Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovak Republic.
Antonie Van Leeuwenhoek. 2012 Feb;101(2):253-65. doi: 10.1007/s10482-011-9629-3. Epub 2011 Aug 21.
In this study, the submerged cultivation of Trichoderma atroviride with protein substrate (bovine serum albumin, casein, ovalbumin, etc.) led to the secretion of protease activity (PA) into the medium. The secretion started within 30 h and rose to the maximum after 72-h cultivation. It continued upon the prolonged cultivation (up to 8 days) with lower secreted PA. Zymography of the secreted protease (SDS-PAGE with 0.2% gelatine), revealing high molecular weight (m.w.) protease(s) (~200 kDa) with high autolytic activity as the only secretory product. Enzymological characteristics of high m.w. proteases elicited by different inducer proteins were surprisingly different, suggesting that mycelia can recognize not only the presence of inducer proteins but also some qualitative characteristics. Low m.w. protease(s), as found from other studies was(were) seen after prolonged cultivation only, as a band with m.w. about 36 kDa. Expression of known Trichoderma spp. genes encoding secreted proteases, prb1 and proA, showed that only prb1 was expressed after 3-4 days of cultivation, i.e., after the early-secretion phase. The secretory activity of the earlier phase was impaired by tunicamycin and brefeldin A and was significantly stimulated by uncoupler. The existence of a biphasic fungal secretory response and the protein quality recognition represent previously unrecognized processes.
在这项研究中，利用蛋白底物（牛血清白蛋白、酪蛋白、卵清蛋白等）对深绿木霉进行液体深层发酵，导致蛋白酶活性（PA）分泌到培养基中。分泌在 30 小时内开始，并在 72 小时培养后达到最大值。在延长培养时间（长达 8 天）后，分泌的 PA 继续分泌，但活性较低。分泌蛋白酶的同工酶电泳（含 0.2%明胶的 SDS-PAGE）显示，只有一种高相对分子质量（m.w.）蛋白酶（~200kDa）具有高自溶活性，是唯一的分泌产物。不同诱导蛋白诱导的高 m.w.蛋白酶的酶学特性差异很大，表明菌丝不仅能识别诱导蛋白的存在，还能识别其某些定性特征。只有在延长培养时间后，才会出现其他研究中发现的低 m.w.蛋白酶（约 36kDa）。对编码分泌蛋白酶 prb1 和 proA 的已知木霉属基因的表达分析表明，只有在培养 3-4 天后才表达 prb1，即在早期分泌阶段。早期分泌阶段的分泌活性受到衣霉素和布雷菲德菌素 A 的抑制，而解偶联剂则显著刺激其分泌活性。这种真菌分泌反应的两相性和蛋白质质量识别的存在代表了以前未被认识的过程。
