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Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley β-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose. [ABSTRACT FROM AUTHOR]

An α-L-rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60°C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p-nitrophenyl-α-L-rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg[SUP-1] respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using [SUP1]H-NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable β-glucosidase. The secondary structure of RamA consists of 27% α-helices and 50% β-sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the Clostridium thermocellum gene licB, coding for a thermoactive β1,3-1,4-glucanase, has been determined. The gene is located downstream, but in opposite orientation to the β-glucosidase gene bglA. A coding region of 1002 bp is flanked by canonical promoter and transcription terminator sequences. The primary translation product of the licB gene has a predicted molecular mass of 37 896 Da. The protein sequence can be divided into several discrete segments: an N-terminal signal peptide, a catalytic region, a segment rich in Pro and Thr residues and a C-terminal reiterated domain. The catalytic region shows close similarity to lichenases of bacilli (52-58% identity) and Fibrobacter succinogenes (35% identity), but is unrelated to barley β-1,3-1,4-glucanases. it consists of two domains, which in the case of the F. succinogenes lichenase are arranged in reversed order to that of C. thermocellum and Bacillus lichenases. The C-terminal reiterated domain of C. thermocellum lichenase is homologous to the duplicated non-catalytic domain of endo-β-l,4glucanases and xylanase Z from the same organism. This domain is considered a characteristic feature of clostridial cellulases organized as multienzyme complex (cellulosome). The β-l,3-1,4-glucanase encoded by the licB gene might therefore be an additional enzyme component of the C. thermocellum cellulosome. [ABSTRACT FROM AUTHOR]