In a microbial (or higher) system, proteins are synthe­sized with the help of an information template in the form of an mRNA molecule and the translatory machin­ery of ribosomes and amino acylated tRNA; however, poly(amino acid)s are synthesized enzymatically without the requirement of an information template.

While proteins owe their functionality to the tertiary structure that they attain, the poly(amino acid)s owe it normally to their physical properties, usually the num­ber of repeating units more dominant in a pool of poly (amino acid)s. Another striking difference between pro­teins and poly(amino acid)s is the fact that one type of a protein will contain an exact number of amino acids, while poly(amino acid)s display wide polydispersity, i. e. in a single organism, the size of the same poly (amino acid) will vary. The fact that these polymers are biocompatible with human physiology and for applica­tions other than pharmacology, the polymer is biode­gradable, makes it an attractive alternative to the widely used polymers obtained through the petrochem­ical route, which are often nonbiodegradable, and end up accumulating in the environment. These poly(amino acid)s have been shown to be useful in multitude of applications, like controlled drug release, preparation of bioplastics, use as antimicrobial additive in food, as well as superabsorbers (replacement of polyacrylate gels) (Obst and Steinbuchel, 2004). They may also be used as a viable source of dipeptide neutraceuticals (Sallam and Steinbuchel, 2010).

There are three identified poly(amino acid)s so far that have been reported to be produced from microbial source; they are cyanophycin, poly-g-glutamate (PGA)
and e-poly lysine (Figure 19.3). In poly(glutamic acid) the amide linkages are formed on the a-amino group to then g-carboxyl group in the polymer backbone, whereas in poly(lysine), the a-carboxyl group is linked to the є-amino group of lysine. In the case of cyanophy­cin, almost equivocal amounts of arginine and aspartic acid are arranged as a polyaspartate backbone, with arginine moieties linked to the b-carboxyl group of almost every aspartic acid residue.

Cyanophycin/Cyanophycin Granule Polypeptide

Cyanophycin is the ideal nitrogen storing molecule, because every repeating unit has about five atoms of ni­trogen, and it is insoluble at physiological conditions in­side the cell protoplasm. Due to its insolubility, it does not cause detrimental shifts in the osmolarity of the cell, hence helps in cell survival (Oppermann-Sanio and SteinbUchel, 2002). Cyanobacteria normally pro­duce cyanophycin when the organism senses a decrease in sulfur, phosphate and significantly by the decrease in nitrogen concentration in the surrounding milieu (Lin et al., 2012). Apart from cyanobacteria, cyanophycin granule polypeptide (CGP) has been found in some strains of Synechococcus sp. (Hai et al., 1999).

FIGURE 19.3 Amino compounds from microbial sources. (For color version of this figure, the reader is referred to the online version of this book.)

Production of Cyanophycin

Cyanophycin is enzymatically produced by the action of cyanophicin synthases on small primers of cyanophy­cin. Because of the slow growth of cyanobacteria in pho­tobioreactors, large-scale production of CGP is hindered by the lower cell densities and thus used to get only low yield of CGP with respect to the cell dry mass (CDM). To solve this problem cyanophycin synthase gene or CphA gene has been identified and cloned into a wide range of organisms, from E. coli to eukaryotic microbes like S. cer — evisiae (Steinle et al., 2008), commercially important strains like Ralstonia eutropha, C. glutamicum, Pseudo­monas spp., etc. (Aboulmagd et al., 2001) and even higher plants like potato and tobacco (Neumann et al., 2005). Recombinant CGP produced were shorter in length (21—35 repeating units), along with a small per­centage of arginine replaced by incorporated lysine. Till some time, the highest CGP production was attained by using Acinetobacter calcoaceticus, with a value of 48% CDM, with the addition of exogenous arginine, along with the addition of other carbon and nitrogen sources (Elbahoul et al., 2005). The economic viability of recom­binant strains is always a problem for large—scale usage, due to the sheer amount of antibiotics that have to be added to the medium. However, the hunt for a commer­cially compatible strain for economical production of CGP led to a method for obtaining high cell densities and high yield of CGP with Ralstonia eutropha. The strain is poly hydroxy butyrate (PHB) negative (the wild type produces PHB) and was devoid of the 2-keto-3-deoxy — phosphogluconate aldolase (eda) gene (Lin et al.,

2012) . The plasmid carrying the cphA gene was con­structed along with the eda gene; for the microbe to sur­vive, the plasmid had to be retained, and in other words, a selective pressure for plasmid retention was achieved, without the use of antibiotic resistance genes, thus making the process economically viable.

In general, the production was optimized with a basic mineral medium, Mineral Salts Medium (MSM), with sufficient supplements of fructose, NH3, K2SO4, MgSO4.7H2O, Fe(III) NH4-citrate, CaCl2.2H2O, and trace elements. A 30 l pilot study gave promising yields of water-insoluble CGP and water-soluble CGP, contrib­uting to 47.5% and 5.8% (w/w) of CDM, and a cell den­sity as high as 57 g/l CDM was obtained (Elbahloul et al., 2005). CGP is normally purified by acid extraction, which involves the solubilization of CGP in acidic solu­tions of pH 1, followed by washes with distilled water, which renders it insoluble again.

Alternative production strategies include the use of molecular farming approach, and expression of the cphA gene in certain specific tissues of selected plants. Potato and tobacco have been successfully transformed with the cphA gene; however, production of CGP within the plant tissues would lead to slow growth and fleshy leaves. Use of the stable cyanophycin synthase for direct synthesis of cyanophycin has been suggested by certain authors as an alternative to the intricately controlled fermentative production of cyanophycin (Hai et al., 2002).

Biodegradability of Cyanophycin

Biodegradation of cyanophycin is observed in all the organisms that naturally produce the polymer, as it serves as a reserve carbon and nitrogen pool. Cyanophy — cin can be depolymerized by intracellular cyanophyci — nase, which also has been isolated from Synechocystis sp. strain PCC6308 (Hai et al., 2002). The cyanophyci — nase gene or the cphE gene has been found to be located downstream of the cphA gene. The cyanophycinase enzyme does not cleave the polymer into arginine and aspartate. Recent studies have shown that cyanophycin is degraded by most of the gut bacteria through the anaerobic route within a time period ranging from 1 day to 7 days (Sallam and Steinbuchel, 2009). The above studies have opened the doors to the use of cyano — phycin directly as nutrient supplement.

Applications for Cyanophycin

Cyanophycin can be hydrolyzed to its constituent amino acids, aspartic acid, and arginine. These amino acids may be utilized directly in food and pharmaceu­tical applications. Cyanophycin can be stripped off of arginine through chemical modifications, so as to pro­duce polyaspartate. Polyaspartate is a polyanionic poly­mer that can be utilized for production of biodegradable surfactants, and can be utilized for applications pertain­ing to polyacrylate (Schwamborn M, 1998). It has been discovered recently that cyanophycin can be degraded by the gut bacteria obtained from a diverse group of or­ganisms ranging from mammals, birds, and fishes. This opens routes for the use of cyanophycin directly as a nutritional substrate, instead of constituent dipeptides or amino acids, which would require additional invest­ments of time and money (Sallam A and Steinbuchel A

2009) . Even though a considerable amount of research has been carried out for the viable production of cyano — phycin, the complete potential for the various bulk chemicals that may be obtained from cyanophycin is not attained yet.