Fermentation Condition Optimization Of Poly-γ-glutamic Acid And Its Structure Characterization

As a kind of macromolecular material,γ-polyglutamic acid is water soluble, biodegradable,nontoxic,harmless towards humans. It is a polymer materials which is made of D-glutamic acid and L-glutamic acid and extracted originally from the capsule of some microorganisms.γ-PGA can be used as absorbent moisturizer,drug delivery materials,antifreeze,flocculant,superabsorbent,biodegradable fiber,animal feed additives and heavy metal adsorbent,widely being used in food, agriculture, medicine,cosmetics and the environmental protection.This paper deals with Synthesis ofγ-PGA by fermentation with Bacillus subtills NOBEL-007 which was screened in our laboratory.Work have been carried out on the fermentation conditions, molecular structure and molecular weight ofγ-PGA,thus provided theoretical basis for the industrial production ofγ- PGA.Single-factor design was used to study the effect of the carbon sources, nitrogen sources,sodium glutamat and inorganic salts onγ-poly glutamic acid production.Then selected the ranges of value of each related factor,making preparation for further optimization.From the single-factor test,we got appropriate fermentation medium: glucose 60g/L,yeast extract 6.0g/L,sodium glutamate 60g/L,NH₄Cl 3.0g/L, CaCl₂ 0.2g/L,K₂HPO₄·3H₂O 3.0g/L,MgSO₄·7H₂O 0.75g/L,MnCl₂·4H₂O 0.04g/L.Using SAS software to design Plackett-Burman experiment, the results showed that the effect of yeast extract,sodium glutamate and CaCl₂ on production ofγ-PGA was the most significant,so these three factors should be optimized emphatically.In order to approach the maximum production area ofγ-PGA and establish an effective fitting equation,we designed steepest ascent experiments according to the analysis of results of the Plackett-Burman experiment.Design Box-Behnken experiment according to the results of steepest ascent. Analysized the results and established regression equation, and then combined the maps of response surface and contour which were droped with Design-Expert software,we get the optimal fermentation medium ofγ-PGA:glucose 50g/L,yeast extract 4.18g/L, sodium glutamate76.89g/L, NH₄Cl 3g/L, CaCl₂ 0.1422g/L, K₂HPO₄·3H₂O 3g/L, MgSO₄·7H₂O 0.75g/L, MnCl₂·4H₂O 0.04g/L,and the theoretical yield ofγ-PGA was 43.39g/L. To verify the prediction results,we found that the Experimental values ofγ-PGA production was 43.26g/L which closed to the theoretical prediction, thus Demonstrated that the credibility of the model is high. Compared to the former 21.26g/L which was not optimized,γ-PGA yield increased by 1.035 times.Identified and analysized fermentation products by using infrared spectroscopy and nuclear magnetic resonance,we comfined that the ermentation products wasγ-PGA actually, and also got its molecular structure.Using paper chromatography and ninhydrin colorimetry to assay the composition and the purity ofγ-PGA,we foundγ-PGA was made of pure glutamic acid,and the purity ofγ-PGA was. 85.13%.The isoelectric point ofγ-PGA was 3.5 measured by turbidimetry/spectro photometry. Finally,SDS-PAGE electrophoresis was used to measure molecular weight ofγ- PGA, the same like other amino acids,γ-PGA also had polymer molecular weight,and the molecular weight range from 100kDto 600kD.From the study on water absorption and water holding capacity ofγ-PGA,and the effect ofγ-PGA on seed germination ratio in rats,we found thatγ-PGA had the same soil water holding capacity as PAM,and its ability to absorb water and increase seed germination rate was better than PAM.And WE also found that water absorption ofγ-PGA in distilled was the strongest,followed by saline, worst in wells and tap,and the similar results can be got by water retention experimentIn soil.The water holding capacity was hignest when the concentration ofγ-PGA was 12g/L.