Ahmed Sallam

Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production

The numerous physiological functions of the nonessential amino acid L-aspartate, the semi-essential amino acid L-arginine, and the essential amino acid L-lysine, made them attractive for a wide range of nutritional and/or therapeutic applications. Furthermore, the administration of these amino acids as mixtures or as dipeptides for higher bioavailability is scientifically approved, and various commercial products of these forms are already available on the market. Although the industrial production of dipeptides is, with few exceptions, in an early stage, several strategies have been established and are compared in this review. Additionally, the recent developments in the technical production of aspartate–arginine and aspartate–lysine dipeptides from the biopolymer cyanophycin produced in microorganisms are discussed. Cyanophycin-derived dipeptides are produced exclusively by biotechnological procedures, probably possess higher bioavailability and may be used as better alternatives to the widely applied amino acid mixtures. Thus, the pivotal advantages and the potential applications of these dipeptides as well as of their constituting amino acids in nutrition and therapy are also discussed. Special emphasis is dedicated to arginine due to its numerous physiological roles in many cardiovascular, genitourinary, gastrointestinal, and immune disorders.

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

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 (CphEal) 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 CphEal 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 CphEal production with only about 30% efficiency in comparison to that with CGP. CphEal was purified utilizing its affinity for the substrate and its specific binding to CGP. CphEal 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 CphEal preparation. Optimum degradation parameters were 100 g liter−1 CGP, 10 g liter−1 crude CphEal powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphEal 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.

Clostridium sulfidigenes sp. nov., a mesophilic, proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from pond sediment.

A novel strictly anaerobic, endospore-forming, rod-shaped, Gram-positive bacterium, designated strain SGB2(T), was isolated from a mixed culture from a pond sediment during screening for sulfate-reducing bacteria capable of degrading cyanophycin (CGP). In this study, the taxonomic characterization of this mesophilic, proteolytic Clostridium isolate and the role which it, and its phylogenetic relatives, may play in peptide degradation and in the sulfur cycle are reported. Strain SGB2(T) was a commensal strain, utilizing CGP degradation products produced by other micro-organisms. Cells were motile until sporulation, forming oval, terminal spores that swell the cells. It showed optimum growth at 34 degrees C, pH 6.6 and in the absence of NaCl. Strain SGB2(T) utilized proteinaceous compounds such as peptone, Casamino acids, gelatin and trypticase soy, in addition to several amino acids and pyruvate. Utilization of many of these compounds was enhanced in the presence of thiosulfate. The isolate was unable to use any of the carbohydrates or alcohols investigated or CGP as carbon and energy sources. Thiosulfate and elemental sulfur were used as terminal electron acceptors. Phylogenetic analysis revealed that strain SGB2(T) belongs to the low-G+C-containing Clostridiales group. It exhibited 99 % 16S rRNA gene sequence similarity to its closest relatives Clostridium thiosulfatireducens Lup21(T) and Clostridium subterminale DSM 6970(T). DNA-DNA hybridization values with these two strains were 39.4 and 42.1 %, respectively. Based on phenotypic, genotypic and phylogenetic characteristics, we conclude that the isolate represents a novel species of the genus Clostridium, Clostridium sulfidigenes sp. nov. The type strain is SGB2(T) (=DSM 18982(T) =ATCC BAA-1538(T)).

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

ABSTRACT 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 β-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 β-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.

Cyanophycin-degrading bacteria in digestive tracts of mammals, birds and fish and consequences for possible applications of cyanophycin and its dipeptides in nutrition and therapy.

Aims:  To determine the susceptibility of cyanophycin granule polypeptide (CGP) to degradation by several mammalian, avian and fish gut flora.

Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1

Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cph E revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.

Biotechnologische Herstellung von Dipeptiden und deren Anwendungen

Although the functions and applications of dipeptides have been poorly studied, compared to proteins or amino acids, their unique properties have long been appreciated. A major obstacle to the industrial production of dipeptides has always been their relatively complicated chemical synthesis. However, novel methods for the economical production of specific dipeptides through biotechnological processes have been developed recently.