| Heparin(HP)is widely used as anticoagulant drug in the clinical.Heparin sulfate(HS)also attracted increasing attentions,because it shows activitie of antiviral,anti-inflammatory,anticancer and so on.The capsule polysaccharide of E.coli K5,heparosan,has backbone similar to that of HP and HS.Therefore,necessary modifications on relative locis of heparosan include isomerization and sulfation could produce artificial HP/HS or heparosansulfated derivates.In this study,heparosan was prepared via E.coli K5 fermentation and then used to produce N-deacetylated and sulfated heparosan(NSNAH)by a chemical method of N-deacetylation and sulfation on GlcNAc of heparosan.Then,two more modifying enzymyes C5-epimerase(C5)and 2-O-sulfotransferese(2OST)were prepared via recombinant E.coli and used to isomerize and sulfate of NSNAH resulting the product 2-O-sulfated NSNAH(NSNA2SHP).~1H-NMR results of NSNA2SHP proved that heparosan was successfully N-deacetylated and sulfated,C5 isomerised coupled with 2-O-sulfated.The antiviral activity test showed that NSAN2SHP could inhibit the replication of hepatitis B virus to some extent.Additionally,△4,5 unsaturated glycuronidase(△4,5)and Heparin lyases(Hep)I,II,II were prepared via recombinant E.coli.These recombinant enzymes were then used to evaluate recovery effect of△4,5on the depolymerized products of HP,HS,and heparosan by Hep Ⅰ，Ⅱ，Ⅲ.The results showed that△4,5 could recover the depolymerized products of HP,HS,and heparosan by Hep Ⅰ，Ⅱ，Ⅲamong which the products of heparosan could be recovered completely.The depolymized products of NSNAH by Hep II also could be recovered completely by△4,5.The results suggested that low molecular weight products could be prepared via HP and HS or heparosan and its sulfated derivates depolymerizing by enzymes with the resultant unsaturated bond recovering by△4,5.The results of this study will provide very important references for the production and potential application of sulfated derivates from heparosan. |
