Biosynthesis of highly pure poly-γ-glutamic acid for biomedical applications

Abstract

The remarkable properties of poly-aminoacids, mainly their biocompatibility and biodegradability, have prompted an increasing interest in these polymers for biomedical applications. Poly-γ-glutamic acid (γ-PGA) is one of the most interesting poly-aminoacids with potential applications as a biomaterial. Here we describe the production and characterization of γ-PGA by Bacillus subtilis natto. The γ-PGA was produced with low molecular weight (10-50 kDa), high purity grade (>99 %) and a D: -/L: -glutamate ratio of 50-60/50-40 %. To evaluate the feasibility of using this γ-PGA as a biomaterial, chitosan (Ch)/γ-PGA nanoparticles were prepared by the coacervation method at pH ranging from 3.0 to 5.0, with dimensions in the interval 214-221 nm with a poly-dispersion index of ca. 0.2. The high purity of γ-PGA produced by this method, which is firstly described here, renders this biopolymer suitable for biomedical applications. Moreover, the Ch/γ-PGA nanocomplexes developed in this investigation can be combined with biologically active substances for their delivery in the organism. The fact that the assembly between Ch and γ-PGA relies on electrostatic interactions enables addition of other molecules that can be released into the medium through changes from acidic to physiological pH, without loss in biological activity.

Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

“…Results c-PGA cytotoxicity in MSC and NC 2D cultures g-PGA is a polyamino acid that was microbially produced in our lab 36 and has been used by us on the basis of its ability to form polyelectrolyte complexes with cationic polymers, such as chitosan (Ch), by electrostatic interaction. 42,49 g-PGA/Ch multilayer films (prepared with [g-PGA] = 0.2 mg/mL) were not cytotoxic for human fibroblasts in culture.…”

“…36 Briefly, g-PGA was produced by B. subtilis in E medium containing 3% L-glutamic acid and 2% citric acid 46,47 at 37°C and 120 rpm for a period of 72 h. The polymer was isolated from the bacterial culture broth following several steps to eliminate the medium components and other bacterial metabolites. Polysaccharides were removed by acidic hydrolysis using 6 M H 2 SO 4 at pH 3.0, proteins were eliminated by incubation with proteinase K, and the resultant solution followed a dialysis step to remove salts and other small molecules.…”

“…18 In particular, g-PGA is obtained from fermentation of several Bacillus strains, 18,35,36 and is constituted by D-and L-glutamic acid residues. 21,36,37 g-PGA biodegradability and nonimmunogenicity make this polymer attractive for tissue regeneration.…”

“…18 In particular, g-PGA is obtained from fermentation of several Bacillus strains, 18,35,36 and is constituted by D-and L-glutamic acid residues. 21,36,37 g-PGA biodegradability and nonimmunogenicity make this polymer attractive for tissue regeneration. 18,21 Besides chemical degradation induced by prolonged exposure to extreme pH and high temperatures, 18 only g-PGA, but not a-PGA, can be degraded by enzymatic/biochemical pathways, giving rise to glutamic acid residues.…”

“…39,41 Our team has previously described g-PGA production by Bacillus subtilis natto and subsequent purification to yield a polymer with low molecular weight (Mw) (10-50 kDa), high purity grade (*99.5%), and a D-/L-glutamate ratio of 50-60/50-40%. 36,42 This high purity level is very attractive since commercially available g-PGA usually has low (c.a. 70%) or unknown purity levels, 33,[43][44][45] which may hinder its application in the biomedical field, and especially when it comes to clinical translation.…”