Reaction #1702039

Reaction equation

γ-linolenic acid

galactose

adenine sulfate

arginine

aspartic acid

glutamic acid

histidine

lysine

methionine

phenylalanine

serine

tyrosine

valine

threonine

→

Arachidonic Acid

Conditions

Workup

1. 1
   Othercultured at 30° C.
2. 2
   Otherfor a day
3. 3
   OtherThe cells were collected
4. 4
   Washwashed with water
5. 5
   workup.ADDITIONTo the lyophilized cells was added 4 ml of chloroform
6. 6
   Otherto recover the supernatant
7. 7
   Otherthe resulting supernatant was recovered together with the supernatant
8. 8
   Otherpreviously recovered
9. 9
   OtherThe solvent was removed by distillation
10. 10
    workup.DISSOLUTIONthe residue was dissolved in a small quantity of chloroform
11. 11
    Otherirradiating with UV rays
12. 12
    Otherrespectively, and each transferred to a test tube

Procedure

These strains were each cultured at 30° C. for a day in 10 ml of the SC-Trp,Leu,Ura liquid medium described above. For these strains, 1 ml of the culture was then cultured at 15° C. for 6 days in 10 ml of SG-Trp,Leu,Ura liquid medium (per liter, 6.7 g of yeast nitrogen base w/o amino acids (DIFCO), 20 g of galactose and 1.3 g of amino acid powders (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine and 6 g of threonine) added with γ-linolenic acid to become 50 μg/ml in duplicate. The cells were collected, washed with water and then lyophilized. To the lyophilized cells was added 4 ml of chloroform:methanol=2:1, which was maintained at 70° C. for an hour. Thereafter, centrifugation was performed to recover the supernatant. To the remaining cells was further added 4 ml of chloroform:methanol=2:1. The mixture was centrifuged and the resulting supernatant was recovered together with the supernatant previously recovered. The solvent was removed by distillation using a speed-vac and the residue was dissolved in a small quantity of chloroform. TLC was performed on a Silica Gel 60 Plate (Merck) under the conditions of hexane:diethyl ether:acetic acid=70:30:1 as the developing solvent to fractionate lipids. The lipids were detected by spraying the primulin solution and irradiating with UV rays. The TG fraction and the PL fraction were scraped off, respectively, and each transferred to a test tube. After the fatty acids were converted to the methyl esters by the hydrochloric acid methanol method, the analysis of fatty acids was performed by gas chromatography.

Source