This study is the first report on the extracellular degradation of cyanophycin (CGP) by Gram-positive bacteria. Three different Gram-positive bacteria were isolated from forest soil that were able to utilize CGP as the sole carbon source for growth. The isolates were assigned to species of the genera Bacillus and Micromonospora. From one of the isolates, which was taxonomically affiliated as Bacillus megaterium strain BAC19, the extracellular CGP depolymerase (extracellular CGPase; CphE(Bm)) was purified to electrophoretic homogeneity by fast protein liquid chromatography and affinity binding to an arginine-agarose column. The purified enzyme was specific for hydrolytic cleavage of CGP, and inhibitor studies indicated that CphE(Bm) is a serine-type peptidase. As CGP degradation products, (beta-Asp-Arg)(2) tetrapeptides in addition to beta-Asp-Arg dipeptides occurred, which were identified by electrospray ionization mass spectrometry analysis. Furthermore, a novel quantitative enzyme assay was developed for kinetic studies on CGP depolymerases. For CphE(Bm), as well as for the extracellular CGPase of Pseudomonas anguilliseptica strain BI (CphE(Pa)) K-M values of 2.2 and 1.0 muM, respectively, for CGP were determined.