Ionic Liquid Pretreatment in Tandem with Recombinant Agarase Cocktail Saccharification of Gelidium amansii for d-Galactose and 3,6-Anhydro-l-Galactose Production

d -Galactose ( d -Gal) and 3,6-Anhydro- l -galactose (3,6- l -AHG) are valuable sugars that can be derived from Gelidium amansii . Herein, ionic liquid (IL) pretreatment was used to deconstruct the cell wall of G. amansii without producing any side-products from sugar degradation. It is combined with enzymatic saccharification using recombinant agarases to effectively produce d -Gal and 3,6- l -AHG from IL-treated G. amansii . ILs were screened for the biomass pretreatment as well as the antisolvents for dissolved biomass retrieval from the IL. [Bmim]Ac is the most effective solvent (99% dissolution) and methanol as antisolvent (78% reconstituted G. amansii ). Using simplex-centroid design, the optimal loading of three β-agarases: Aga2, AgaA7, and Aga50D for the first hydrolysis was determined to be at equal fractions (0.33). Addition of α-neoagarobiose hydrolase AhgI finally produced maximum yields of 56.5% for d -Gal and 33.7% for 3,6- l -AHG. Given the industrial importance of d -Gal and the high market price of 3,6- l -AHG, results demonstrate the potential of combined IL pretreatment and enzymatic hydrolysis using recombinant agarases as a green process for sugar production from red macroalgae.