Physiological and nutritional determinants of protease secretion byClostridium sporogenes:characterization of six extracellular proteases
✍ Scribed by Clive Allison; George T. Macfarlane
- Publisher
- Springer
- Year
- 1992
- Tongue
- English
- Weight
- 524 KB
- Volume
- 37
- Category
- Article
- ISSN
- 1432-0614
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✦ Synopsis
Proteases were the principal secretory proteins of Clostridium sporogenes and were optimally produced after active growth at 37 ° C. Glucose, ammonia and peptides repressed protease production. Protease formation was maximal in cultures grown at pH 6.5, but proteolytic activity exhibited a pH optimum of 7.0-8.0. Protease activity in culture filtrates was stimulated by divalent metal ions (Ca 2÷, Mn 2÷ and Co 2÷) and was strongly inhibited by ethylene diaminetetraacetate (EDTA) and thimerosal. Non-denaturing polyacrylamide gel electrophoresis and H P L C gel filtration demonstrated the presence of six major proteases of low molecular mass (approx. 15-35 kDa). The enzymes were partially purified from non-denaturing gels. Each hydrolysed azocoll and azocasein, but differed in their activity against a range of native collagen substrates. All six enzymes degraded human placental collagen (Type IV) but only one had a broad substrate specificity, being able to hydrolyse the more recalcitrant collagens (Types I, II and III). Experiments with individual proteases showed that their activities were strongly inhibited (40-85%) by 5 mM EDTA, indicating t h a t they were metalloproteases. The enzymes exhibited different susceptibilities to inhibition by either 3 mM phenylmethylsulphonylfluoride (PMSF), 5 mM thimerosal, or 10 mM cysteine, which respectively inhibit serine, thiol and metalloproteases.