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United States Department of Agriculture

Agricultural Research Service

Title: Expression of Cloned Polysaccharidase Genes from the Rumen Anaerobic Bacterium Ruminococcus Flavefaciens in Lactic Acid Bacteria

item Ekinci, Sait - ABERDEEN, UK
item Scott, Karen - ABERDEEN, UK
item Mercer, Derry - ABERDEEN, UK
item Whitehead, Terence
item Flint, Harry - ABERDEEN, UK

Submitted to: European Congress on Biotechnology
Publication Type: Abstract Only
Publication Acceptance Date: October 26, 1995
Publication Date: N/A

Technical Abstract: Ruminococcus flavefaciens is a strictly anaerobic Gram-positive bacterium that plays an important role in the utilization of cellulose and hemicellulose in the rumen. A number of polysaccharidase genes have been characterized from this species, including gene coding for a family of xylanases that show unusual complex domain structures. We have recently investigated the expression of R. flavefaciens genes originally cloned in E. coli in Gram-positive hosts using the shuttle vector pVA838. The endA gene, which encodes a cellulase fragment, and the xynD gene, which encodes a xylanase, were expressed from their own promoters in Lactococcus lactis and in the rumen species Streptococcus bovis. In S. bovis, the cloned activities were recoverable from young cells, but tended to decline in older cultures when glucose was provided as energy source. The xynD gene codes for a polypeptide that has separate catalytic domains conferring xylanase and beta-(1,3-1,4)-glucanase activity. This enzyme is predicted to be approximately 90kDa, but is subject to extensive proteolysis in E. coli. On the other hand, in L. lactis most of the cloned xylanase and beta-(1,3-1,4)-glucanase activity was associated with the full-size, bifunctional XynD enzyme which was visualized through zymogram techniques following SDS PAGE and renaturation. S. bovis and Enterococcus faecalis gave patterns of xynD expression different from those in E. coli and L. lactis. In conclusion, lactic acid bacteria appear to be promising hosts for studying structure/function relationships in polysaccharidases from Ruminococci and for expressing cloned genes from this group of strict anaerobes.

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