Araki T, Lu Z, Morishita T
Department of Chemistry of Fishery Resources, Faculty of Bioresources, Mie University, 1515 Kamihama Tsu 514, Japan
J Mar Biotechnol. 1998 Aug;6(3):193-7.
A systematic method was designed for the isolation of a large number of protoplasts from an agarophyte alga Gracilaria verrucosa using agarase from a marine bacterium Vibrio sp. PO-303 and commercial enzymes (Cellulase Onozuka RS and Macerozyme R-10). Pretreatment of the tissue with 5% papain at 22 degreesC for 30 min before digestion with polysaccharide-degrading enzymes increased the protoplast yield. Suitable pH and temperature for the polysaccharide-degrading enzyme reaction were 6.5 and 22 degreesC, respectively. Mannitol (0.7 M) was found to be an excellent osmotic stabilizer. When the tissue (1 g, fresh wt.) of G. verrucosa pretreated with 5% papain solution (20 mM MES buffer, pH 7.5, containing 0.7 M mannitol) was digested with an enzyme mixture consisting of 4 units of agarase, 4% Cellulase Onozuka, 2% Macerozyme, and 0.7 M mannitol in 20 mM MES buffer (pH 6.5) with gentle agitation for 150 min at 22 degreesC, 1.03 x 10(8) protoplasts were obtained.
设计了一种系统方法，用于从石花菜属藻类龙须菜中分离大量原生质体，该方法使用来自海洋细菌弧菌属PO-303的琼脂酶和商业酶（纤维素酶Onozuka RS和离析酶R-10）。在用多糖降解酶消化之前，将组织在22℃下用5%木瓜蛋白酶预处理30分钟，可提高原生质体产量。多糖降解酶反应的适宜pH值和温度分别为6.5和22℃。发现甘露醇（0.7M）是一种优良的渗透稳定剂。当用5%木瓜蛋白酶溶液（20mM MES缓冲液，pH7.5，含0.7M甘露醇）预处理的龙须菜组织（1g鲜重），在20mM MES缓冲液（pH6.5）中，用由4单位琼脂酶、4%纤维素酶Onozuka、2%离析酶和0.7M甘露醇组成的酶混合物在22℃下温和搅拌消化150分钟时，可获得1.03×10⁸个原生质体。
