Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System

The effects of water activity (aw), 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 (rT/H) values of 1.21 and 1.43, respectively. The lowest rT/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.


Introduction
Although highly abundant, human milk oligosaccharides (HMOs) are unique to human breast milk (10-15 g/L). They are widely known to play an important role in infant health by acting as prebiotics, modulators of gut motility, as well as they prevent enteric infections by blocking the adhesion of pathogenic bacteria [1,2]. Chemical structures of HMOs follow a similar blueprint, for example, 3-fucosyllactose can be formed when the hydroxyl group at C-3 of glucose at the reducing end of a lactose molecule is substituted by fucose while the most abundant 2'-fucosyllactose is obtained by the substitution of the hydroxyl group at C-2 of galactose [3].
In vivo biosynthesis of fucosylated oligosaccharides is catalyzed by specific transferases found in the Golgi apparatus of the mammary alveolar cells [4]. Since these enzymes are difficult to express and purify, as well as require expensive nucleotide-activated substrates, which add to the high cost of their production, their usage for the in vitro synthesis of fucosylated oligosaccharides is limited [5,6]. In contrast, glycosidases with transfucosylation activity like α-L-fucosidases (E.C. 3.2.1.51) are more available and relatively inexpensive with broad substrate specificity which is an advantageous characteristic in the synthesis of fucosylated oligosaccharides [7,8]. Under conventional conditions, fucosidases inherently catalyze the glycosidic bond hydrolysis [6]. However, when an acceptor molecule other than water is present in the reaction media, it can attack the nucleophilic glycosyl-enzyme intermediate, and the transglycosylation reaction can proceed (Fig. 1). The most recent reports have described successful protocols to obtain fucosylated oligosaccharides in the α-L-fucosidase-assisted enzymatic reactions [9][10][11][12].
Owing to the fact that fucosidase-catalyzed synthesis of carbohydrates can be controlled kinetically, it has been possible to shift reaction equilibrium in favor of transglycosylation by manipulating reaction conditions [13]. The implemented strategies rely on the suppression of water activity (a w ) in the media by either the addition of high concentrations of acceptor substrate [14] or the incorporation of organic cosolvents [15][16][17].
The main limitation to these approaches lies in the fact that enzymes may show lower activity in the presence of organic cosolvents compared to an aqueous medium while solubility of nonpolar substrates and products is increased [13,16]. However, successful Fig. 1 Scheme of reaction mechanism of α-L-fucosidase. If is the acceptor is a glucosyl molecule, transglycosylation occurs, meanwhile if the acceptor is water hydrolysis occurs applications of miscible organic solvents in enzymatic reactions, such as dimethyl sulfoxide (DMSO) [18,19], acetone [20,21], or acetonitrile [22], have been reported.
Thus, this work aimed at studying the effects of water-miscible organic solvents in the reaction system on transglycosylation activity of α-L-fucosidase from Thermotoga maritima to produce fucosylated oligosaccharides.

α-L-Fucosidase Activity Assay
α-L-Fucosidase activity assay was carried out for 10 min at 60 °C. Briefly, 50 µL of α-Lfucosidase from T. maritima was added to 450 µL of pNP-Fuc (3.5 mM) in phosphate buffer (0.1 M, pH 8) and the reaction progress was analyzed every minute. The reaction was stopped by adding 50 µL of NaOH (1 M). Released pNP was quantified by measuring absorbance (Shimadzu UV-1800, Tokyo, Japan) at 410 nm. One unit of α-L-fucosidase is defined as the amount of enzyme required to release 1 mmol of pNP under the assay conditions.

