Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus

• Published in: Applied microbiology and biotechnology 1997-10, Vol.48 (4), p.473-479
• Main Authors: Fontes, C.M.G.A, Clarke, J.H, Hazelwood, G.P, Fernandes, T.H, Gilbert, H.J, Ferreira, L.M.A
• Format: Article
• Language: English
• Subjects: Aerobic bacteria; Amino Acid Sequence; Analysis; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - physiology; Base Sequence; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; cela gene; Cellulase; Cellulase - chemistry; Cellulase - genetics; Cellulase - isolation & purification; Cellulase - physiology; Cellulose; Cellulose - metabolism; Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; genbank/af003696; Genes; Genes, Bacterial; Gram-Negative Aerobic Rods and Cocci - enzymology; Gram-Negative Aerobic Rods and Cocci - genetics; Hydrolysis; Inactivation; Microbiology; Microorganisms; Miscellaneous; Mission oriented research; Molecular Sequence Data; Nucleotides; Peptides; Proteases; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; soil biology; Soil Microbiology; soil science; Substrate Specificity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530051082

The widespread presence of cellulose-binding domains in cellulases from aerobic bacteria and fungi suggests the existence of a strong selective pressure for the retention of these non-catalytic modules. The complete nucleotide sequence of the cellulase gene, celA, from the aerobic soil bacterium Cellvibrio mixtus, was determined. It revealed an open reading frame of 1089 bp that encoded a polypeptide, defined as cellulase A (CelA), of Mr 41 548. CelA displayed features characteristic of an endo-beta-1,4-glucanase, rapidly decreasing the viscosity of the substrate while releasing only moderate amounts of reducing sugar. Deletion studies in celA revealed that removal of 78 nucleotides from the 5' end or 75 from the 3' end of the gene led to the complete loss of cellulase activity of the encoded polypeptides. The deduced primary structure of CelA revealed an N-terminal signal peptide followed by a region that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase family 5. These data suggest that CelA is a single-domain endoglucanase with no distinct non-catalytic cellulose-binding domain. Analysis of the biochemical properties of CelA revealed that the enzyme hydrolyses a range of soluble cellulosic substrates, but was inactive against Avicel, xylan or any other hemicellulose. CelA was resistant to proteolytic inactivation by pancreatic proteinases and surprisingly, in view of its mesophylic origin, was shown to be thermostable. The significance of these findings in relation to the role of single-domain cellulases in plant cell wall hydrolysis by aerobic microorganisms is discussed.