Purification and properties of the proteinase produced in vitro by Verticillium dahliae

Verticillium dahliae, a vascular parasite of sunflower, was grown in a medium supplemented with sunflower cell walls as a sole nitrogen source. The culture filtrate contained three proteinases. The largest amounts were associated with the proteinase PIII fraction, while proteinases PI and PII fractions were in very small quantities. The properties of the purified proteinases were similar and it seemed likely that PI and PII fractions resulted from autolysis of the PIII fraction. The PIII proteinase showed a molecular weight of 12 750 and optimal action at pH in the 7.5–9.5 range. Activity was that of an endopeptidase which hydrolyzed casein, haemoglobin, gelatin, and N-benzoyl-DL-arginine-p-nitroanilide and was much less active on N-tosyl-L-arginine methyl ester and did not hydrolyze N-benzoyl-L-tyrosine ethyl ester. Ethylenediaminetetraacetate and calcium had no effect on the PIII proteinase which was inhibited by HgCl2, phenyl-methyl-sulfonyl fluoride, N-p-tosyl-L-lysine-chloromethyl ketone and L-1-tosyl-amide-2-phenyl-ethyl-chloromethyl ketone. This indicated that the active site in the PIII proteinase probably contained free sulfhydryl groups.

Proteolytic Inactivation of Thermonuclease Activity of Staphylococcus aureus During Recovery from Thermal Injury

Staphylococcus aureus cells were injured thermally by exposure to 55°C for 15 min and allowed to recover for various lengths of time at 37°C in Trypticase Soy Broth. During recovery, thermostable nuclease (TNase) production was measured using a turbidimetric-spectrophotometric method. Production increased during recovery until approximately 2 h after injury when the amount of TNase began to decrease unexpectedly. Protease(s) was thought to be degrading the TNase, and positive results of gelatin agar diffusion tests and heat inactivation experiments supported this hypothesis. Protease inhibitor studies with ethylene diamine tetraacetate (EDTA) and phenyl methyl sulfonyl fluoride (PMSF) confirmed the involvement of protease(s) in the observed decrease in TNase activity. Implications of TNase inactivation in screening of foods for enterotoxigenic staphylococci are discussed.