SUMMARY: NCDO 2217 produced three major proteases, P, P and P of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease: arylamidase ratios of these enzymes indicated that they were probably endopepti-dases. Experiments with protease inhibitors suggested that P and P had characteristics of serine and metalloproteases respectively and that P was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn, Ca and Mg, but was strongly inhibited (about 95%) by Cu and Zn. The temperature optimum for protein hydrolysis was 43°C. Proteolysis was temperature sensitive, however (90% reduction at 60°C) and was maximal at alkaline pH, with two broad peaks at pH 7.9 and pH 8.8. Cell fractionation showed that P was located intracellularly and in the periplasm, whereas P and P were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 M-NaC1 suggested that ionic interactions were involved in the association of these enzymes with the membranes.


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