CELL-BOUND PEPTIDASE ACTIVITIES OF TREPONEMA-DENTICOLA ATCC-33520 IN CONTINUOUS CULTURE

The oral spirochaete Treponema denticola ATCC 33520 was grown a t a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min-1 (mg dry wt)-1 and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH 9, and a specific activity of 0.3 nmol min-1 (mg dry wt)-1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min-1 (mg dry wt)-1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity. The cell-bound peptidase activities are of potential importance for invasion and multiplication in the junctional epithelium and the destruction process in periodontal pockets with an anaerobic and alkaline environment.