Molecular Cloning of Clostridium Thermocellum DNA and the Expression of Further Novel Endo- ,4-glucanase Genes in Escherichia Coli

Abstract

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in Escherichia coli HBlOl and a Lac-mutant thereof. Twentyeight clones with carboxymethylcellulase (CMCase) activity were selected from two libraries by means of the Congo Red plate assay. Restriction enzyme analysis indicated that the CMCase+ clones contained a total of 13 unique DNA inserts. Hybridization of recombinant plasmids with chromosomal DNA confirmed the physical maps in all but one case and was further used to demonstrate the absence of homology between the Hind111 restriction fragments of similar size which occurred in many of the clones. Without exception, CMCase+ E. coli clones expressed endoglucanase activity, but differed with respect to the amount and nature of the enzyme activity produced; additionally, some clones had exoglucanase activity which, in at least one case, was not attributable to the production of a second enzyme. For a few selected clones, the partially purified CMCase was analysed by electrophoresis. A temperature profile characteristic of a thermostable enzyme was demonstrated for the endoglucanase of one of the most active clones. Based on the evidence presented here, it is probable that the 13 unique DNA fragments described do not contain any of the C. thermocellum endoglucanase genes previously cloned.

Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85

“…However, in our hands, a molecular weight of 50,100 was obtained by gel filtration of purified EG1 on Bio-Gel P-150 (unpublished data). EG2 bound to Sephadex G-100, and in this respect, it was similar (2,24,26,27,29,30). Second, the endoglucanases are subject to proteolytic cleavage, giving more than one component of some of the endoglucanases in the extracellular culture fluid.…”

Cellulose degradation by Clostridium thermocellum: From manure to molecular biology

“…Apart from the previously cloned endoglucanase genes celA and celB, the second set of clones contained eight new genes encoding five endoglucanases, two xylanases, and one /3-glucosidase [18]. Using the same approach, several other groups generated independent collections of cel genes [19][20][21], whose relationship to genes in other collections was not always clear. Therefore, we collaborated with Hazlewood and colleagues in Cambridge, and after comparison of the two collections it was established that they contained a total of 15 endoglucanase cel genes, two xylanase xyn genes and one /3-glucosidase bgl gene [22].…”

Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46

“…Total DNA from B.fibrisolvens strain A46 was extracted and purified as described by Romaniec et al (1987). Largescale preparations of plasmid DNA were made from E. coli by alkaline lysis and purified by caesium chloride density-gradient centrifugation (Maniatis et al, 1982).…”

“…Agarose gel electrophoresis, transformation of E. coli and the modification of DNA using restriction enzymes and T4 DNA ligase were done essentially as described by Gilbert et al (1987). Southern blot hybridization was performed according to Romaniec et al (1987).…”