Microbial Degradation of Poly(amino acid)s

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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...

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confidence: 99%

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

Sallam

Alene

Przybilla

et al. 2009

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“…CGP accumulation is promoted by several conditions, including phosphorus limitation (46), sulfur limitation (5), low temperature, low light intensity, or a combination of these factors (32), and in the presence of translational or transcriptional inhibitors (3,13). CGP functions as a temporary nitrogen, energy, and possibly carbon reserve (12,26).…”

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confidence: 99%

Anaerobic and Aerobic Degradation of Cyanophycin by the Denitrifying Bacterium Pseudomonas alcaligenes Strain DIP1 and Role of Three Other Coisolates in a Mixed Bacterial Consortium

Sallam

Steinbüchel

2008

Four bacterial strains were isolated from a cyanophycin granule polypeptide (CGP)-degrading anaerobic consortium, identified by 16S rRNA gene sequencing, and assigned to species of the genera Pseudomonas, Enterococcus, Clostridium, and Paenibacillus. The consortium member responsible for CGP degradation was assigned as Pseudomonas alcaligenes strain DIP1. The growth of and CGP degradation by strain DIP1 under anaerobic conditions were enhanced but not dependent on the presence of nitrate as an electron acceptor. CGP was hydrolyzed to its constituting ␤-Asp-Arg dipeptides, which were then completely utilized within 25 and 4 days under anaerobic and aerobic conditions, respectively. The end products of CGP degradation by strain DIP1 were alanine, succinate, and ornithine as determined by high-performance liquid chromatography analysis. The facultative anaerobic Enterococcus casseliflavus strain ELS3 and the strictly anaerobic Clostridium sulfidogenes strain SGB2 were coisolates and utilized the ␤-linked isodipeptides from the common pool available to the mixed consortium, while the fourth isolate, Paenibacillus odorifer strain PNF4, did not play a direct role in the biodegradation of CGP. Several syntrophic interactions affecting CGP degradation, such as substrate utilization, the reduction of electron acceptors, and aeration, were elucidated. This study demonstrates the first investigation of CGP degradation under both anaerobic and aerobic conditions by one bacterial strain, with regard to the physiological role of other bacteria in a mixed consortium.

“…Furthermore, CGP proved to be highly resistant to many proteases and arginase (45,47). Therefore, CGPases probably evolved as specialized enzymes for the purpose of degrading CGP under certain environmental conditions (30,32,39). CGP is a widespread biopolymer that represents a valuable source of nitrogen, carbon, and energy and probably occurs in many habitats (see the spectrum of CGP-producing organisms mentioned above).…”

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confidence: 99%

Degradation of Cyanophycin by Sedimentibacter hongkongensis Strain KI and Citrobacter amalonaticus Strain G Isolated from an Anaerobic Bacterial Consortium

Obst

Krug

Luftmann

et al. 2005

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Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endosporeforming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gramnegative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to ␤-Asp-Arg and ␤-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the ␤-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.