Reducing Of Impurities F,A,D,B And Analysis Of The Influence Factors In The Fermentation Of Spramycin By Streptomyces Ambofacines

Spiramycin belongs to the macrolides antibiotics.It is produced by Streptomyces ambofacines and is wildly used in the pharmaceutical industry.However,it is found that low titer,poor composition and abnormal fermentation always happened in the production processes.This paper is a series of studies on these problems.First of all,we compared the influence of Tween 85,glycerol,n-propyl alcohol and choline chloride on the fermentation of spiramycin in the shake flask culture.It was found that adding glycerol can accelerate the synthesis of spiramycin effectively.It was proved that adding 0.4%glycerol at 14 h can improve the titer by 26.2%in 15L fermentor.At the same time,impurities of F and D fell from 2.7%and 3.7%to 2.0%and 2.8%,respectively.Impurities A and B can fulfill the requirement of European pharmacopoeia.It was found that the flux of glycolysis and TCA cycle increased by adding glycerol.Besides,the process of primary metabolism is accelerated,making metabolism enter stable and production stage early.Secondly,it was found that ammonium nitrate is the crucial factors which caused abnormal fermentation.When the concentration of ammonium nitrate is more than 0.2%(50 mmol/L)in basic medium,abnormal fermentation is happened such as thickness and redness.Through the process study,it was found that hight concentration of ammonium nitrate may lead to the unbalance of carbon and nitrogen metabolism.At the same time the flux of glycolysis and TCA cycle is suppressed.Moreover,it was found that adding 1.2%glycerol at 24 h can avoid the abnormal fermentation,improve the titer and reduce impurities content.Finally,by comparing two kinds of dextrin in the fermentation process,it was found that dextrin Z was more effective to produce spiramycin than dextrin Y,but dextrin Z lead to more accumulation of impurities F,A and D.The hydrolysis rate of dextrin Y was slower than dextrin Z,and the total sugar remained unchanged in the late fermentation stage.It was guessed that the structure of these two kinds of dextrins is different.It was found that dextrin Z can be hydrolyzed more slowly and continually by amylase,so more glucose is released,and leads to more glucose.We measured the gene expression of lactone ring synthesis(srm10),carbonhydrate synthesis(srm19,srm20,srm33)and methylation(Srm7 and srm21).It was proved that srm10 gene expression of dextrin Z is higher than dextrin Y.but srm19,srm7,srm21 and srm20/srm33 are higher than dextrin Z.