Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.
Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.
复制标题
糖化酵母 STA2 编码的胞外葡糖淀粉酶的生化和免疫学特征。
DOI：
10.1016/0003-9861(86)90410-8
复制
发表时间：
1986
影响因子：
3.9
通讯作者：
Marmur,J
中科院分区：
文献类型：
--
作者：
Modena,D;Vanoni,M;Englard,S;Marmur,J
在糖化酵母中，三个非连锁基因（STA 1、STA 2、STA 3）中的每一个编码葡糖淀粉酶（α-1，4葡糖葡糖水解酶，EC 3.2）。1.3)让酵母在淀粉上生长通过乙醇沉淀、Trisacryl M DEAE色谱和HPLC凝胶过滤，从培养基中纯化由STA 2基因编码的酶（葡糖淀粉酶II）至接近均质。葡糖淀粉酶II由两个相同的亚基组成，其平均大小为300 kDa。在变性条件下，天然二聚体酶容易解离成单体。变性酶的酶促去糖基化产生中间的部分糖基化形式和56-kDa完全去糖基化的蛋白质。葡糖淀粉酶通过从不同寡糖的非还原端切割α-1，4键释放葡萄糖单元，但仅具有几乎不可检测的α-1，6水解活性。发现纯化的酶的最适pH为5.1。该酶对麦芽六糖的亲和力（Km = 0.98 mM，V Km = 2.39）大于对麦芽三糖（Km = 2.38，V Km = 0.68）或麦芽糖（Km = 3.20，V Km = 0.39）的亲和力。已经提出了针对葡糖淀粉酶II的多克隆和单克隆抗体。多克隆抗体以剂量依赖性方式特异性抑制酵母葡糖淀粉酶II活性，但也发现免疫印迹其他酵母糖蛋白。这种寡糖特异性反应可以通过添加过量的甘露聚糖而不影响葡糖淀粉酶反应性来竞争。多克隆抗体与其他淀粉分解酶的交叉反应性与进化距离密切相关。证据表明，已经获得了对碳水化合物或蛋白质表位具有特异性的单克隆抗体。
In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (α-1, 4 glucanglucohydrolase, EC 3.2. 1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving α-1, 4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable α-1, 6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (K m= 0.98 mM, V K m= 2.39) than for maltotriose (K m= 2.38, V K m= 0.68) or maltose (K m= 3.20, V K m= 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.