An endoglucanase gene, engB, from ClostridYum cellulovorans, previously cloned into pUCl9, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichiu coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosomebinding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulolytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.

By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium ceflulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl 13-1,4-cellobiosidase activity. The two proteins were very homologous (80%o) up to the Pro-Thr-Thr region which divided the protein into -NH2-and -COOH-terminals. The -COOH-region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocelum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH-region of EngD, which had homology to the cellulose-binding domains of Celulomonas fimi exo-and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.Cellulose degradation is an important part of the biological recycling of carbon. Since the initial investigation of the mechanism of cellulose degradation more than 30 years ago, much progress has been achieved, especially with aerobic fungal cellulases. The degradative process can be different for different species, especially between anaerobic and aerobic organisms. With cellulolytic organisms such as Tichoderma reesei, Neocallismastix frontalis, Pseudomonas fluorescens, Cellulomonas fimi, Fibrobacter succinogenes, and Ruminococcus flavefaciens, it is not certain whether the enzymes essential for the degradation of crystalline cellulose function individually or whether they are present on the cell or substrate surface as a complex (1,15,20). There is evidence that in Clostridium thernocellum as well as in Clostridium strain C7, the enzymes are present in a complex, termed the "cellulosome" (3, 18,19,22,23). There is evidence that the enzymes present in Clostndium cellulovorans are also present in a complex. It has been shown that the native cellulase complex of C. cellulovorans is a multiprotein complex of large molecular mass. The enzymic proteins are probably organized on a nonenzymic scaffolding protein which has been cloned and sequenced (25,27,29). The aim of our investigation was to find out whether our cloned enzymes were part of the cellulase complex.Many cellulolytic enzymes have been found to contain a catalytic domain and a binding domain (5, 8-10, 16, 31, 34). It was found that the specificity of C. cellulovorans endoglucanases EngB and EngD was conferred by the NH2-terminal domain and that the COOH-terminal domains of both genes had homology to enzymes from other species of cellulolytic bacteria (6, 13). Thus, it would also be interesting t...

The nucleotide sequence of engD, an endo-beta-1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No. M37434). The COOH-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi. EngD and EngB, another C. cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH2-termini, in contrast to their carboxyterminal domains which show no homology. EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC. Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions. Chimeric proteins that contained the NH2-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH2-terminus showed no cellobiosidase activity. Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH2-terminus and EngD COOH-terminus.

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A method was developed to allow detection of the probiotic Bifidobacterium lactis LAFTI B94 in human clinical samples. A new probe, Laf94p, was developed to accomplish colony hybridization of B. lactis B94. PCR detection of B94 was also achieved using the species-specific (B. lactis) primer pair. These tests and probes allowed detection and quantification of B94 in the human intestinal flora. The sensitivity of the probe was assessed by monitoring faecal levels of B94 in humans who were fed the culture. In this trial, five volunteers were fed with the probiotic. The presence of B94 was assessed daily. Viable B94 could be detected at high levels (as high as 1.8 x 10(9) cfu g(-1) wet weight) during the feeding period. Four weeks after the feeding stopped, B94 could still be detected in one subject. These results indicate that B94 survives in the human gastrointestinal tract.