Breeding Of γ-Poly Glutamic Acid-Producing Bacillus Licheniformis, Optimization Of Fermentation Conditions And Product Purification

γ-Poly glutamic acid (γ-PGA) is a macromolecular peptide that consists of D- and L-glutamic acid and is polymerized by γ-glutamyl bonds. γ-PGA is water soluble, biodegradable and biocompatible. Therefore, potential applications of γ-PGA and its derivatives have been of interest in a broad range of industrial fields such as food, cosmetics, medicine and water treatment.In this paper, the screening of high γ-PGA producing mutant was investigated. The liquid fermentation in shaking flask and fed-batch fermentation in 3.7L fermentator of the mutant was optimized, and isolation and preliminary purification of γ-PGA was finished.Bacillus licheniformis CICC10099 was mutagenized by ⁶⁰Co γ-irradiation. The results showed 200 Gy was the optimum irradiation dosages with high positive mutation rate of 13 % and death rate of above 90 %. A new mutant Bacillus licheniformis S16 was isolated after ⁶⁰Co γ-irradiation mutation and had good genetic stability of high γ-PGA producing after several generation cultures. Compared with the yield of the original strain CICC10099, γ-PGA yield of the mutant S16 was increased from 9.8 g·L^-1 to 16.9 g·L^-1, and the increase rate was 72.4 %.Single-factor designs were used to determine the ranges of value of each related factors which may effect the production of γ-PGA. Then a Plackett-Burman design was used to evaluate the influence of related factors. Glycerol and MnSC₄·H₂O influenced γ-PGA production positively while K₂HPO₄ and liquid volume negatively. The concentrations of Glycerol, MnSO₄·H₂O and K₂HPO₄ were further optimized using central composite designs and response surface analysis. The optimized condition allowed the γ-PGA concentration to be increased to 19.23g·L^-1.Batch liquid fermentation of γ-PGA was conducted in 3.7L bioreactor. At agitation speed 500r·min^-1, aeration 1.10VVM, temperature 37℃ and pH 7.5, when pH was not controlled, the pH of broth continuously decreased, which negatively effected the growth of strain and production of γ-PGA; while pH is controlled at 6.5, the maximum biomass and concentration of γ-PGA were bigger than the uncontrolled course, and at fermentation time 80h γ-PGA concentration reached 23.49 g·L^-1.Purification of γ-PGA mainly included five steps: removing cell by centrifugation, first ethanol precipitation, dialysis, second ethanol precipitation and dried at 40 °C under vacuum. The purified sample was completely hydrolyzed in 6M HC1 at 110 °C for 36h. Paper chromatography of showed that there was only one stain, which equaled to that of pure L-glutamic acid. The concentration of L-glutamic acid of the hyrolysate was determined by HPLC, and the concentration of pure γ-PGA in the purified sample was 95.7% through calculation. The purified sample showed an absorption peak at 228nm and took 95.46% of the whole peak areas when it was analyzed by HPLC at 220nm.