CLONING, EXPRESSION, SEQUENCE-ANALYSIS AND BIOCHEMICAL-CHARACTERIZATION OF AN AUTOLYTIC AMIDASE OF BACILLUS-SUBTILIS 168 TRPC2
CLONING, EXPRESSION, SEQUENCE-ANALYSIS AND BIOCHEMICAL-CHARACTERIZATION OF AN AUTOLYTIC AMIDASE OF BACILLUS-SUBTILIS 168 TRPC2
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DOI：
10.1099/00221287-137-8-1987
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发表时间：
1991-08-01
期刊：
影响因子：
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通讯作者：
FOSTER, SJ
中科院分区：
文献类型：
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作者：
FOSTER, SJ
通过活性的功能测定，将自溶素结构基因克隆到来自枯草芽孢杆菌168 trpC 2基因组DNA的表达文库的3 kb EcoRI DNA片段上。 该片段的测序显示了5个开放阅读框，其中一个中心编码的溶解酶，发现通过亚克隆活性映射和其同源性最近测序的自溶素基因。 该蛋白的推导序列为272个氨基酸，分子量为29957 Da。当在大肠杆菌DH 5-α中表达时，蛋白被加工成21 kDa的形式，通过复性SDS-PAGE估计。 自溶素是一种N-乙酰胞壁酰-L-丙氨酸酰胺酶，其活性是MgCl 2依赖性的（最佳20 mM）和LiCl敏感性。 该酶可以结合和水解广泛的肽聚糖底物分离革兰氏阳性菌;结合也是氯化镁依赖性的。 最初的定位实验将自溶素基因定位在B上的aroD附近。subtilis 168染色体。
By use of a functional assay for activity, an autolysin structural gene was cloned on a 3 kb EcoRI DNA fragment from a lambda-gt11 expression library of Bacillus subtilis 168 trpC2 genomic DNA. Sequencing of the fragment showed five open reading frames, the central one of which encoded the lytic enzyme as found by subclone activity mapping and its homology to a recently sequenced autolysin gene. The protein had a deduced sequence of 272 amino acids and a molecular mass of 29957 Da. When expressed in Escherichia coli DH5-alpha, the protein was processed to a 21 kDa form, as estimated by renaturing SDS-PAGE. The autolysin was an N-acetylmuramyl-L-alanine amidase and its activity was MgCl2-dependent (20 mM optimum) and LiCl-sensitive. The enzyme could bind to and hydrolyse a wide range of peptidoglycan substrates isolated from Gram-positive bacteria; the binding was also MgCl2-dependent. Initial mapping experiments located the autolysin gene near aroD on the B. subtilis 168 chromosome.