Screening of High Producing Strain for γ-poly Glutamic Acid and Optimization of Fermentation Conditions

Abstract: γ-Polyglutamic acid(γ-PGA) 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. Soγ-PGA will be widely used in medicine, food and agriculture fields. The paper concentrates on the screening and identification the strains producingγ-PGA, the optimization of medium and cultivation conditions. Main results are as follows:1. The strain of HD11 producingγ-PGA was selected from lobster sauce. With the characters of gram-positive, the cell size of 1.7~2.1μm×0.5~0.8μm, having spores and the optimum temperature of 37℃, the strain of HD11 was identified as Bacillus and named as Bacillus sp. HD11 by morphological, physiological and biochemical identification analysis.2. The screening of high yield strains producingγ-PGA was studied. Bacillus sp. HD11 was used as original strain forγ-PGA production, which was treated with multi-mutagenicity of ultraviolet, diethyl sulfate, and nitrosoguanidine. Bacillus sp. HD-F9 was isolated, and theγ-PGA yield attained 10.72 g/L increasing by 56.3 %, and the mutant showed genetic stability during 10 generation.3. The liquid fermentation conditions of the strain HD-F9 were optimized by single-factor experiment. The optimal culture conditions were as follows: initial pH 7.5, temperature 37℃, seed age 10 h, inoculum 3 %(V/V), 40 mL medium in 250 mL shake flask, rotation speed of 200 r/min, and culture time 48 h.4. The liquid fermentation medium of the strain HD-F9 was studied. Firstly, the single-factor experiments of carbon, nitrogen, monosodium glutamate, NaCl and metal ions were carried out, then the orthogonal experiment of the main components of the medium was designed. Finally, the response surface methodology was used to deeply optimize the fermentation medium. The results showed that the strain HD-F9 was glutamate-dependent bacteria, and the optimal fermentation medium were 60.03 g/L glucose, 8.1 g/L yeast, 41.7 g/L monosodium glutamate, 2.5 g/L KH2PO4, 0.31 g/L MgSO4. The final yield ofγ-PGA reached 14.56 g/L increasing by 35.82 %.5. Using the strain of HD-F9, the batch culture and fed-batch culture was preliminarily studied in 7 L fermentor. The results showed thatγ-PGA is secondary metabolite.γ-PGA would be quickly degraded for lack of carbon source,γ-PGA degrading enzyme may existed in the strain of HD-F9. Therefor controlling the glucose concentration is important to the synthesis ofγ-PGA. The production ofγ-PGA was seriously affected by large foam, so defoaming is the key step in the fermentation ofγ-PGA …
Key words: γ-Polyglutamic acid; Screen; Mutagenicity; Fermentation; Optimization

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