Physiological and nutritional determinants of protease secretion by Clostridium sporogenes: characterization of six extracellular proteases

Abstract

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.

Bacterial extracellular zinc-containing metalloproteases.

“…In many instances, only the physiological and nutritional factors affecting extracellular protease production have been studied (9,145). However, in several organisms regulatory proteins involved in transcription of metalloprotease genes were identified.…”

“…Studies on the effects of various divalent metal ions on enzyme activity of the clostridial collagenases have suggested differences in the mechanisms of catalysis (10,11,113). Six proteases, each differing in activity against various collagen substrates and sensitivity to inhibitors, produced by C. sporogenes had characteristics of metalloproteases (9). Proteases secreted by the potential pathogen C. bifermentans were predominantly of the metalloprotease type, and although a wide variety of proteins were hydrolyzed, none of the enzymes degraded collagen (145).…”

Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

“…This suggests that the bacterial proteins in CM have caused this inhibitive effect. In particular, this could be due to the extracellular proteases of C. sporogenes, one of which is collagenase, that degrade the tumor tissue 51 52 .…”

Soil organic matter turnover over decadal to millennial timescales

“…In this context, amino acids are likely utilised for microbial biomass synthesis and as an N source (Schimel and Bennett, 2004), especially in N-limited soil such as this Alfisol (Jones et al, 2016;Jones and Dalal, 2017). This increase in the use of amino acids is also reflected in higher protease activity (an enzyme related to protein catabolism) with sample age, suggesting longer desiccation period induced a greater need for additional sources of N compounds in the sandy soil (Allison and Macfarlane, 1992;Glenn, 1976). The identified differences in physiological profiles in the two soil types could be partly attributed to variation in the genetic composition of microorganisms (Griffiths et al, 2011; and the habitat characteristics e.g.…”