Screening Of Poly-γ-glutamic Acid High-yield Strains And Study On Molecular Weight Regulation

Poly-y-glutamic(γ-PGA)is an amino acid polymer linked by glutamic acid monomers through γ-amide bonds,and has excellent water absorption and biodegradability.Features can be applied to all aspects.However,the application ofγ-PGA is closely related to its relative molecular weight.The γ-PGA characteristics and application range of different relative molecular weight are different.Therefore,the regulation of the relative molecular weight of γ-PGA is necessary.The molecular weight of γ-PGA,in addition,is the degradation of γ-PGA to a specific molecular weight in vitro.In this paper,a strain of Bacillus siamensis HJ-3 was screened asγ-PGA producing bacteria,and the relative molecular weight of γ-PGA was regulated from fermentation conditions and in vitro enzymatic degradation.The main research contents were as follows:(1)This paper screened a high-yield γ-PGA strain HJ-3 with stable genetic characteristics and could be used for fermentation to produce γ-PGA.By observing the bacterial morphology,physiological and biochemical results of the strain HJ-3,16SrDNA and gyrB gene determination and phylogenetic tree analysis,the strain HJ-3 was identified as Bacillus siamensis,and no production of y-bacteria was previously observed.The PGA report was the first to discover that Bacillus siamensis could produce γ-PGA.The basal fermentation test of Bacillus siamensis HJ-3 found that Bacillus siamensis HJ-3 was a glutamate-independent strain,but the addition of sodium glutamate to the medium significantly increased γ-PGA production.(2)The effects of different fermentation conditions on the production of γ-PGA by Bacillus siamensis were investigated.The effects of different temperatures(33 0C,35℃,37℃,39℃,41℃)on the relative molecular weight of γ-PGA were investigated.It was found that the relative molecular weight of γ-PGA produced by Bacillus siamensis could be regulated by regulating temperature.However,It was found that Bacillus siblings could produce the high molecular weight of γ-PGA at 33℃;the low molecular weight of γ-PGA could be produced at 35℃.However,the yield of γ-PGA was too low at 33℃ and 35℃.So it was not recommended to use temperature to control the relative molecular weight of γ-PGA.Different pH values(6.0,6.5,7.0,7.5,8.0,8.5)were selected to investigate the effect on the relative molecular weight of γ-PGA.It was found that the lower the pH value in the range of pH6.0-8.5,the larger the relative molecular weight of γ-PGA.It was indicated that the relative molecular weight of γ-PGA could be regulated by pH.Different surfactants(SDS,X-100,CTAB,Tween 80)were selected to investigate their effects on the relative molecular weight of γ-PGA.It was found that the addition of surfactant had little effect on the relative molecular weight of γ-PGA.The effects of different sodium chloride concentrations(2.5%,5%,7.5%,10%)on the relative molecular weight ofγ-PGA were investigated.It was found that the relative molecular weight of γ-PGA increased first and then decreased with the increase of sodium chloride addition,but it was always higher than the contrast.The relative molecular weight of γ-PGA was the highest when the addition amount was 5%.(3)It was found from the fermentation conditions that the addition of sodium chloride was suitable for regulating the relative molecular weight of γ-PGA.to further investigate the effect of ions on the relative molecular weight of γ-PGA,and continue to explore the sodium salt(sodium nitrate,sodium sulfate,chlorination Sodium,sodium carbonate,sodium sulfite)and chloride salts(ammonium chloride,potassium chloride,magnesium chloride,calcium chloride)regulate the relative molecular weight of γ-PGA.The results showed that the effect of sodium salt on the relative molecular weight of γ-PGA was sodium sulfate,while sodium chloride and sodium nitrate were basically similar,indicating that chloride ion and nitrate ion were basically the same.The tendency of sodium chloride and potassium chloride in the chloride salt was the same,but the potassium ion had a greater influence than the sodium ion.lt was found that the low concentration of inorganic salts could reduce the relative molecular weight of γ-PGA.The high concentration of inorganic salts could inhibit the synthesis of the low molecular weight of γ-PGA and improve the relative molecular weight of γ-PGA.(4)From the genome sequencing and protein alignment analysis,the γ-PGA metabolic pathway related genes of Bacillus siamensis HJ-3,including glutamate racemase,synthetic gene and degradation gene,Pgds degrading enzyme γ-PGA relative molecular weight.The relative molecular weight of PGA had a great influence.Therefore,the Pgds degrading enzyme in Bacillus siamensis HJ-3 was heterologously expressed,and its function was verifi ed and used to regulate the relative molecular weight of γ-PGA in vitro.The degradation of Pgds in Bacillus siamensis HJ-3 was found.The enzyme was capable of degrading γ-PGA below 75 kDa,so Pgds degrading enzymes could be used to regulate the relative molecular weight of γ-PGA.In summary,Bacillus siamensis HJ-3 was a γ-PGA producing strain,which was a glutamate-independent γ-PGA producing strain,and the yield could be greatly improved after the addition of sodium glutamate.The relative molecular weight ofγ-PGA produced by Bacillus siamensis could be regulated by fermentation conditions.The inorganic salt was more suitable for regulating the relative molecular weight ofγ-PGA,and different concentrations of inorganic salts were added to regulate the relative molecular weight of γ-PGA;and the low molecular weight of γ-PGA could be degraded in vitro by Pgds degrading enzyme in Bacillus siamensis HJ-3 to obtain the high molecular weight of γ-PGA.