Biotechnological Process for Production of β-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization

Abstract

ABSTRACT A triphasic process was developed for the production of β 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 β-aspartate-arginine and β-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.