Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Abstract

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...

Characterization of Xylanolytic Enzymes in Clostridium cellulovorans : Expression of Xylanase Activity Dependent on Growth Substrates

“…We were not successful in determining the N-terminal amino acid sequence of the polypeptide corresponding to X2, since it occurred in very low quantities. In previous work, we have cloned the genes for C. cellulovorans EngB and EngD, which have not only CMCase activity but also xylanase activity (9,10,14). Although the deduced molecular masses for EngB (48 kDa) and EngD (50 kDa) are close to that of X2, X2 has no CMCase activity (data not shown).…”

The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases

“…The enzyme complex was reported to be very hydrophobic and formed membrane protuberances on the cell surface (12). Previously, we reported the cloning of the engB (1) and engD (7) genes and the characterization of endoglucanase B (EngB) and EngD, which are associated with CbpA in the enzyme complex (2). Furthermore, we reported the cloning and sequencing of the gene for the largest nonenzymatic subunit, CbpA (16).…”

Characterization of an Endo-Processive-Type Xyloglucanase Having a β-1,4-Glucan-Binding Module and an Endo-Type Xyloglucanase from Streptomyces avermitilis

“…CBM2a has an extra loop containing eight amino acids that is not found in CBM2b. The amino acid sequence of CBM2 of SaGH74A showed high similarity with that of CBM2a in C. fimi and C. cellulovorans (10,24), and three Trp residues in CBM2a which are involved in substrate binding are completely conserved in CBM2 of SaGH74A (26). The results of the binding assay and affinity gel electrophoresis in our study showed that the CBM2 of SaGH74A had an affinity for soluble ␤-1,4-glucan chains but not for insoluble and crystalline substrates.…”