| Г-polyglutamic acid (γ-PGA) is a biodegradative polypeptide synthesized by microorganism. Г-PGA produced by microbes usually consists of thousands of glutamate monomers and its molecular weight (Mw) is 2 ×105~ 1 × 106 Dalton. PGA produced by different bacterium strains has different molecular weight. F-PGA can be degradated to glutamate that is endogenic material, so it doesn't have cumulative, toxic and side effect. In PGA, alpha-amino groups are joined with y-carboxyl groups by amide linkage. So there are plenty of carboxyl groups on the side, chain. This is the reason why PGA has good water solubility and is prone to be modified. Basing on these characteristics, PGA is fit for being used as drug delivery system.This article roundly discusses the survey about the application and development of γ-PGA as slow and controlled release drug carrier and systemically studies the preparation method of γ-PGA-ferment-hydrolyze method.The main researches we did are: (1) Filtration and identification of high-yield strain of γ-PGA; (2) Production of γ-PGA with high efficiency; (3) Separation and purification of γ-PGA; (4) Preparation of γ-PGA used as slow and controlled release drug carrier.1. Filtration and identification of PGA's high-yield strain.Using separation culture medium as a specific "sieve", we got a high-yield strain and named it as Bacillus subtilis NX-2. Its biological shape and property: bacillus subtilis, gram-positive, the cell size is 0.7-1.0×1.3~2.0μm, the spore size is 0.7-0.9×1.0-1.5μm, columniform, middle outgrowth, thin wall. This bacterium can grow at 15℃~50℃ and the optimum temperature is 30℃~40℃.2. Production of γ-PGA with high efficiency.In the article we studied the effect of some fermentation factors such as carbon source, nitrogen source, pH, ion, temperature, rotate rate ect. using viscosity, absorbance and yield as criterion. We found: (1) 0.5μ yeast extract is the best nitrogen resource; (2) Glucose, maltose, glycerol, fructose and sucrose etc. can all be carbon source, maltose is the best; (3) If glutamate not added into the medium, NX-2will not synthesize PGA. When 3% glutamate is added, γ-PGA can largely cumulated in the fermentation liquid and the glutamate will not decrease; (4) The production of PGA which is cultured in shake flask in laboratory under the optimum conditions. The cost of the whole produce progress is low. The optimum fermentation conditions we confirmed are that 0.05% MgSO4 7H2O and 0.2% K2HPO4 are added into the medium as mineral element; 0.5% yeast extract as nitrogen source; 2% glucose as carbon source; 3% L-glutamate sodium; pH is 7.0; temperature is 37℃, rotate rate is 220rpm.3. Separation, purification and identification of PGA.The fermentation liquid with high viscosity containing γ-PGA was centrifugated to eliminate the thalli. 4 times volume industry alcohol was added to the supernatant, and the γ-PGA precipitated. The ppd. PGA was dissolved in deionized water, dialyzed to eliminate the little molecules and dried in vacuum at 60℃ to get the white crystal.The product was analyzed and identified by element analysis, UV, IR, H'-NMR, C14-NMR etc. and it was proved to be γ-PGA.We determined the molecular weight of γ-PGA by electrophoresis. Utilizing the characteristic of the carboxyl group in side chain, we chose alkaline methylene blue (0.5%/3% acetic acid), which can conjugate with carboxyl group as dye, polyacrylamide (1%) as gel carrier.. The electrophoresis buffer solution is 90mmol-L-1 tris/boracic acid buffer solution (pH8.5) and the sample buffer solution is 63.5 mmol-L-1 tris/boracic acid buffer solution (pH6.8). The determination result was that the Mw of the produced PGA was 2xl05~3.6xl05Da, the degradated PGA 2×104~6×l04Da.4. Preparation of low-molecular weight γ-PGA.We took method of hydrolyzing macromolecular PGA with acid and heat to get low-molecular weight γ-PGA. Thinking for the follow up purification step, the hydrochloric acid's concentration added to the solution...
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