Screening Of The Strains Producing Poly-γ-Glutamic Acid

Poly-γ-glutamic acid is an extracellular macromolecular peptide that consists of D- and L-glutamic acids throughγ-glutamyl bonds.γ-PGA is water-soluble, biodegradable, edible and non-toxic toward human and the environment. Therefore, potential applications ofγ-PGA and its derivatives have been of interest in the past few years in a broad range of industrial fields such as food, cosmetics, medicine and water treatment.γ-PGA and its derivatives offer a wide range of unique applications including being used as biopolymer flocculants, thickener, humectant, drug carrier, sustained-release material, highly water absorbable hydrogels, heavy metal absorber. The paper concentrates on the screening and identification the producingγ-PGA strain, the optimization of culture medium and cultivation conditions and the separation and purification ofγ-PGA from the fermentation broth. Main results are as follows:A bacterial strain L536 was isolated from the bean sauce and the viscosity of the culture broth achieved 0.41Pa·s. The product of the strain L536 was a homopolymer of glutamic acid, which was demonstrated by the paper chromatography analysis and amino acid composition analysis. With the following identifications of colony morphology, physiological and biochemical characteristics and 16S rDNA gene sequence, the strain L536 was identified as Bacillus.amyloliquefaciens, named B.amyloliquefaciens L536. The research on B.amyloliquefaciens producing polyglutamic acid has not been reported in domestic so far.The culture medium and the fermentation conditions of the strain L536 producingγ-PGA were optimized by single-factor experiment and orthogonal experiment. The optimum culture conditions were as follows (g/L): sodium citrate 18, sodium glutamate 40, soybean flour 20, NaCl 15, NH4Cl 7, MgSO4·7H2O 0.9, K2HPO4 0.5, MnSO4·H2O 0.2, CaC12·2H2O 0.2, initial pH 7.0, temperature 37℃, inoculation amount 3%(V/V), rotation speed of rocking bed 200r/min, broth's volume in shake flask 35mL/250mL and culture time 48h. Under the optimum condition, the viscosity of the culture broth achieved 1.6Pa·s, 3 times the value to the initial, and the production ofγ-PGA was from 15g/L to 22.57g/L.γ-PGA was purified by ethanol precipitation,dialysis and drying under vacuum. The concentration of pureγ-PGA was 93%.The characterization of'γ-PGA was investigated by paper chromatography, UV and IR.Paper chromatography of the purifiedγ-PGA showed that there was only one stain, which indicated the polymer was composed of glutamic acid. The UV scanning demonstrated the purifiedγ-PGA showed an absorption peak at 204nm. The IR chromatograph of the sample basically tallied with that ofγ-PGA standard sample prepared by Nanjing Industry University. It indicated the polymer wasγ-PGA.