Production Of γ-Polyglutamic Acid By Fermentation

As a kind of biological macromolecules, ?-polyglutamic acid (?-PGA) iswater-soluble, biodegradable, edible and non-toxic toward humans and theenvironment . The polymer is made of L- and D-glutamic units linked through the-amine and the ?-carboxylic acid groups. ?-PGA and its derivative offer a wide rangeof unique applications including being used as thickener, humectant, bitterness,relieving agent, cryoprotectant, sustained release materials, drug carrier curablebiological adhesive, biodegrade fibers, highly water absorbable hydrogels, biopolymerflocculants heavy metal absorber and animal feed additives . It have been studied inmany country because its potential application. This article deals with biosynthesis of?-PGA by fermentation with Bacillus subtilis PGAN-12 which was screened in ourlaboratory. Work has been carried out on the nutrient requirement for cell growth andimproving conditions for ?-PGA productivity.(1) High purity???-PGA was prepared by ultrafiltration during which smallmoleculecompounds were removed . The purity of ?-PGA was confirmed by GPC and HPLC.Then the concentration of ?-PGA in broth was determined on the above refined?-PGA. Size-Exclusion Chromatography was used to analysis the concentration of?-PGA in broth directly. Determination of ?-PGA in broth was on BEKMANCOULTER System Gold 125/166, Pharmacia Biotech SuperoseTM6 Size-ExclusionChromatography column . Data collection and process use 32 Karat 5.0 versionchromatography software. Elution was 0.15M NaCl+0.05M phosphate buffer (pH7.2). Tri-dilute water was filtered by 0.45m soluble ultrafilter membrane, UVwavelength?=210nm . The flow rate of the pump was 0.4ml/min. Compared withformer methods, it's convenient and high-accuracy .(2)PGAN-12 could produce a large amount of ?-PGA in the medium containingglucose and sodium glutamate. The seed medium contained: glucose 20g/L,yeastextract 5g/L,K2HPO4.3H2O 2 g/L,MgSO4 0.05g/L; sodium glutamate 10g/L, andculture time of seed was 16h; Inoculation size was 1%. The suitable carbon source IIIwas glucose, however, when organic acids in TCA cycle including critic acid wereused as carbon sources, PGAN-12 produced no ?-PGA. The optimum nitrogen sourcewas yeast extract. PGAN-12 is a ?-PGA producing strain depending on L-glutamicacid in the medium, In the presence of 70g/L sodium glutamate, the highest ?-PGAconcentration reached up to 18.32g/L, while 30g/L sodium glutamate was added,14.2g/L?-PGA was obtained with highest yield of 62.1%. Through orthogonalexperiments of sodium glutamate, glucose, yeast extract, the optimum medium wasobtained . It contains : glucose 40g/L,yeast extract 8g/L,K2HPO4.3H2O 2 g/L,MgSO40.05g/L,sodium glutamate 40g/L. As for fermentation condition , optimum initial pHwas 7.5, the same as nature pH; The 500mL flash contained 80mLmedium withcultivation at 32℃and at 220rpm. There was 18.5 g/L ?-PGA in this medium after 24hof fermentation.(3)This article deals with biosynthesis of poly –?-glutamic acid by fermentation inour lab ,we studied on fed-batch in order to improve production of ?-PGA. Throughtime course of ?-PGA syntheses in flash, glucose was found to be a main substratefor ?-PGA synthesis. Based on this , comparison of several different fed-batchmedium, the optimum one was gained, it contains: glucose 70g/L,yeast extract 20g/L,K2HPO4.3H2O 40 g/L, MgSO4 0.1 g/L,sodium glutamate 70g/L, feeding timewas 18h ,and production of ?-PGA reached 23.8g/L after 28h when glucose wasdepleted from the culture medium .(4) In the course of ?-PGA fermentation by Bacillus subtilis PGAN-12, metal ionsCa2+,Mn2+,Mo6+ ,Mg2+,Co2+,Fe3+ ,K+ in the medium had certain effect onsynthesis and stereochemical composition of ?-PGA. It was found that the yield of?-PGAwas greatly increased by Ca2+,Mn2+,Mo6+ ,Mg2+,Co2+,K+ in their optimalconcentration, Through the combination of those metal ions, an appropriate...