CHARACTERIZATION OF A METALLOPROTEASE INHIBITOR PROTEIN (SMAPI) OF SERRATIA-MARCESCENS

As suggested by Y. Suh and M. J. Benedik (J. Bacteriol, 174:2361-2366, 1992), Serratia marcescens ATCC 27117 produced very small amounts (0. 8 U ml(-1)) of an inhibitor protein (SmaPI) that shows an inhibitory a ctivity against extracellular 50-kDa metalloprotease (SMP) of S. marce scens and that iS localized in the periplasm of cells at the optimal g romth temperature of 25 degrees C, A recombinant S. marcescens harbori ng plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml (-1) that is also localized in the periplasm of cells at 25 degrees C. However, a large amount of SmaPI (86 U ml(-1)) was extracellularly pr oduced at the supraop- timal growth temperature of 37 degrees C from t he recombinant S. marcescens(pSP2), We purified SmaPI from the culture supernatant of S. marcescens(pSP2) grown at 37 degrees C, and some bi ochemical properties were characterized. SmaPI had a pi value of about 10.0 and was a monomeric protein with a molecular mass of 10,000, Sma PI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues), The inhibitor was stable in boiling water f or up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation, SmaPI inhibited SMP by formation of a noncov alent complex with a molar ratio of 1:1 and showed a high protease spe cificity, which inhibited only SMP among the various proteases we exam ined.