Studies on Okaramine-Production promoting Factor in Soyabean

单纯青霉（Penicillium simplicissimum） ATCC 90288生长在豆渣（全大豆的不溶性残留物）上，产生具有独特结构的杀虫吲哚类生物碱豆胺。该结构促使我们澄清了鱿鱼胺的生物起源。菌株ATCC 90288在Czapek培养基中不产生okamines，但在Czapek培养基中添加okamines的甲醇提取物促进了okamines的产生，强烈提示okamines中有某种因子促进了真菌对okamines的产生。为了明确该因子，首先建立了检测该因子的测定系统。采用高效液相色谱法测定菌株菌丝中乳胺A和乳胺B的产量。利用该检测系统，对大白菜中的促生因子进行了分离，得到了如下结果：1)依次用乙酸乙酯和正丁醇提取山核桃醇提取物，水渣具有促进活性，说明该因子为水溶性物质。2)水残液在Dowex 1上层析，活性组分（0.5-4 NHCl洗脱物）在Dowex 50W上进一步层析。0.5*4 N NH_4OH洗脱液表现出活性。在Sephadex LH-20上进行柱层析纯化，得到了部分纯化的派系，其活性在浓度为30 ppm左右。该馏分似乎含有几种对茚三酮试剂呈阳性的化合物。3)尿苷作为一种活性物质已被分离出来，它对单纯青霉ATCC 90288产足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.