Studies on Okaramine-Production promoting Factor in Soyabean

简单青霉菌ATCC 90288在大豆不溶性残渣上生长时，能产生具有独特结构的吲哚生物碱。这种结构促使我们阐明了奥卡拉胺的生物发生。菌株atcc 90288在查氏培养基中不产生阿卡拉胺，但添加到查氏培养基中的欧卡拉甲醇提取物促进了阿卡拉胺的产生，这有力地表明奥卡拉中的某一因子促进了该真菌产生阿卡拉胺。用高效液相色谱仪测定了该菌株菌丝体中的促生素A和B的产生量。利用该检测体系，我们尝试从欧卡拉中分离出促进因子，得到如下结果：1)欧卡拉的偏心醇提取物依次用乙酸乙酯和正丁醇提取，水中残渣表现出促进活性，表明该因子是水溶性物质。2)水中残留物在Dowex 1上进行层析，活性组分(0.5-4 NHCl洗脱物)在Dowex 50W上进一步层析，0.5×4 N_4NH_4OH洗脱液显示活性。用Sephadex LH-20柱层析对这些活性组分进行纯化，得到部分纯化的活性组分，活性浓度约为30ppm。3)尿苷已作为一种活性物质被分离出来，它对简单青霉ATCC 90288产生鸦片胺有微弱的促进作用。

Penicillium simplicissimum ATCC 90288, when grown on okara (the insoluble residue of whole soyabean), produces insecticidal indole alkaloid okaramines, which has a unique structure. The structure prompted us to clarify the biogenesis of okaramines. The strain ATCC 90288 did not produce okaramines in a Czapek medium, but the methanol extract of okara supplemented to the Czapek medium promoted the production of okaramines, strongly suggesting that a certain factor in okara promoted the production of okaramines by the fungus.To clarify the factor an assay system for the detection of the factor was first established. The produced amounts of okaramines A and B in mycelia of the strain were measured by HPLC.Using this assay system, we tried to isolate the promoting factor in okara, and some results are obtained as follows.1) The metanol extract of okara was extracted with ethyl acetate and n-butanol successively, and the aqueous residue exhibited the promoting activity, indicating that the factor was water soluble substance.2) The aqueous residue was chromatographed on Dowex 1, and the active faction (0.5-4 NHCl eluates) was further chromatographed on Dowex 50W.The 0.5*4 N NH_4OH eluates showed the activity. Purification of these factions by colume chromatography on Sephadex LH-20 afforded a partially purified faction, which exhibited the activity in the concentration of ca. 30 ppm. This fraction seemed to contain several compounds which are positive to Ninhydrin reagent.3) Uridine has already isolated as an active substance which weakly promotes the production of okaramines by Penicillium simplicissimum ATCC 90288.