Graduate Thesis Or Dissertation
 

Biological preparation of ¹⁴C labeled aflatoxin

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  • 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.
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file details SCHOENHARDGRANT1972.pdf 2017-10-20 Pubblico Scaricare