Bile

The present study was performed to determine whether the degree of lecithin hydrophobicity regulates bile metastability and, therefore, affects the process of cholesterol crystallization. Supersaturated model bile (MB) solutions were prepared with an identical composition on a molar basis (taurocholate/lecithin/cholesterol, 73:19.5:7.5; total lipid concentration 9 g/dl) except for the lecithin species; egg yolk phosphatidylcholine, soybean phosphatidylcholine, 1-palmitoyl-2-linoleoyl-sn-phosphatidylcholine, dilinoleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine. Each MB solution was incubated and sequentially examined. Video-enhanced contrast microscopy demonstrated that the rate of vesicular aggregation and fusion correlated with the degree of lecithin hydrophobicity, and that the rate of cholesterol crystal nucleation correlated with the degree of lecithin hydrophilicity. In MBs containing less hydrophobic lecithin, needle-like crystals developed and transformed into mature plate-like crystals, whereas classical plate-like crystals were consistently observed in MBs composed of hydrophobic lecithin. Laser-diffraction particle size analysis demonstrated that the increase in lecithin hydrophobicity enlarged the vesicle dimension, enhancing its cholesterol-holding capacity. Correlation between vesicular cholesterol packing density and lecithin hydrophobicity suggests that the process of bile cholesterol nucleation and growth is regulated, in part, by acyl chain unsaturation in lecithin. Since the composition of biliary lecithins is responsive to dietary manipulations, this study provides new insights into the prevention of cholesterol gallstones.

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