The Factors Affecting The Yield And Molecular Weight Of ?-polyglutamic Acid In Fermentation And Its Mechanism

Poly-?-glutamic acid??-PGA?is an extracellular polymer,and it synthesized by microorganisms.?-PGA is a kind of homogeneous polymer amino acids,which consists of glutamate acid units through?-glutamine bond polymerization.It has many features such as strong water absorption,biodegradability,biocompatibility,nontoxicity.Therefore,it has wide application prospect in industry,food,medicine,environment,agriculture.daily chemical industry and other fields.?-PGA as a polymer,molecular weight is one of the important structural parameters.Different uses of?-PGA require different molecular weights.Low molecular weight?-PGA is particularly required in the pharmaceutical industry.The microorganisms generally produced?-PGA which is greater than 1000 kDa.Therefore,it is very important to control the molecular weight of?-PGA.At present,low molecular weight?-PGA is mainly purified by fermentation and then subjected to molecular weight control.But this way is time consuming and energy consuming.It is an effective and low-cost production of low molecular weight to obtain?-PGA with relatively low molecular weight by fermentation.In this study,Bacillus licheniformis ATCC9945a was used as the ferme ntation strain.The factors affecting the yield and molecular weight of?-PGA were screened out from fermentation time,fermentation temperature,inoculation amount,adding fungicide and adding different metal salt by shake flask fermentation.The high Fe³⁺concentrations in the medium would not only decrease the molecular weight of?-PGA,but also increase the?-PGA yield.We attempted to elucidate the mechanisms associated with the observed results through physiological analysis,transcriptional level investigation,and enzymatic activity analysis.Result:we found out that the fermentation time,fermentation temperature,inoculation amount,adding fungicide and Fe Cl₃ concentration could affect?-PGA yield and molecular weight.For the fermentation time from 24-168 h,the yield of?-PGA was keep on increasing,while the molecular weight was decreasing.The inoculation amount reached10%,the yield of?-PGA was higher than that of inoculation amount of 1%and 5%,and the molecular weight decreased rapidly.The fermentation temperature was increased to 42?C,the yield of?-PGA was higher than that of 30 or 37?C,and the molecular weight decreased faster.Fermentation culture medium adding 0.0005-0.01 g/L potassium sorbate cans reduce?-PGA molecular weight.The higher the amount of potassium sorbate,the molecular weight of?-PGA is lower,and the yield of?-PGA production is lower.After fermentation to 48 h,the addition of 0.001 g/L glutaraldehyde can also be fermented to low molecular weight?-PGA,but the yield of?-PGA is only 25.32±1.49 g/L,which is 13%lower than that without addition.In cultures containing amended with 7.4 mmol/L FeC l₃,the weight average molecular weight of?-PGA reached 3.18±0.3×10⁵ Da,decreased by 76%than that of the culture containing no FeC l₃?1.31±0.14×10⁶ Da?.Cultures containing increasing concentrations of FeC l₃?0-7.4 mmol/L?showed a corresponding increase in?-PGA yield from 17.36±3.51 g/L to 24.32±1.73 g/L.Meanwhile,in cultures containing 7.4mmol/L FeC l₃,increased carbon sources utilization was related to?-PGA production and not cell growth,and the improved transcriptional level of?-PGA synthetase genes?pgsA,pgsB and pgsC?and hydrolase genes?ggt and cwl O?.The result of?-glutamyltransferase?GGT?enzymatic activity was in agreement with the results of the transcriptional level analysis.The addition of CoCl₂,CuC l₂ and ZnC l₂ all did not increase the yield of?-PGA and decrease the molecular weight.The concentration of CaC l₂,MgSO₄ and MnSO₄ in the fermentation medium had no obvious effect on the yield and molecular weight of?-PGA.The concentration of K₂HPO₄ in the fermentation medium also had no significant effect on the molecular weight of?-PGA.K₂HPO₄ concentration decreased,?-PGA production also decreased.But the concentration of K₂HPO₄ decreased,the yield of?-PGA also decreased.Conclusion:increasing the concentration of FeC l₃ in culture,prolonging fermentation time,increase fermentation temperature and inoculation quantity could produce low molecular weight?-PGA in fermentation phase.High FeCl₃ concentrations could enhance expression of?-PGA synthetase genes?pgsA,pgs B and pgsC?and increase carbon sources utilization,which may contribute to improved production of?-PGA.However,high FeC l₃concentration also could improve the degradation genes ggt and cwl O expression,which caused a reduction in the?-PGA molecular weight.This study would further improve the fermentation production of low molecular weight?-PGA.