Synthesis of Fucosylated Oligosaccharides
Enzymatic synthesis of fucosylated oligosaccharides was performed in phosphate buffer (0.1 M, pH 8) for 120 min at 90 °C. The reaction mixture consisted of α-L-fucosidase (0.0065 U/mL), pNP-Fuc (3.5 mM) as a donor substrate, and D-lactose (438 mM) as an acceptor substrate. The reaction progress was analyzed every 30 min until stopped by adding 50 µL of NaOH (1 M). The synthesized fucosylated oligosaccharides were quantified as described in the "Carbohydrate Quantification" section. Released pNP was quantified by measuring absorbance (Shimadzu UV-1800, Tokyo, Japan) at 410 nm.

Effects of pH, Temperature, and Enzyme Concentration on Transglycosylation Activity of α-L-Fucosidase
The effect of pH on transglycosylation activity was determined in the pH range from 5 to 10 for 1 h at 60 °C. Briefly, 50 µL of α-L-fucosidase (0.0065 U/mL) from T. maritima was added to 400 µL of phosphate buffer (0.1 M) containing pNP-Fuc (3.5 mM) and D-lactose (438 mM). The reaction progress was analyzed at 10, 20, 40, and 60 min until stopped by adding 50 µL of NaOH (1 M). The synthesized fucosylated oligosaccharides were quantified as described in the "Carbohydrate Quantification" section. In a similar way, the effect of temperature on transglycosylation activity was determined. Experiments were carried out in a temperature range from 60 to 95 °C. The effect of enzyme concentration was determined similarly, employing 0.013, 0.026, 0.052, and 0.11 µg/mL at pH 8 and 90 °C. The conversion yield was defined as the ratio of the synthesized fucosylated oligosaccharides to the initial concentration of donor substrate (pNP-Fuc). One unit of α-L-fucosidase was defined as the amount of enzyme required to release 1 µmol of pNP per minute under the conditions described.

Estimation of a w
In this study, a w was estimated for DMSO, acetone, and acetonitrile in different proportions using a combination of the models reported by Bell et al. [23] and García-Garibay et al. [24]. Firstly, Wilson coefficients were calculated considering the molar volume of water as 18 mL/mol, DMSO as 71.3 mL/mol, acetone as 74.05 mL/mol, and acetonitrile as 52.86 mL/mol, with a gas constant (R) of 8.314 J/mol K at a temperature of 333 K (temperature of the enzymatic reaction). Calculated intermediate values were DMSO (Λ ws= 6.0306 and Λ sw= 0), acetone (Λ ws= 0.4619 and Λ sw= 0.1298), and acetonitrile (Λ ws= 0.3589 and Λ sw= 0.1483). Later, the coefficients of water activity for each water:solvent media were calculated, and finally a w of the system were determined.

Effect of a w on the Stability and Hydrolytic Activity of α-L-Fucosidase
The effect of a w on enzyme stability was evaluated by the addition of organic cosolvent to the reaction system during 180 min at 60 °C. The test solvents were acetone (a w 0.97), acetonitrile (a w 0.96), or DMSO (a w 0.99). Briefly, 50 μL of α-L-fucosidase was mixed with 10 μL of cosolvent and 40 μL of phosphate buffer (0.1 M, pH 8). Reaction progress was monitored every 30 min. The enzyme activity was measured spectrophotometrically through the release of pNP as already described. Residual hydrolytic activity was determined as the percentage of enzyme activity compared to control.
The effect of a w on the α-L-fucosidase hydrolytic activity was evaluated by varying the volume of cosolvent in phosphate buffer (0.1 M, pH 8). The reactions were carried out at 60 °C for 10 min. In brief, 50 μL of α-L-fucosidase from T. maritima was added to the reaction mixture containing 3.5 mM pNP-Fuc, and different concentrations of test cosolvents in the reaction volume of 450 μL as specified in Table 1. The reaction was monitored every minute until stopped by adding 50 µL of NaOH (1 M). The hydrolytic activity was measured spectrophotometrically through the release of pNP as already described. Residual hydrolytic activity was determined as the percentage of enzyme activity compared to control.

