Study On Preparation Of γ-PGA And Its Hydrogel

Poly(y-glutamic acid) (y-PGA) is an unusual anionic polypeptide that is made of D-and L-glutamic acid units connected by amide linkages between a-amino and y-carboxylic acid groups. y-PGA is water soluble, biocompatible, biodegradable, independent of oil resources and non-toxic toward humans and environment. Therefore, potential applications of y-PGA and its derivatives have been of interest in the past few years in the fields of medicine, food, cosmetics, plastics and water treatment. The preparation of y-PGA was investigated systematically in this paper. One bacterium, which could produce a large amount of y-PGA, was screened and isolated. The biosynthetic mechanism of y-PGA was studied and the strain showed some unique characteristics. By this strain, y-PGA.could be produced at high concentration with high productivity and yield, similar to the best result which have been reported.The preparation and application of γ-PGA hydrogel was also carried out due to its promising commercial value. ?The major work of this thesis was showed as follows:Bacillus subtilis NX-2 with high γ-PGA productivity was isolated from soils using the isolation medium containing L-glutamic acid. The patents had been applied to protect this strain and method to produce γ-PGA. This strain was deposited in China General Microbiogical Culture Collection Center, named Bacillus subtilis NX-2 with the accession number of CGMCC No.0833.Assimilability to carbon source by NX-2 was unique. NX-2 produced no y -PGA without additional carbon source. Glucose, sucrose, maltose, starch or glycerol could be the suitable carbon sources. In the case of glucose or sucrose, very high yield of y -PGA ,about 30 g/1, was attained. Lactose was good for NX-2 cell growth, but had little effect on y -PGA production. However, Y -PGA could not be produced when citric acid was used as carbon sources, which is different from the other bacteria reported before. Glycerol was also a good carbon source. When 30 g/1 of glycerol was added, 24.3 g/1 y -PGA was accumulated.The effect of L-glutamic acid on the production of Y -PGA by B. subtilis NX-2 was investigated. In the absence of glutamic acid, no Y -PGA was produced. Exogenous glutamic acid was essential for the production of Y -PGA. The amount of y-PGA increased upon addition of glutamic acid to the medium. When 70 g/1 of L-glutamic acid was added to the medium, the highest y-PGA concentration of 41 g/1 was obtained. But apparent conversion rate to L-glutamic acid substrate was relatively low, only 58% in this case. The amount of y-PGA reached 30.2 g/1 in the presence of 30 g/1 L-glutamic acid after the short incubation time of 24 h, resulting in the high apparent conversion rate of 101% and high productivity of 1.26 g/l/h. From experimental results, it was assumed that that glutamic acid unit in Y -PGA came from two sources: one is from L-glutmaic acid added to the medium and the other is from glucose through the de novo pathway of glutamic acid synthesis.Optimization on the production condition of y-PGA by NX-2 was carried out. The suitable medium was as follows (g/1): glucose 30,yeast extract 5, L-glutamic acid 30, KHaPO0.5, and MgSO4 0.1. Under this condition, the maximum Y -PGA concentration of" 30.2g/l was obtained in 24h with a productivity of 1.26g L-1 h-1, which was higher than other strains reported before.The y-PGA biosynthesized by B. subtilis NX-2 was characterized by FT-IR, HR/MAS, HR/MAS NMR and Y -glutamyltranspeptidase enzymatic hydrolysis to confirm that the sample was y-PGA which contained the structure as follows:y-PGA hydrogel had been prepared from microbial y-PGA and ethylene glycol glycidyl ether in aqueous medium. The optimal conditions were as follow: y-PGA concentration was 12%, the ratio of weight of ethylene glycol glycidyl ether to y-PGA was 18.75%, and pH5.0.The reaction was carried out at 60 for 44-48h. y-PGA hydrogel, which could absorb 950 times of water of its weight, was obtained with 95% gel yield. The mechanism of crosslink reaction was summarized for the first time...