Clone And Expression Of Three Key Enzymes In The Enzymatic Synthesis Pathway Of Biolngical Heparin

Heparin shows anticoagulant activity both in vivo and in vitro, which is the most effective and widely used anticoagulant drugs at present. The study of heparin is now focused on the in vitro enzymatic synthesis of biological heparin. Uridine diphosphate glucuronic acid (UDP-GlcAc) and3’-phosphoadenosine-5’-phosphosulfate (PAPS) are the important glycosyl and sulfate donors, respectively, in the chemoenzymatic synthesis pathway of ultra low molecular weight heparin (ULMWH), both of which are too expensive to be used practically. Based on these facts, this study aims to solve the source problems of UDP-GlcAc and PAPS and thereby lay the material foundations for ULMWH industrial preparation.For UDP-GlcAc, an elaborately designed synthesis pathway was put forwarded, which used uridine triphosphate (UTP) and glucose-1-phosphate (Glc-l-P) as two initial substrates and employed uridine diphosphate glucose pyrophosphorylase (UGPase) and uridine diphosphate glucose dehydrogenase (UGD). As to PAPS, aryl sulfotransferase (ASTIV) is able to regenerate it by using p-nitrophenyl sulfate (PNPS) as a sulfate donor.The UGPase coding gene galU and UGD coding gene ugd were cloned by PCR from E.coli K12(MG1655) genome, whereas the cDNA of rat ASTIV was synthesized after the codon optimization. Three expression vectors pET-28a(+)-galU, pET-28a(+)-ugd and pET-28a(+)-srat astIV were constructed and transformed into E.coli BL21(DE3), respectively. The reconstituted strains were cultured and induced to express the target proteins in soluble forms. By using affinity chromatography, the UGPase was obtained with a purity of97.4%and a recovery rate of50.6%and the UGD was obtained with a purity of93.2%and a recovery rate of72.4%whereas the ASTIV was obtained with a purity of95.3%and a recovery rate of50.7%.The expression conditions (including the induction timing, induction temperature, inducer concentration and induction time) of the three proteins were optimized. After optimization, the expression quantity of UGPase was0.67g/L with a specific activity of8.72U/mg,0.71g/L with a specific activity of87.5mU/mg for UGD whereas80mg/L with a specific activity of42.5mU/mg for ASTIV. Analyses of HPLC indicated the substrate conversion rate for UGPase was96.7%and37.4%for the UGPase and UGD two enzymes coupled reaction. Furthermore, the influences of reaction conditions (including the pH, temperature, metal ions and ion strength) on UGPase and UGD were studied. Treated with alkaline phosphatase (CIAP) for configuration conversion, the specific activity of ASTIV increased to85.0mU/mg, which is doubled.In conclusion, this study has established an in vitro enzymatic synthesis pathway of UDP-GlcAc as well as achieved the expression and purification of rat ASTIV in E.coli expression system. These results are meaningful for solving the raw material source problems in ULMWH enzymatic synthesis pathway both in theory and application.