The β-fructofuranosidase (Xd-INV) from the basidiomycota yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is unique in its ability to synthesize neo-fructooligosaccharides (neo-FOS). In order to facilitate its industrial application, the recombinant enzyme expressed in Pichia pastoris (pXd-INV) was immobilized by entrapment in polyvinyl alcohol (PVA) hydrogels. The encapsulation efficiency exceeded 80%. The PVA lenticular particles of immobilized pXd-INV were stable up to approximately 40 • C. Using 600 g/L sucrose, the immobilized biocatalyst synthesized 18.9% (w/w) FOS (59.1 g/L of neokestose, 30.2 g/L of 1-kestose, 11.6 g/L of neonystose and 12.6 g/L of blastose). The operational stability of PVA-immobilized biocatalyst was assayed in a batch reactor at 30 • C. The enzyme preserved its initial activity during at least 7 cycles of 26 h.

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

During the synthesis of prebiotic carbohydrates such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), or isomaltooligosaccharides (IMOS), D-glucose is released as a side-product of the transglycosylation process. It is desirable to remove glucose from these sugar mixtures due to its caloric contribution and its effect on caries and diabetes. In this work, we have investigated the use of immobilized Komagataella phaff ii (formerly Pichia pastoris) for elimination of D-glucose and D-fructose in several sugar syrups. K. phaff ii cells were immobilized in calcium alginate beads to facilitate the separation of the yeast cells from the reaction medium and reuse of the biocatalyst. The immobilized yeasts were successfully reutilized for at least 20 cycles (of 195 min) to remove D-glucose (62.3 g/L) and D-fructose (5.5 g/L) in a FOS syrup, without affecting the concentration of oligosaccharides. Excellent selectivity was also found for elimination of D-glucose (57.2 g/L) in IMOS syrups. The methodology is versatile and easy to scale-up, as demonstrated in the removal of D-glucose (97.5 g/L) and D-fructose (142 g/L) for the purification of heteroglucooligosaccharides synthesized by Metschnikowia reukauf ii α-glucosidase. In addition, D-glucose (50 g/L) was selectively removed by K. phaff ii beads in the presence of D-galactose (50 g/L) for at least 20 cycles of 150 min and applied to GOS purification.