The Expression And Application Of Chondroitin Polymerase From Escherichia Coli K4

Bacterial extracellular polysaccharide have many important biological functions,and it have many applications in the prevention and treatment of human diseases,which have attracted wide attention.Glucuronic acid(GlcA)is an important component of many bacterial extracellular polysaccharides,which plays an important role in the formation,structural and conformational stability,bioactivity,and many other properties of extracellular polysaccharides.The unique structure of GlcA,which contains a free carboxylic group at the 6-position,makes the chemical synthesis of GlcA-containing extracellular polysaccharide is difficult.Besides,the extracellular polysaccharides isolated from biological sources have structurally heterogeneous.Therefore,it is an effective complementary way for chemical synthesis methods to synthesize the GlcA-containing extracellular polysaccharides and their repeating units in vitro.This is the focus of the current research.Glucouronosyltransferase(GlcA-T)is crucial for the biosynthesis of GlcA-containing extracellular polysaccharides.At present,there are few studies on GlcA-Ts.Among them,the chondroitin polymerase(K4CP)from Escherichia coli K4 strains is a bifunctional glycosyltransferase,which has both GlcA-T activity and N-Acetylgalactosaminyltransferase(GalNAc-T)activity.Therefore,in this study,we used K4CP as the tool enzyme to carry out the enzymatic synthesis of the GlcA-containing extracellular polysaccharides repeating unit in vitro.In order to lay the foundation for the study of GlcA-T and the enzymatic synthesis of GlcA-containing extracellular polysaccharides and their repeating units in vitro.Firstly,we expressed K4CP from Escherichia coli K4 strains.According to reports,the enzyme(K4CPA57)that was deleted the 57 amino acids from the N-terminal of K4CP also has GlcA-T activity.Therefore,we constructed K4CP-pET-28a(+)and K4CPA57-pET-28a(+)expression vector,transformed it into BL21(DE3)bacteria after sequencing correctly and induced expression in vitro.SDS-PAGE and Western-blot results demonstrate the expression of target protein.Secondly,we purified the K4CP and K4CPA57.By Ni2+ affinity chromatography and ultrafiltration purification,SDS-PAGE showed the purity of K4CP and K4CPA57 reached as high as 90%.The BCA protein assay results showed that the expression level of K4CP and K4CPA57 was 3 mg/L and 110 mg/L respectively.Thirdly,we studied the activity of K4CP and K4CPA57.Both K4CP and K4CPA57 can use hyaluronic acid hexasaccharide as substrate and UDP-acetylgalactosamine(UDP-GalNAc)as donor,transfering GalNAc attached to hyaluronic acid hexasaccharide to form the corresponding heptasaccharide products,respectively.Then UDP-glucuronic acid(UDP-GlcA)was added to the above reaction system,to form the corresponding octasaccharide products,respectively.The results showed that both K4CP and K4CPA57 have GlcA-T and GalNAc-T activity.Fourthly,we studied the substrate specificity of K4CP and K4CPA57.Study of the acceptor substrate specificity of K4CP showed that K4CP can identify hyaluronic acid tetrasaccharide,hyaluronic acid hexasaccharide,hexasaccharide derivative(NH2-6)of Streptococcus pneumoniae type 3 capsular polysaccharide repeating unit and heptasaccharide derivative(NH2-7)of Streptococcus pneumoniae type 3 capsular polysaccharide repeating unit.Study of the donor substrate specificity of K4CP showed that K4CP can identify UDP-GalNAc,UDP-acetylglucosamine(UDP-GlcNAc)and UDP-GlcA.Study of the acceptor substrate specificity of K4CP?57 showed that K4CP?57 can identify hyaluronic acid hexasaccharide,NH2-6 and NH2-7.Study of the donor substrate specificity of K4CP?57 showed that K4CP?57 can identify UDP-GalNAc and UDP-GlcA.Fifthly,we use K4CP synthetized NH2-7 in vitro.K4CP catalyzed the reaction of 2.5 mg NH2-6 and enough UDP-GalNAc.After the reaction,we purified 1.52 mg product with yield of 60.8%.ESI-MS and nuclear magnetic resonance(NMR)analysis results showed that the product is NH2-7.Our research showed that K4CP and K4CPA57 can be used for enzymatic synthesis of GlcA-containing oligosaccharides in vitro.Our research lay the foundation for the enzymatic synthesis of the derivatives of bacterial extracellular polysaccharide repeating unit as well as bacterial extracellular polysaccharide in vitro.