Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C₅-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin.
Keywords: 2-O-sulfotransferase; 2OST; 3′-phosphoadenosine-5′-phosphate; 3′-phosphoadenosine-5′-phosphosulfate; 4-nitrophenyl sulfate; AST-IV; Aryl sulfotransferase IV; C(5)-epi; C(5)-epimerase; ESI; HHCOSY; HMQC; Heparosan; Hp; Immobilized enzymes; LC/MS; N-sulfo heparosan (NSH), N-sulfo, N-acetyl heparosan; N-sulfo, N-acetyl, 2-O-sulfoheparin; NACHp; NMR; NSNA2SHp; NSNAH; PAP; PAPS; PNPS; arylsulfotransferase IV; cGMP; current good manufacturing practices; electrospray ionization; heparin; heteronuclear multiple-quantum coherence; liquid chromatography/mass spectrometry; non-anticoagulant heparin; nuclear magnetic resonance; proton–proton correlation spectroscopy.
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