Screening Of High Producing Strain For γ-Poiy-Glutamic Acid

Poly-γ-glutamic acid which can be produced by microorganisms, is a compound ofequal poly amino acid that consists of glutamic acid through γ-glutamyl bonds. γ-PGAis water-soluble, biocompatible, edible and non-toxic toward human and theenvironment. Thus, γ-PGA and its derivatives have a wide range of unique applicationsin medicine, food, agriculture and cosmetics. The paper concentrates on the screeningby mutation, anion exchange column chromatography of γ-PGA, structural analysis, andthe research about batch fermentation of γ-PGA. Main results are as follows:A strain, designated Bacillus amyloliquefaciens DU-44, producing γ-PGA with ahigh yield was successfully obtained after Bacillus amyloliquefaciens L536was treatedby UV, DES or DES-UV. In addition, it was genetically stable. The yield of γ-PGA wasincreased from20g/L to35g/L.Carbon and nitrogen sources for the mutant strain were optimized by single-factorexperiment. The optimum carbon and nitrogen sources were as follows (g/L): sodiumcitrate20, soybean flour extracting80,(NH4)2SO44.6. Dissolved oxygen was a keyfactor to fermentation of γ-PGA, which limited the yield of γ-PGA. The optimal rotationspeed and broth’s volume in shake flask were200r/min and35mL/250mL respectively.Under the optimum condition, the yield of γ-PGA was enhanced from35g/L to45g/L,increasing by28.6％more than before.In order to further known the characteristics of Bacillus amyloliquefaciens andimprove the γ-PGA yield in the course of batch fermentation, the effects of differentfermentation conditions, such as pH, culture temperature, rotating speed and aerationrate were investigated. The optimum technological conditions were determined asfollows: pH7.0, culture temperature37℃, rotation speed400r/min, the aeration rate1.0vvm. Under the optimum condition, the yield of γ-PGA was enhanced from32g/L to40g/L, increasing by25％more than before.γ-PGA was purified by anion exchange column chromatography. Through staticaladsorption test and dynamic test of the anion resin, the optimum technologicalconditions were determined as follows: sample pH value was8.0, sample quantity was27.3mg/g*resin, eluant was0.7M NaCl, eluting velocity was1.0mL/min, elutingtemperature was20℃. At last, the purity of γ-PGA was from86.7％to96.2％.The structure of γ-PGA was investigated by paper chromatography, UV, IR and NMR.γ-PGA was only composed of glutamic acid, which absorption peak at210nm bythe UV scanning. The IR spectrum of the the sample was similar to that of γ-PGAstandard sample made by Nanjing Industry University. At last, the sample proved to beγ-PGAby NMR.