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.