Production And Molecullar Weight Analysis Of Capsule Polysaccharide From E.coliK5 And Its ?waaR Mutant

Heparin?HP?,a kind of highly sulfated glycosaminoglycan,was widely used as anticoagulant.Heparan sulfate?HS?is less sulfated than the heparin with less anticoagulant activity.The oligosaccharide of the E.coli K5 capsule is a chain with repeated two-sugar unit of gluconic acid and acetylglucosamine,which is similar to the polysaccharide chain of heparin and haparan sulfate.HP and HS could be produced via chemo-enzymatic modification of heparosan.Thus,E.coli K5 is a potential strain for the production of heparosan in large scale,as a precursor of heparin and heparan sulfate.To produce E.coli K5 capsule polysaccharide with higher purity in large scale,the effect of fermentation medium on heparosan yield was first evaluated.Glucose addition significantly improved the yield of capsule polysaccharide.Organic nitrogen source also has role on the heparosan production.Based on the optimal medium,the effect of feeding strategy on E.coli K5 fermentation in a 5L bioreactor was further evaluated.Some limited factors,such as DO and NH₃·H₂O showing key effect on the growth and heparosan yield of E.coli K5 in the 5-liter bioreactor were found.A more economical method,?NH₄?₂SO4 precipitation,for the separation of the capsule polysaccharide from the fermentation both was determined after comparison.Besides,The gene waaR encodes the?-1,2–glycosyl transferase which will affected stability of the polysaccharide that on the capsule.A mutant of E.coli K5 with the deletion of waaR gene was evaluated for its heparosan yield and molecular weight distribution.Gel permeation chromatography and gel electrophoresis showed lower molecular weight of heparosan was produced in the mutant?E.coli K5?waaR?than that in wild type?E.coli K5?.It suggested that the mutant is potential for the preparation for low molecullar weight heparosan and its derivates such as HS or HP.