Model-Based Complete Enzymatic Production of 3,6-Anhydro-l-galactose from Red Algal Biomass

3,6-Anhydro-l-galactose (l-AHG) is a bioactive constituent of agar polysaccharides. To be used as a cosmetic or pharmaceutical ingredient, l-AHG is more favorably prepared by enzymatic saccharification of agar using a combination of agarolytic enzymes. Determining the optimum enzyme combination from the natural repertoire is a bottleneck for designing an efficient enzymatic-hydrolysis process. We consider all theoretical enzymatic-saccharification routes in the natural agarolytic pathway of a marine bacterium, Saccharophagus degradans 2-40. Among these routes, three representative routes were determined by removing redundant enzymatic reactions. We simulated each l-AHG production route with simple kinetic models and validated the reaction feasibility with an experimental procedure. The optimal enzyme mixture (with 67.3% maximum saccharification yield) was composed of endotype β-agarase, exotype β-agarase, agarooligosaccharolytic β-galactosidase, and α-neoagarobiose hydrolase. This approach will reduce the time and effort needed for developing a coherent enzymatic process to produce l-AHG on a mass scale.