Fermentation Of Bacillus Subtilis To Produce ?-polyglutamic Acid

Poly ?-glutamic acid(?-PGA)is a kind of a large number of biosynthesis of amino acid polymer by microorganisms.It is composed of D-or L-glutamic acid by gamma amide bond connection.poly ?-glutamic acid has a good application prospect who is a biopolymer material in the field of pharmaceutical,environmental protection,agricultural,food and cosmetic industries.In order to improve the production of ?-PGA,this paper first choosed a strain which could produce a large number of ?-PGA.And then through the optimization the fermentation and transformation strain to enhance the production of y-PGA.By identification,we caller the strain Bacillus Subtilis jxz-9.Through the experiment of single factor analysis and orthogonal experiment,determine the optimum fermentation medium components.the optimal media and conditions:sucrose 40g/l;peptone 5g/l;L-glutamic acid 80g/l;NH4CI 5g/l;K2HP04 7.5g/l;MgS04 0.25g/l;culture temperature 37?;shaking speed 200rpm;10%inoculation quantity;PH 7.0;the fermentation period 30h.Then from the strain,we improve the yield of y-PGA by the means of genetic engineering.?-PGA molecular mass is between 100 kd-1000 kd,AS adding of ?-PGA in the fermentation process,fermented liquid viscosity also increases.Oxygen is an important limiting factor in the production of y-PGA by the glutamic acid independent strain B.S JXZ-9.because the culture broth of y-PGA is high viscous.The vgb gene who encode Vitreoscilla hemoglobin(VHb)was introduced into B.S JXZ-9 to overcome the low concentration of dissolved oxygen.This paper will integrate Vitreoscilla VGB gene into the B.S-JXZ-9,in order to realize the low cost high yield synthesis of poly ?-glutamic acid.A problem still exists in the fermentation process:the substrate utilization problem.Research has found that DL-glutamate utilization rate is highest but DL-glutamate as substrate is too expersive,it was restricted in application.Over expression of glutamate racemase in this paper,not only reduces the cost,but also provides polyglutamic acid yield.In order to realize large-scale industrialized production of y-PGA,We should utilize 5L fermenter.Fermentation experiments carried out in 5 L fermenter.At first we choose a strain called B.S JXZ-9 which can produce a large number of product.From the external environment and the ability of the strain producing poly glutamic aci,we optimize fermentation condition:And found a optimal medium composition.Then recombinant plasmid pKS-vgb was constructed by cloning vgb into the Bacillus expression vector pBluescrpt?KS(-)and transformed into B.S NS-9.The expression of VHb was comfirmed by CO-difference spectra analysis.We construct a engineering bacteria which could over express glutamate racemase.It have a great positive effect on y-PGA production.At last In the process of fermentation production of y-PGA,We discovered the Mn2+affect the ratio of D-and L-glutamic acid and researched its mechanism.We found that the Mn2+ could change the stereo chemical structure of the poly glutamic acid by regulating glutamate racemase activity.Then we investigated the effect of glutamate racemase of ?-PGA fermentation.fermentation experiments carried out in 5 L Fermenter and found that,agitation,aeration and pH have profound impact on ?-PGA production.