Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum

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Abstract
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.