Multi-Step Enzymatic Production and Purification of 2-Keto-3-Deoxy-Galactonate from Red-Macroalgae-Derived Agarose.

2-keto-3-deoxy sugar acids, which have potential as precursors in medicinal compound production, have gained attention in various fields. Among these acids, 2-keto-3-deoxy-l-galactonate (KDGal) has been biologically produced from D-galacturonate originating from plant-derived pectin. KDGal is also found in the catabolic pathway of 3,6-anhydro-l-galactose (AHG), the main component of red-algae-derived agarose. AHG is converted to 3,6-anhydrogalactonate by AHG dehydrogenase and subsequently isomerized to KDGal by 3,6-anhydrogalactonate cycloisomerase. Therefore, we used the above-described pathway to produce KDGal from agarose. Agarose was depolymerized to AHG and to agarotriose (AgaDP3) and agaropentaose (AgaDP5), both of which have significantly higher molecular weights than AHG. When only AHG was converted to KDGal, AgaDP3 and AgaDP5 remained unreacted. Finally, KDGal was effectively purified from the enzymatic products by size-exclusion chromatography based on the differences in molecular weights. These results show that KDGal can be enzymatically produced and purified from agarose for use as a precursor to high-value products.