Effect of Organic Media on the Transglycosylation Reaction
The effect of acetone (a w 0.97), acetonitrile (a w 0.96), and DMSO (a w 0.99) on the α-Lfucosidase transglycosylation activity was evaluated for 180 min at 60 °C. Reaction volume of 450 μL was kept constant while varying the organic cosolvent concentration in phosphate buffer (0.1 M, pH 8) as specified in Table 1. The reaction mixture consisted of 50 μL of α-Lfucosidase from T. maritima (0.0065 U/mL), pNP-Fuc (3.5 mM), and D-lactose (438 mM). The reaction progress was analyzed every 30 min until stopped by adding 50 µL of NaOH (1 M). The synthesized fucosylated oligosaccharides were quantified by HPLC as described in the "Carbohydrate Quantification" section. The hydrolytic activity was evaluated under the same conditions of transglycosylation, through the pNP released as already described. The rate of transglycosylation to hydrolysis (r T//H ) was expressed as the concentration ratio of the synthetized fucosylated oligosaccharides to the released pNP.

Carbohydrate Quantification
The quantification of the synthesized fucosylated oligosaccharides and the released fucose was performed on the HPLC (LabAlliance, State College, PA, USA) with an ion-exclusion column Rezex RNO-Oligosaccharides Na + (4%) (60 × 10 mm; particle size 12 µm) (Phenomenex; Amstelveen, Netherlands) and oven temperature of 75 °C. The HPLC was equipped with a SOFTA 300S light scattering detector (Chrom Tech, MN, USA) with a nitrogen flow of 62.5 psi, spray chamber temperature of 10 °C, and a drift tube temperature of 45 °C. Samples were filtered through 0.22-µm Millipore Durapore membranes prior to assay by HPLC and were eluted with Milli-Q water at a flow rate of 0.3 mL/min. A calibration curve of 2'-fucosyllactose was used to estimate the amount of the product formed. Additionally, a standard 2'-fucosyllactose, D-lactose, and L-fucose were used to determine their retention times and identify the reaction products.

Statistical Analysis
All experiments were performed in triplicate and results are reported as a mean value ± standard deviation. For statistical analysis, the statistical software IBM SPSS Statistic version 25.0 for Windows (IBM, NY, USA) was used to carry out a one-way analysis of variance (ANOVA) followed by the Tukey's test for comparing all pairs of groups. A p value of 0.05 was considered statistically significant.

Synthesis of Fucosylated Oligosaccharides
The synthesis of fucosylated oligosaccharides was carried out using pNP-Fuc and D-lactose as donor and acceptor substrates, respectively. Figure 2b shows a typical HPLC chromatogram of transglycosylation products. Retention time of the obtained fucosylated oligosaccharide (7.7 min) coincided with that of 2'-fucosyllactose standard (Fig. 2c). In addition, Fig. 2b reveals the presence of fucose and fucosylated oligosaccharides which is indicative that the enzyme catalyzed both the hydrolysis of pNP-Fuc and transglycosylation, respectively.
More detailed observations of the reaction progress of typical transglycosylation reaction are shown in Fig. 3. The highest concentration of fucosylated oligosaccharide (0.76 mM) was detected after 60 min. As can be observed, in parallel to the pNP-Fuc hydrolysis, some of the released fucose was enzymatically transferred to lactose leading to the formation of fucosylated oligosaccharide while the remaining fucose was observed as free fucose in the reaction medium. Similar results have been described by other authors; Lezyk et al. [10] reported an activity of 0.0038 UE/mL in the synthesis of 2'-fucosyllactose using α-L-fucosidase from T. maritima at a concentration of 51 μg/mL, employing 20-mM pNP-Fuc and 25-mM D-lactose at pH 5.0 and 30 °C. On the other hand, Okuyama et al. [25] reported an activity of 0.004 UE/mL in the synthesis of galactosyl oligosaccharides by using a retaining α-galactosidase mutant of Bacteroides thetaiotaomicron (BtGH97b) at a concentration of 4.8 µmol/mL. The activity obtained in this study (0.0127 UE/mL) was higher than those reported by the aforementioned authors and using lower concentration of α-L-fucosidase (0.013 μg/mL). Figure 4a shows the transglycosylation activity of α-L-fucosidase from T. maritima. The highest activity was found in the pH range from 7 to 10 since no statistically significant difference was calculated for this pH interval (p < 0.05). Additionally, the enhanced transglycosylation activity was observed with the increasing pH from 5 to 8, which resulted in the improved yields from 6.07 to 40.67%, respectively. It is remarkable how transglycosylation reaction yields can be increased by working at optimum conditions. The obtained data are comparable with those reported for other glycosidases from thermophilic sources when reaction conditions were optimized. In accordance with our results, Wu et al. [26] reported improved yields in galactooligosaccharides (GOS) synthesis by changing pH from 4 to 6 in a reaction catalyzed by β-glycosidase from Sulfolobus solfataricus. Similarly, Ji et al. [27] determined pH 6 as the optimum for the GOS production catalyzed by a recombinant β-galactosidase from T. maritima expressed in Escherichia coli in contrast to pH above 7 at which the enzyme activity was remarkably reduced. In another study, the improved yields of GOS were linked to the pH rise from 5 to 5.5 using β-mannosidase from Pyrococcus furiosus as a catalyst [28].

