Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13

Abstract

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in λ EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5·5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and β-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.