Microthecin (2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one), which has anti-microbial activity, is one of the end products of an alternative glycogen and starch degrading pathway, the anhydrofructose pathway. It is formed from 1,5- anhydro-D-fructose (AF), a product of a-1,4-glucan lyase (EC 126.96.36.199) from glycogen and starch by aldos-2-ulose dehydratase (AUDH; EC 4. 2.1.110). In the current study, the yield and purity of microthecin was examined with respect to pH and buffers using AUDH purified from the fungus Phanerochaete chrysosporium. It was found that AUDH had a Km of 5.4 and 4.9 mM towards AF and its natural substrate glucosone, respectively, while its Vmax with AF was 5.5 times higher than that with glucosone. Higher molar conversion of 90% was obtained in a reactor with pH controlled around 5.0 and 248C with de-ionized water as the solvent. Microthecin was found to be most stable in de-ionized water at a pH around 7.0 and stable in freeze-dried form. Under acidic conditions microthecin was degraded to 2?-furyl-2-hydroxymethylketone (FHMK). A 13C-NMR method was established to simultaneously monitor the reaction components including AF, microthecin and its intermediates. A mechanism of microthecin formation from AF via the intermediate ascopyrone M (APM) and the degradation of microthecin to FHMK are proposed based on the NMR data obtained.