To prepare 11α-hydroxylation of progesterone by A. Ocraceous.

Aim: To prepare 11α-hydroxylation of progesterone by A. Ocraceous.

Requirements:
Culture of Asprgillus ocraceous
Growth medium: Dextrose 1%
Corn steep liquor 0.5%
Yeast Extract 0.5%
Dipotassium H.PO4 0.05%
Water 100ml
pH 6.5(before stabilization)
Tween 80 (1:5000) solution in H2O progesterone

Procedure:
Fully sporulated culture of A. Ocraceous was taken and small amount of tween 80 solution was added spores were scrapped and transfer aceptically in to the growth medium and incubated on rotary shaker for 24 hr.
Progesterone was weighed and taken in a conical flask, wetted with small amount of tween80 and sterilized by steam in air 1000c for 30 min. (because progesterone forms clumps at ordinary sterilization temperature)
24hr culture is added aseptically to the sterile progesterone solution and this was incubated at room temperature on rotary shaker. The culture was harvested with chloroform (3 1/2vol) after 24hrs and the extract were dehumidified with sodium sulphate.
Thin layer chromatography run with spots of chloroform layer and 11α-progesterone solution and chloroform: acetone 7:3 solvent system. After sufficient running 11α-progesterone is referent. The chromatography is allowed to air dried and spread with 50% sulphuric acid in methanol solution. This is dried in hot air oven at 1000c for 15min. the resulting spots were pressured and the amount of convasion was noted.

Result: From the TLC we observed that the Rf value standard culture is higher than blank culture

Interpretation: The residue obtained with test organism were dissolve in 0.5 ml purified ethanol and in standard organism the residue was not dissolved so the Rf value of standard is greater than blank.