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The effects of water activity (a w ), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water-organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10 and temperature 90-95 °C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (r T/H ) values of 1.21 and 1.43, respectively. The lowest r T/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from T. maritima as biocatalyst., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The present study evaluated the influence of water activity and lactose concentration on the synthesis of galactooligosaccharides (GOS), by means of a hyperthermophilic beta-glycosidase in an organic system. The production of GOS gradually grew as water activity increased in the reaction system; later, their synthesis decreased as water activity increased. The authors used the response surface methodology to study how different water activities and different concentrations of lactose influenced the synthesis of GOS and their length. In every case, the variable that proved to have the greatest effect on GOS synthesis was water activity. Maximum GOS3 synthesis was reached at a water activity interval of 0.44-0.57, with lactose concentrations of 0.06%-0.1%, while GOS4 and GOS5 maxima were reached at water activity intervals of 0.47-0.57 and 0.49-0.60, respectively. The research showed that higher water activity was required to synthesize GOS of greater length. Synthesis of GOS would then depend on the flexibility of the enzyme, which in turn would depend on water activity of the reaction system. This hypothesis was supported by experiments in which the reaction temperature was modified in order to change the flexibility of the enzyme, thus leading to longer GOS.

Regular intake of fucosylated oligosaccharides has been associated with several benefits for human health, particularly for new-borns. Since these biologically active molecules can be found naturally in human milk, research efforts have been focused on the alternative synthetic routes leading to their production. In particular, utilization of fucosidases to perform stereoselective transglycosylation reactions has been widely investigated. Other reasons that bring these enzymes to the spotlight are their role in viral infections and cancer proliferation. Since their involvement in the pathogenesis of these diseases have been widely described, fucosidases have become a target in newly developed therapies. Finally, activity disorders of biologically important fucosidases can lead to health problems such as fucosidosis. What is common for both mechanisms is the interaction between the enzyme and substrates in and around the active site. Therefore, this review will analyse different substrate structures that have been tested in terms of their interaction with fucosidases active sites, either in synthesis or inhibition reactions. The published results will be compared from this perspective. [ABSTRACT FROM AUTHOR]

In recent years, the synthesis of human milk oligosaccharides, particularly fucooligosaccharides, has been investigated. These oligosaccharides provide protection against gastrointestinal diseases for newborns, among other functionalities. α-L-fucosidase from Thermotoga maritima of GH29 family has been reported to produce fucooligosaccharides by means of a transfucosylation reaction. However, the interaction between acceptor and donor substrates, and the enzyme has been little studied. For that reason, and due to the importance of fucooligosaccharides, the present study proposes a theoretical synthesis route employing α-L-fucosidase from T. maritima, lactose as acceptor substrate and pNP-fucose as fucosyl donor. A molecular docking study was carried out using crystallographic structure of α-L-fucosidase from Protein Data Bank. By simulating the theoretical synthesis with T. maritima fucosidase, it was observed that lactose interacts with the enzyme in regions other than the active site. However, it is approaching the active site when the fucose is in the position to form the enzyme-substrate complex. This finding may explain the transfucosylation yield documented in the literature when the proportion of fucosyl acceptor substrate increases concerning that of the donor substrate. [ABSTRACT FROM AUTHOR]