Partial characterization of structure and function of a xylanase gene from the rumen hemicellulolytic bacterium Eubacterium ruminantium

A gene encoding for xylanase activity in the rumen hemicellulolytic bacterium Eubacterium ruminantium was cloned into pBR322 in Escherichia coli (E. coli ). The primary clone had a 5.7 kb insert produced by Eco RI partial digestion. Subcloning followed by sequencing allowed for the discovery that this enzyme has a glycosyl-hydrolase family 10 catalytic domain with a family 9 carbohydrate binding module at C-terminus and a region partially homologous to a family 22 carbohydrate binding module at N-terminus. Cloned xylanase is specifically active against xylan and oligoxyloside to produce xylobiose and xylotriose, showing optimal pH and temperature at 7.0 and 50°C, respectively. Molecular size of the xylanase (91 kDa) was confirmed by zymogram analysis of the E. coli clone, which agreed with the predicted size from the DNA sequence. Functions of the two modules at C- and N-termini were evaluated by using xylanase variants with and without the respective module and the C-terminal module was found to be functional in binding to acid-swollen cellulose and insoluble oat-spelt xylan, whereas the N-terminal module was inactive for binding them.