Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.
Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.
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唾液链球菌胞外葡聚糖内切酶的分子克隆。
DOI：
10.1128/jb.173.23.7423-7428.1991
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发表时间：
1991
影响因子：
3.2
通讯作者：
Bleiweis,AS
中科院分区：
文献类型：
--
作者：
Lawman,P;Bleiweis,AS
我们报告了在大肠杆菌中克隆了编码来自唾液链球菌 PC-1 的胞外内切葡聚糖酶（α-1,6-葡聚糖水解酶，EC 3.2.1.11）的基因。来自指定葡聚糖酶活性的唾液链球菌 PC-1-Lambda ZAP II 基因组文库的重组体被鉴定为蓝色葡聚糖琼脂上被透明区域包围的噬菌斑。其中一个克隆 PD1 具有 6.3 kb EcoRI 片段插入片段，该片段编码具有葡聚糖酶活性的 190 kDa 蛋白质。该重组菌株还产生了两种具有葡聚糖酶活性的低分子量多肽（90 和 70 kDa）。从唾液链球菌浓缩培养液中回收天然葡聚糖酶，作为单个 110-kDa 多肽。 PD1 噬菌体裂解物和 PC-1 培养上清液提取物分别用于测量重组形式和天然形式葡聚糖酶的底物特异性。通过薄层色谱法对这些反应产物的分析揭示了重组特定酶产生的预期异麦芽糖产物，但无法解析天然酶的较大多糖产物。此外，唾液链球菌既不利用底物也不利用葡聚糖水解产物进行生长。
We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.