ISOLATION AND PURIFICATION OF FLAVOBACTERIUM ALPHA-1,3-GLUCANASE-HYDROLYZING, INSOLUBLE, STICKY GLUCAN OF STREPTOCOCCUS-MUTANS
ISOLATION AND PURIFICATION OF FLAVOBACTERIUM ALPHA-1,3-GLUCANASE-HYDROLYZING, INSOLUBLE, STICKY GLUCAN OF STREPTOCOCCUS-MUTANS
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DOI：
10.1128/jb.124.3.1489-1501.1975
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发表时间：
1975-01-01
影响因子：
3.2
通讯作者：
MISAKI, A
中科院分区：
文献类型：
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作者：
EBISU, S;KATO, K;MISAKI, A
对变形链球菌、血链球菌和链球菌无细胞提取物合成的多糖的理化性质进行了研究。以及他们对葡聚糖酶的敏感性。在所检查的多糖中，不溶性葡聚糖对现有的葡聚糖酶制剂具有相当的抵抗力，而由变形链球菌 OMZ 176 产生的不溶性粘性葡聚糖对牙菌斑的形成可能很重要，其抵抗力最强。通过对土壤样本进行富集培养，使用 OMZ 176 葡聚糖作为唯一碳源，分离出一种生物体，该生物体在含有 OMZ 176 葡聚糖的不透明琼脂平板上产生被清晰的裂解区包围的菌落。该生物体被鉴定为黄杆菌属菌株，并命名为 Ek-14 细菌。 EK-14 细菌在胰蛋白酶大豆肉汤中生长，从培养物上清液中浓缩能够水解 OMZ 176 葡聚糖的酶，并通过二乙氨基乙基纤维素 (DE-32) 柱负吸附和羧甲基纤维素 (CM-32) 柱梯度洗脱层析进行纯化。该酶是一种碱性蛋白质，等电点为pH 8.5，分子量为65,000。其最适pH为6.3，最适温度为42℃。纯化的酶以还原糖的形式释放OMZ 176葡聚糖总葡萄糖残基的11％，并溶解约一半的底物葡聚糖。发现产物是异麦芽糖、黑糖和黑三糖，以及一些低聚糖。纯化的酶以内溶方式裂解α-1,3-葡聚糖，并且对含有α-1,6、α-1,4、β-1,3、β-1,4和/或β-1,6键作为主要键的葡聚糖无活性。
Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.