Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization.
A triphasic process was developed for the production of beta 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 degrees 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 degrees C and at pH 7 to 8.5. Phase III comprises degradation of CGP to beta-aspartate-arginine and beta-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 degrees 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.
Sallam
Ahmed
A
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
Kast
Alene
A
Przybilla
Simon
S
Meiswinkel
Tobias
T
Steinbüchel
Alexander
A
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004151:Dipeptides / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D044202:Pseudomonas alcaligenes / Q000201:enzymology
D001426:Bacterial Proteins; D004151:Dipeptides; D010940:Plant Proteins; C024250:cyanophycin; D010447:Peptide Hydrolases; C120585:cyanophycinase