3.4. Enzymatic Quantification of Free Sugar Monomers

In this work, the polysaccharides were synthetized by three microalgae and their compositions enzymatically characterized. The limitation of enzymatic kits is that they require previous knowledge of the polysaccharide composition in order to choose appropriate kit(s). The polysaccharide composition of the microalgae was found in the literature, and their monomeric sugar composition was determined using the reference chromatographic method (HPAEC-PAD). Each simple sugar was quantified with the following enzymatic kits (Megazyme) (the nature of the enzyme or the combination of enzymes used for the quantification is described in parentheses):

l-fucose K-FUCOSE 02/17 (L-fucose deshydrogenase)

d-galactose/l-arabinose K-ARGA 04/17 (galactose mutarotase + β-galactose deshydrogenase)

d-glucose K-GLUHK-110A/K-GLUHK-220A 07/14 (hexokinase, glucose-6-phosphate dehydrogenase)

l-rhamnose K-RHAMNOSE 02/15 (L-rhamnose dehydrogenase)

Glucuronic/Galacturonic acids K-URONIC 04/16 (uronate dehydrogenase)

d-xylose K-XYLOSE 04/16 (xylose mutarotase, β-xylose dehydrogenase)

These kits comprised buffers, coenzymes (NAD⁺ and NADP⁺), and one or two enzymes (as described above in parentheses). The targeted sugars accounted for more than 90% of the composition of the polysaccharides under analysis.

After acid hydrolysis and neutralization, the simple sugars were quantified following the protocols developed by Megazyme. Each quantification required 10 µL of hydrolysate. The volumes of buffer, coenzymes, and enzymes depended on the kit used. The final volume in each well was between 242 and 282 µL. Quantification was done by reading the absorbance at 340 nm on a Perkin Elmer EnSpire Multimode Plate reader with a Greiner microplate (96 wells).