Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum.

Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins. The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis. P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu. P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin. P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-[3-(2-furyl)acryloyl]-Gly-Leu-NH2. In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin. P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin.