Selected parameters that affect crude toxin production in both
primary culture (growing cell culture) and resting culture were
examined during efforts to produce sufficient crude toxin for purification.
The parameters included carbohydrate and label source and
concentration, cell concentration, incubation time and temperature
and shaker speed. A higher yield of crude toxin was obtained with
0.005 M glucose plus 0.002 M acetate than with 0.08 M glucose
alone. Acetate-1-¹⁴C gave a higher specific activity of crude toxin
than glucose-6-¹⁴C. Maximum yields and incorporation were
obtained with washed cells from 72 hour primary culture incubated
with nitrogen free replacement medium for 12 hours at 30°C on a
rotary shaker at 200 rpm.
A number of chromatography supports and solvent systems
were examined in an attempt to purify crude toxin. Activated silica gel H eluted from a column with a gradient of chloroform:
methanol from a metering pump gave fractions of pure aflatoxin B₁
Comparison of the ratios of the ultraviolet radiation absorbances
of the toxin to pure reference aflatoxin B₁ was a criteria for
chemical purity. Retention of specific activity after chromatography,
hydrogenation to tetrahydrodeoxoaflatoxin B₁ and preparation of the
hemiacetal and epimeric acetates indicated the label was incorporated
in aflatoxin B₁.
A successful method for the preparation of ¹⁴C labeled aflatoxin
B₁ from resting cultures of Aspergillus parasiticus ATCC
15517 in a defined synthetic medium containing 0.02 M glucose and
0.005 M acetate is described. After purification ¹⁴C labeled aflatoxin
B₁ of specific activity 744 μci per mmole was obtained from
2 mci acetate-1-¹⁴C of specific activity 1 mci per mmole. / Graduation date: 1972
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/26910 |
| Date | 07 January 1972 |
| Creators | Schoenhard, Grant Louis |
| Contributors | Sinnhuber, Russell O. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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