Isolation and characterization of lipid globules from oleaginous yeast, rhodotorula gracilis NRRL Y-1091

Abstract

The preparative isolation of lipid globules from $\underline{Rhodotorula}$ $\underline{gracilis}$ NRRL Y-1091 grown in nitrogen limited condition was carried out by flotation method with rate-zonal centrifugation in Ficoll linear density gradient. For the recovery of intact lipid globules, such experimental conditions as cell disruption method, gradient density and centrifugal force were selected. Phase contrast micrograph showed that $\underline{Rh}$. $\underline{gracilis}$ grown under nitrogen limited condition formed intracellular lipid globules. The lipid globules in whole cells and those isolated from whole cells were stained by treatments with such cationic dyes as neutral red or Toluidine Blue 0. Lipophilics dyes such as Sudan black or Nile Blue a stained both lipid globules in whole cells and those isolated from cell lysate. Lipid globules disrupted when suspended in diluted osmotic stabilizer(Sorbitol + mannitol) buffer solution containing at a concentration between 0.8 M and 0.7 M, but below 0.7 M, lipid globules increased in volume, and again lipid globules ruptured below 0.1 M. The lipid in lipid globules was composed mainly of neutral lipids (87.8%, W/W) predominantly as triacylglycerols (71.8% of neutral lipids). Phospholipids were detected in lipid globules. The quantity of sterolesters included in lipid globules were detected in a considerable amount (19% of neutral lipid). The major components of phospholipids in lipid globules and in whole cells phosphatidylcholine (38.6%) and phosphatidylserine (42.8%), respectively. The major fatty acid components of neutral lipid of whole cells and lipid globules are palmitic, oleic, and linoleic acid. The degree of fatty acid unsaturation of neutral lipid in whole cells was higher than that in lipid globules.