F. heparinum is a gram-negative, nonpathogenic soil bacterium, which produces enzymes that degrade glycosaminoglycans (GAGs).  These lyases act upon GAGs through eliminative mechanism, leading to depolymerisation of the chains and formation of  unsaturated fragments. When  in the culture media glucose is substituted by GAGs as the sole source of carbon and nitrogen, the bacteria are able to induce the production of enzymes that degrade GAGs to its basic disaccharides units. Addition of heparin, heparan sulfate or their disaccharides to the medium leads to the induction of three enzymes, known as heparinase (Hepase) and heparitinases (Htase) I and II (Dietrich et al., JBC 248, 6408,1973; Nader et al., JBC 265, 16807, 1990),  recently referred as heparinases I, II and III, respectively (Su et al., Appl. Environ. Microbiol. 62, 2723, 1996). The present work aims to study the induction of Hepase III (supposedly Htase I) and the molecular and biochemical analyses of this enzyme, that shows a unique specificity. Htase I cleaves exclusively N-acetyl or N-sulfo-glucosaminide-glucuronic acid linkage on heparan sulfate. Sulfation of the glucosamine residue at C-6 is impeditive for enzyme activity. Initially, a growth curve was done in minimum media in the presence of glucose. In parallel, an induction curve in the presence of heparin was performed for different periods of time. The activity of the induced enzymes was assayed using chondroitin sulfate, heparan sulfate and heparin. The products formed were analyzed by agarose and polyacrylamide gel electrophoresis and paper chromatography followed by silver nitrate detection as well as HPLC profile. The activity  is detected after 4 hours of induction. Total RNA was also extracted from each sample and mRNA were enriched by removal of rRNA with magnetic beads (Ambion). mRNA was converted to cDNA with reverse transcriptase, using random primers, and amplified by PCR with specific primers to hepase III. A product of 1980 pb was detected, in a time dependent manner, up to 6 hours of induction. These results showed a significant increase on mRNA , which express hepase III after induction. The molecular weight and activity of enzyme were also detected by PAGE and zymogram, previously impregnated with heparan sulfate, showing a sharp and distinct band of 65kDa.
(supported by: CAPES, CNPq).