Studies on Okaramine-Production promoting Factor in Soyabean

简青霉ATCC 90288在奥卡拉（整个大豆的不溶性残留物）上生长时，产生具有独特结构的杀虫吲哚生物碱奥卡拉胺。该结构促使我们澄清okaramines的生物起源。菌株ATCC 90288在Czapek培养基中不产生奥卡胺，但是添加到Czapek培养基中的奥卡拉的甲醇提取物促进了奥卡胺的产生，强烈地表明奥卡拉中的某种因子促进了真菌奥卡胺的产生。本研究利用高效液相色谱法测定了该菌株菌丝体中奥卡胺A和奥卡胺B的产生量，并利用该测定体系对奥卡拉中的促生长因子进行了分离，得到以下结果：1）奥卡拉的甲醇提取物依次用乙酸乙酯和正丁醇提取，水提物具有促生长活性，2）水相残留物经Dowex 1柱层析，活性组分（0.5 ~ 4 NHCl洗脱液）经Dowex 50 W柱层析，0.5 × 4NNH_4OH洗脱液显示活性。通过Sephadex LH-20柱层析纯化这些组分，得到部分纯化的组分，其在约100 μ g/ml的浓度下显示出活性。30 ppm。该级分似乎含有几种对茚三酮试剂呈阳性的化合物。3）尿苷已被分离为活性物质，其微弱地促进简青霉ATCC 90288产生奥卡胺。

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.