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We detected biosynthetic activity for aflatoxins G1 and G2 in cell extracts of Aspergillus parasiticusNIAH-26. We found that in the presence of NADPH, aflatoxins G1 and G2 were produced fromO-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B1, aflatoxin B2, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G2 from dihydro-O-methylsterigmatocystin was suppressed whenO-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G1 was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G1 and G2. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from eitherO-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.

The effects of various macronutrients on growth and anthocyanin formation in callus cultures of roselle (Hibiscus sabdaritfa L.) were investigated. Of the nutritional factors examined the type and concentration of carbon and nitrogen sources and phosphate concentration showed marked effects on the growth and anthocyanin production. Utilization of an optimized medium based on the results obtained in the present investigation resulted in a 2.5 fold increase in the anthocyanin content Potential exists for application of a two-stage culture method for the production of anthocyanin pigment Introduction Calyces of roselle (Hibiscus sabdariffa L.) contain cyanidin and delphinidin glycosides1) and have been used for making jelly, jams, beverages and food colorants2). Cultured roselle cells might potentially be a suitable source for large scale production of anthocyanin pigments. In a previous paper3) we reported that callus tissues derived from seedlings of roselle could accumulate anthocyanin pigments tentatively identified as cyanidin-3-monoglucoside (major pigment) and cyanidin-3-xylosylglucoside. Anthocyanin formation in the callus markedly enhanced by 2, 4-D and inhibited by gibberelic acid.The present paper describes the effects of macronutrients on cell growth and anthocyanin production of roselle callus as well as an optimized growth and production medium based on the results obtained in this study. Materials and MethodsPlant material and culture method. Callus tissues derived from seedlings of roselle were subcultured at 1-month intervals on Linsmaier and Skoog (LS) basal agar medium4) supplemented with l p M 2, 4-D and l p M kinetin at 25 under 3, 000 lux illumination (16 hr/day). For investigating the influence of various macronutrients, callus tissues (ca 0.2 g) were transferred onto 20 ml of test medium in 50 ml Erlenmeyer flasks and incubated at 25 under illumination for four weeks before harvest. All the test media contained both 2, 4-D and kinetin at 1 uM level.Extraction and quantitative analysis of anthocyanin. Fresh callus tissues were homogenized with 1% methanolic HCl in a mixer. The homogenate was allowed to stand overnight at 4 and then filtered. Absorbance of the filtrate was measured at 530 nm and the anthocyanin content was calculated as a percentage of fresh weight of callus using the molecular extinction coefficient (log E 4.47) for cyanidin-3-monoglucoside5). Results and DiscussionEffects of carbon sourceAlthough sucrose as a carbon source can support both growth and secondary metabolite production in callus and suspension cultures of plant cells, other carbon sources can also be effective. The effects of different carbon sources at 3% concentration on the growth and anthocyanin production of roselle callus are shown in Fig, 1. As regards anthocyanin formation glucose was as effective as sucrose, whereas fructose and maltose could support anthocyanin formation to a limited extent. In contrast, sucrose was most effective for cell growth; glucose, fructose and maltose were much inferior to suc...

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During research on the Maillard reaction between xylose and lysine (Lys), we detected a major peak showing anabsorbance maximum at about 280 nm by diode-array-detection (DAD)-HPLC. In this study, the peak was isolated and identified as 4-hydroxy-5-methyl-3(2H)-furanone (HMFO). This compound accounted for 60 _ 80% of the total area of HPLC peaks (280 nm), and about 20 mg/100 mL of HMFO was produced from a solution containing 200 mg/100 mL of xylose. HMFO was produced not only from xylose, but also from arabinose and ribose, and ribose was the best precursor. When HMFO was heated in a buffer solution with or without Lys, it was decomposed or polymerized, and colored and colorless polymers appeared. Diacetyl and methylglyoxal were the major decomposed dicarbonyl compounds from HMFO; these dicarbonyl compounds are considered to be the major precursors for polymers formed from HMFO.