CHARACTERIZATION OF A CELLULOSE-BINDING, CELLULASE-CONTAINING COMPLEX IN CLOSTRIDIUM-THERMOCELLUM
CHARACTERIZATION OF A CELLULOSE-BINDING, CELLULASE-CONTAINING COMPLEX IN CLOSTRIDIUM-THERMOCELLUM
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DOI：
10.1128/jb.156.2.828-836.1983
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发表时间：
1983-01-01
影响因子：
3.2
通讯作者：
BAYER, EA
中科院分区：
文献类型：
--
作者：
LAMED, R;SETTER, E;BAYER, EA
从C.描述了热纤。通过两步程序从培养上清液中分离CBF，所述两步程序包括纤维素亲和层析和Sepharose 4B凝胶过滤。经免疫电泳、聚丙烯酰胺凝胶电泳（PAGE）、凝胶过滤和分析性超滤测定，分离的CBF是均一的。CGF与. apprx形成复合物。2.1百万兆瓦EM分析负染色制备的隔离CBF显示一个颗粒，复杂的四级结构的多亚基实体。该分子是. apprx。18 nm大小。虽然尿素不能将复合物分解成其组成部分，但在十二烷基硫酸钠存在下的PAGE将CBF复合物分解成14条多肽带。免疫沉淀实验证实，这些多肽形成相同的复合物的一部分。只有1个CBF亚基（MW 210，000）具有抗原活性。通过使用凝胶覆盖测定技术，至少8个剩余的CBF相关的多肽组分显示出纤维素分解活性。显然，CBF包含离散的多亚基复合物或一组密切相关的复合物，其除了纤维素结合的性质之外还表现出单独的抗原性和多种纤维素酶活性。
The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from C. thermocellum is described. The CBF was isolated from the culture supernatant by a 2-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis [PAGE], gel filtration and analytical ultracentrifugation. The CGF formed a complex with .apprx. 2.1 million MW. EM analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule was .apprx. 18 nm in size. Although urea failed to break the complex into its component parts, PAGE in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides formed part of the same complex. Only 1 CBF subunit (MW 210,000) was antigenically active. By using a gel-overlay assay technique, at least 8 of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. Evidently, the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding.