Study On Isolation And Identification Of γ-polyglutamic Acid Bacteria And Its Biosynthesis Conditions

The purpose of this study was to isolate and,select high-yieldingγ-polyglutamic acid bacteria from multiple samples.The target strain could be identified and nominated as Bacillus subtilis natto QY-09 through individual and colony morphology,physiological and biochemical characteristics.This study explored the culture medium and fermentation conditions. That was the concentration of carbon source,nitrogen source,sodium glutamate,NaCl,inorganic ions as well as the initial pH,temperature, species age,inoculum size,dissolved oxygen.We had established the curve of the fermentation process to study the relationship of bacteria growth and synthesis ofγ-PGA.The results showed that the culture medium and fermentation conditions had a greater impact on theγ-PGA yield and production costs.The strain,Bacillus subtilis natto QY-09,was a glutamate-dependent one,whose suitable medium was glucose 50g/L,soy peptone 40g/L,sodium glutamate 40g/L,NaC1 30g/L,MgSO₄ 0.5g/L,CaCl₂ 0.3g/L,K₂HPO₄ 3.0g/L,KH₂PO₄ 4.5g/L and suitable biotin.Fitting culture conditions were as follows:initial pH7.0,temperature 37℃,seed age 15h, inoculation 3%(V/V),the volume of liquid 40mL/250mL for 56h.The yield ofγ-PGA was up to 32.7g/L.We could see that the fermentation process ofγ-PGA synthesis and cell growth was not completely synchronized from the curve.The physical and chemical properties,structural characteristics and molecular weight of the purified products(96.10%purity) were discussed. The results showed it was soluble in water,insoluble in ethanol,acetone and other organic solvents.It was composed of glutamic acid without other amino acids or proteins.We established UV spectrophotometry to determinate theγ-PGA content.Its linear equation was A= 0.493 c + 0.0108, correlation coefficient r 0.9981,the average recovery 99.5%,and relative standard deviation 1.0%.The study of molecular weight ofγ-PGA through GPC and SDS-PAGE had shown that the products had different molecular weight in different fermentation time.The molecular weight ofγ-PGA was between 409 and 473 kDa at 56h.Furthermore its molecular weight distribution became gradually wide since 56h.