Streptomyces aureofaciens TH-3 secretes a protease termed 'kibilysin'. for which we showed unique substrate specificity and preference for Tyr. Pro, and Leu at the P-1 position using fluorescence energy transfer substrate (FRETS) combinatorial libraries. Using (7-methoxycoumarin-4-yl) acetyl-Lys-Pro-Leu-Gly-LeuD-23-diamino propionic acid (2,4-dinitrophenyl)-Ala-Arg-NH2, we confirmed that kibilysin digests the substrate between Pro and Leu. Its gene was cloned and sequenced. The primary structure of the enzyme showed 40,66, and 61% identity, respectively. with those of thermolysin from Bacillus thermoproteolyticus, and metalloendopeptidases from Streptomyces cinamoneus TH-2 and S. griseus. Its deduced amino acid sequence contained an HEXXH consensus Sequence for zinc binding, which is a common motif of the peptidase family M4. Moreover, we succeeded in over-expression of kibilysin using Streptomyces lividans. (C) 2008 Elsevier Inc. All rights reserved.