Oil bodies and oil-associated proteins from coconut (Cocos nucifera L.): Characterization and a modified method for isolation

College

College of Science

Document Type

Archival Material/Manuscript

Publication Date

2007

Abstract

Background: Oil bodies are small spherical organelles which are deposition sites of plant storage lipids composed mainly of triacylglycerols (TAGs) covered by a protein- phospholipid complex. They are synthesized during the early to middle stages of seed development and are used as energy reserves for germination. Oleosins are a unique class of proteins found in the oil bodies of diverse plant species. They are small, largely hydrophobic proteins embedded in the phospholipid coat of oil bodies. They comprise up to 2–8% of the total seed proteins and are responsible for the oil body size and stability. Oleosins are suggested to play roles including the stabilization of oil bodies preventing their aggregation and as receptor for the binding of lipase to mobilize TAGs during germination and post-germinative growth. The solid endosperm of the seed of coconut (Cocos nucifera L.) consists of about 45–55% oil with high lauric acid content and medium chain triacylglycerol profile. Here, we explored the presence of coconut oil bodies and oleosins, which, by far, have not been explored in coconut. We aimed to improve existing methods for their isolation, determine oil body composition, and characterize oil body components.

Materials and Methods: Coconut oil bodies were isolated from 11-12 month old coconut samples (Laguna Tall variety) using both liquid nitrogen-powdered solid endosperm and coconut milk extracted from the solid endosperm as starting material. The oil bodies were purified using two methods. The first method consisted of sucrose density gradient washing, collection of the oil body pad, and floatation centrifugation. An alternative method involved urea washing and recovery by centrifugation. Purified oil bodies were observed and measured under a light microscope. Thin layer chromatography was employed to determine oil body lipid composition. Oil body-associated proteins were separated from lipids by two washes of diethyl ether, removing the ether layer while leaving the protein layer in the middle each time. Alternatively, the aqueous and interfacial layers were washed with chloroform:methanol (2:1 v/v) and centrifuged, followed by collection of protein-containing precipitate. Purified proteins were characterized using SDS-PAGE and two-dimensional electrophoresis. SDS-PAGE-resolved proteins were electroblotted onto PVDF membrane and the protein of the desired molecular weight was subjected to N-terminal sequence analysis.

Results: Of 1055 oil bodies measured, up to 76% had a diameter in the range of 1-4 μm (Figure 1a,b). The average diameter of these oil bodies is 2.54 μm consistent with published sizes of plant seed oil bodies. Up to 10 percent of the oil bodies had diameter several times larger (10 μm) than the normal size (1-2 μm). The oil bodies consisted of 97% TAGs, the rest being phospholipids (phosphophatidylethanolamine and phosphatidylcholine) and the oil body storage protein, oleosin. The purified oleosin

showed a single 14400 Mr band on SDS-PAGE (Figure 1c). Two-dimensional electrophoresis also resolved one band of 14400 with an isoelectric point of 9-10 (not shown). N-terminal amino acid sequence analysis of the 14.4 kDa band gave GEERR or GEEER, indicating the presence of isoforms. Additional urea washings of the coconut oil bodies followed by ether delipidation showed two oleosin bands of 14400 and 11000 Mr on SDS-PAGE (Figure 1c). While smaller than the reported size of oleosins from other organisms (15–26 kDa), we believe these to be oleosins as they withstood the stringent purification steps.

Conclusion/Perspectives: Coconut oil bodies were successfully purified from powdered coconut meat and milk by floatation centrifugation, buffer washing with and without KCl, and buffer with urea. Our results will contribute to the understanding of the biochemistry of the accumulation and storage of proteins and oils in coconut. The discovery of oil bodies and oleosins in coconut intensifies the possibilities of improving the quality of the coconut oil to make it more competitive in the world market. Oleosins are also potential materials in the pharmaceutical industry. Knowing the characteristics of oil body-associated proteins will lay the ground for its applications in the genetic improvement of coconut through biotechnology. Our work is the first report on the isolation and characterization of oil bodies and oleosins of the oil-rich solid endosperm of coconut.

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Disciplines

Agriculture

Keywords

Coconut oil; Plant proteins

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