McLellan W L, McDaniel L E, Lampen J O
J Bacteriol. 1970 Apr;102(1):261-70. doi: 10.1128/jb.102.1.261-270.1970.
An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.
基于从酵母细胞壁释放甘露聚糖，已开发出一种改进的磷酸甘露聚糖酶（制备酵母原生质体所需的一种酶）测定方法。描述了一种大规模培养环状芽孢杆菌以最大量生产该酶的方法。含有分泌酶的培养基被浓缩，并且该酶通过硫酸鱼精蛋白处理、硫酸铵分级分离、在P - 100上进行凝胶过滤和等电密度梯度电泳进行纯化。尽管该酶被纯化至表观均一性，但它仍含有不能通过大小或电荷分离的海带多糖酶活性。这两种酶活性在两性电解质电泳上也表现出两个等电点（pH 5.9和6.8）。海带多糖酶对酵母葡聚糖无活性。该酶制剂显示可从酵母中去除甘露聚糖而不去除葡聚糖。电子显微镜观察支持这种甘露聚糖是酵母细胞壁外层的观点。当补充相对少量的蜗牛酶时，磷酸甘露聚糖酶将从酵母产生原生质体。这种活性存在于蜗牛酶中，但当单独使用蜗牛酶时似乎是限速的。磷酸甘露聚糖酶已被证明可用于研究酵母细胞壁中聚合物的大分子组织。
