Analysis of genes and enzymes involved in the degradation of cellulose and proteins by Butyrivibrio fibrisolvens H17c

Bibliography: pages 147-169.Butyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate