Thermodynamics of metal interactions with chitin-related biopolymers by isothermal titration calorimetry and production of 2-keto-3-deoxy-D-manno-octulosonic acid from D-glucose in vivo

摘要

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), is used as a biomaterial and for the removal of metals in water purification. Given the role of metal binding to carbohydrates in both biological and industrial settings, isothermal titration calorimetry (ITC) was used to determine the thermodynamics of binding between chitin-related carbohydrate substrates and metal ions. The binding interactions between chitin and common water contaminants mercury, copper, iron, nickel, chromium, lead, zinc, cadmium, silver and cobalt have been studied. The strongest binding has been found towards mercury and the weakest to cobalt with a Kb of 1.16*105 M-1 and 3.96*103M-1, respectively. The formal charge of the heavy metal changed the binding strength in an inverse fashion. The chitin-heavy metal cation interactions were all determined to occur in an enthalpically driven manner. The degree of binding of a series of small chitin fragments to divalent copper ion using ITC have also been tested. The binding strength of GlcNAc has been found to be the weakest among the substrates tested with a Kb of 3.8*103 M-1. Penta-N-acetylchitopentaose (GlcNAc)5 has provided the strongest metal interactions with a Kb of 22.1*103 M-1. All experiments afforded enthalpically driven and favorable interactions. Gibbs free energy of reaction values were all measured to be negative, which is indicative of spontaneous reactions. These results demonstrated that increasing numbers of GlcNAc units enhance the binding strength for divalent copper cation, but the magnitude of the effect points to statistical binding rather than chelation-based multivalency. KDO (2-keto-3-deoxy-D-manno-octulosonic acid) is an 8-carbon sugar that is essential for the growth of Gram-negative bacteria. The production of reasonable quantities of KDO would allow studies to understand the chemistry and biology of this key sugar with the potential to develop anti-infective therapeutics against Gram-negative bacteria. The first synthesis of 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) using glucose through pathway engineering of Escherichia coli was designed as a low cost strategy to obtain KDO. Although a transporter protein for the sugar is unknown, KDO could be isolated from the fermentation broth. An optimum yield of 334 mg KDO per liter of cultured cells was obtained using glucose as a carbon source.