A triphasic process was developed for the production of  dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphE al ) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter ؊1 citrate, pH 6.5, and 37°C are ideal parameters for CphE al production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter ؊1 CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter ؊1 induced CphE al production with only about 30% efficiency in comparison to that with CGP. CphE al was purified utilizing its affinity for the substrate and its specific binding to CGP. CphE al turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to -aspartate-arginine and -aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphE al preparation. Optimum degradation parameters were 100 g liter ؊1 CGP, 10 g liter ؊1 crude CphE al powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphE al powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.Cyanophycin granule polypeptide (CGP), or multi-L-arginylpoly(L-aspartic acid), was discovered in cyanobacteria about 130 years ago (6). The branched polymer consists of a poly(aspartic acid) backbone with arginine moieties linked to the -carboxyl group of each aspartic acid by its ␣-amino group (34, 42) and accumulates during the transition from the exponential to the stationary growth phase (23, 40) and under limiting conditions, including low temperature, low light intensity, and phosphorus or sulfur limitation (44). CGP functions as a temporary nitrogen, energy, and possibly also carbon reserve (10, 21). Because CGP contains five nitrogen atoms in every building block, it is an ideal intracellular nitrogen reserve (43). Most genera of cyanobacteria (5,22,23,43,48) and some heterotrophic bacteria (12, 16, 51) harbor a cyanophycin synthetase gene (cphA) and synthesize CGP. The polymer is insoluble at neutral pH as well as at physiological ionic strength (4). In cyanobacteria, CGP has a molecular mass of 25 to 100 kDa (41), while in recombinant strains it exhibits a molecular mass of 25 to 30 kDa and a lower polydispersity and contains lysine, which partially replaces arginine (2, 50).CGP degradation (intra-or extracellularly) leads mainly to the release of dipeptides. Intracellular degradation of CGP is catalyzed b...