Production by Clostridium acetobutylicum ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9

Abstract

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.

Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium acetobutylicum

“…The activity of the purified C. acetobutylicum cellulosome against CMC, 0.115 U/mg (28), is about 0.6% of the activity of the GH44 C. acetobutylicum EG against CMC, 18.9 s Ϫ1 (Table 2) or 20.0 U/mg, these values measured at a lower temperature and pH. C. acetobutylicum ATCC 824 Cel9G, a GH9 EG, whose encoding gene was found in the same cellulosomal gene cluster as the gene encoding the GH44 EG studied here (25), has a specific activity of 7.4 U/mg against CMC (20). The experiments were conducted at different pHs, and the cellulosome does contain noncatalytic proteins, which may contribute to some of its lower observed activity, since activity is based upon mass instead of molarity.…”

Substrate-Induced Production and Secretion of Cellulases by Clostridium acetobutylicum

“…coli M15 harboring either pWUR70 or pWUR70 was tested on agar plates as described previously (16). None of these strains showed CMC degradation on plates even after prolonged incubation (results not shown).…”

“…On glucose, cellobiose, xylose, or lichenan, C. acetobutylicum grew well, and after 4 days of fermentation the insoluble lichenan residue had disappeared completely. Growth of the cultures was monitored by optical density determinations at 600 nm, except for lichenan-grown cultures, for which the amount of protein in cell extracts was determined by obtaining growth curves comparable to those described previously (16).…”

Genome-Wide Gene Expression Analysis of the Switch between Acidogenesis and Solventogenesis in Continuous Cultures of Clostridium acetobutylicum

“…Unfortunately C. acetobutylicum is not known to grow on cellulose despite the presence of putative cellulosome-encoding genes that have been identified in the genome [Sabathe et al , 2002] . While some of these genes have been shown to be expressed in small amounts, their function remains an unanswered question [Lopez -Contreras et al , 2003.…”