Enzymatic and molecular properties of the Clostridium tertium sialidase

Clostridium tertium metabolizes sialoglycoconjugates via a secreted sialida se [EC 3.2.1.18] and an intracellular acylneuraminate pyruvate lyase [EC 4. 1.3.3]. The sialidase was enriched 1,900-fold from the culture medium with a specific activity of 0.7 U per mg protein. It exhibits a temperature opti mum of 50 degrees C and tolerates mercury ions at relatively high concentra tions (50% inhibition at 5.2 mM Hg2+). The sialidase gene was detected on t wo restriction fragments (HincII, HindIII) of chromosomal DNA and their cor rect recombination resulted in an active enzyme expressed by Escherichia co li, The structural gene is represented by 2,319 bp encoding a protein of 77 3 amino acids with a molecular mass of 85.4 kDa. The first 28 amino acids p ossess the character of a signal peptide, The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases. It has 42.6% identi cal amino acids when compared with the sialidase of Clostridium septicum an d 64,8% with a sialidase gene amplified from Macrobdella decora. A further open reading frame was detected 30 bp downstream from the sialidase gene, w hich exhibits significant homology with acylneuraminate pyruvate lyases. Fo r the first time, both genes were found in close proximity on a bacterial c hromosome, probably being part of one operon.