STUDIES ON NATURAL AND EDIBLE BIOPOLYMERS. ISOLATION, CHARACTERIZATION AND CHEMICAL MODIFICATIONS OF POLY(Γ-GLUTAMIC ACID) (Γ-PGA) FROM BACILLUS SUBTILIS

Poly(γ-glutamic acid) (γ-PGA) is a unusual anionic, water-soluble homopolyamide composed of D- and/or L-glutamic acid monomers, connected by amide bonds between α-amino and γ-carboxyl groups, mainly produced by Gram-positive bacteria of the genus Bacillus. γ-PGA is hygroscopic and water-soluble; in the salt form is able to form gels containing water in more than 3,000 times its weight. It is fairly resistant to hydrolysis, although it is degraded in basic medium at high temperature; it appears to be insensitive to common proteases such as pepsin or tripsin. γ-PGA is completely innocuous and edible. The popular food known as natto in Japan or dan-douchi in China, a soybean fermented compound that contains about 1% of γ-PGA, has formed part of the diet of Asian people for hundreds of years. Due to its biodegradability and to the intrinsic absence of toxicity as well as to the classification as GRAS (Generally Regarded As Safe) of the main producers, i.e. Bacilli, γ-PGA represents one of the naturally occurring polymers of interest for the production of environmentally friendly derivatives. Although having been known and even used for industrial applications for a long time, and considerable efforts having been spent in investigating its chemical and biological properties, much lies ahead to be elucidated about this polymer biosynthesis and conformational aspects. In addition, its potential applications appear to be still almost unexploited in the Western world. This Ph.D. thesis deals with the bioproduction, characterization and derivatization of γ-PGA from Bacillus subtilis in order to obtain novel biomaterials useful for biopackaging, biomolecule immobilization, and as drug carriers. In particular, the problems we have addressed were: the optimization of productivity, by means of genetic engineering technologies; the characterization of the polymer, with regard to its stereochemical composition and molecular weight, which deeply affect its chemical-physical properties and reactivity; the development of effective methodologies for chemical modification of the polymer, in view of preparing derivatives of pharmaceutical and technological interest. Finally, we also established a synthetic approach for the preparation of standard samples with controlled stereochemistry necessary for conformational studies.