How to make your own fumagillin

Fermentation: 680 litres of an aqueous medium containing, weight per
volume, glucose 6 %, corn 50 steep solids 2 %,, and calcium carbonate
0.5 %, are inoculated with spores of a fumagillin-producing strain of
fumigatus and are incubated during 3 days at 24 C with aeration and
stirring 200 litres of this inocu 55 lum are added to 470 litres of a
medium at p H 5 0-5 5 containing (after addition of the inoculum),
weight per volume, glucose 6 %, sodium nitrate 3 %, potassium
dihydrogen phosphate, 0 1 %, potassium chloride O 05 %, 60 magnesium
sulphate heptahydrate 0 001 %, ferrous sulphate heptahydrate 0 001 %,
and yeast autolysate 0 04 % The mixture is contained in a fermenter
fitted with an agitator and a sparger for aeration and is incubated 65
with agitation and aeration during 4 days at 24 C It is then filtered
and the filtrate is estimated by ultra-violet absorption
spectrophotometry to contain 4 1 grams of fumagillin per 100 litres 70
Isolation 18 litres of the filtered broth at p H 6 5 are stirred and
about 250 grams of barium chloride are added gradually until there is
no further precipitation 50 grams of 75 kieselguhr are then added and
the mixture is filtered The filtrate is extracted with two successive
1 8 litre portions of butyl acetate.

The total extract is extracted with three successive 3 6 litre
portions of buffer solution 80 of p H 9 (containing per litre 3 3
grams of boric acid, 4 1 grams of potassium chloride and 0 92 gram
sodium hydroxide) Phosphoric acid is added to the total buffer extract
until the p H of the mixture is 5 It 85 is then extracted with three
successive one litre portions of butyl acetate The combined butyl
acetate extracts are dried over magnesium sulphate and evaporated to
dryness under reduced pressure in an atmo go 90 763,482 763,482 sphere
of nitrogen The yellow solid residue is crystallised from acetone and
0 37 gram of colourless crystals are obtained which contain 98 % of
fumagillin as estimated by ultra-violet absorption spectrophotometry.

When the process of the above Example is repeated using an equal
volume of the same fermentation broth, but omitting from the sequence
of operations the treatment of the broth with barium chloride, there
is obtained 0 29 gram of colourless crystals estimated by ultra-violet
absorption spectrophotometry to contain 93 % of fumagillin.

[Don't try this at home]

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