Effect of pH and Temperature on the α-L-Fucosidase Transglycosylation Activity
It is noteworthy that conversion yields of enzymatic transglycosylation increase in alkaline media. Sulzenbacher et al. [29] and Tarling et al. [30] suggested that pH changes of the reaction medium affected the pK a of the key amino acids involved in the catalysis: Asp224 and Glu266. Abdul Manas et al. [13] concluded that ionization of this key amino acid was a determinant factor in favoring interactions with either water, which would lead to hydrolysis, or sugar acceptor resulting in the improved yield of transglycosylation product as observed in the present work.
As for the effect of temperature, the highest transglycosylation activity was observed at 95 °C (Fig. 4b). Furthermore, the product yield at 95 °C was three times higher than that obtained at 60 °C (32.01 and 10.58% respectively). Similar results were observed by Zeuner et al. [31] who reported a sixfold increase of the product yield (from 0.9 to 5.4%) of N-acetyllactosamine catalyzed by β-galactosidase from P. furiosus when the Fig. 3 Kinetics of transglycosylation reaction catalyzed by α-L-fucosidase from T. maritima. Reaction was performed at 90 °C using 3.5-mM pNP-Fuc as donor substrate, 438-mM D-lactose as acceptor substrate, and 0.0065 U/mL of α-L-fucosidase. All reacts were dissolved in 0.1-M phosphate buffer at pH 8 temperature was raised from 40 to 90 °C. In another work, the concentration of GOS in the presence of β-galactosidase from Bacillus circulans was three times greater at 60 °C than at 25 °C (5 and 15 g/L, respectively) [32]. Moreover, the research group of Fourage et al. [33] reported a 2.6-fold increase of yield in the pNP-Fuc-Fuc synthesis (from 16 to 42%) catalyzed by β-glycosidase from Thermus thermophilus, in response to the temperature rise from 37 to 75 °C. Figure 4c shows the results obtained after varying the concentration enzyme. The highest transglycosylation activity was found at 0.013 µg/mL. When adding more enzyme, the reaction predominant was hydrolysis. Similar results were reported by other authors. Bridiau et al. [34] reported that a lower enzyme concentration (28.5 UE/mL) obtained higher transglycosylation yield in the N-acetyl-lactosamine synthesis, using the β-galactosidase from B. circulans. Moreover, Abdul Manas et al. [35] reported that transglycosylation was dominant at an enzyme concentration of 10 U and lower in the malto-oligosaccharide production from Bacillus lehensis G1. In another study, α-L-fucosidase from T. maritima was employed at a concentration of 0.13 µg/mL to catalyze the synthesis of fucosyllactose, using 3.5-mM pNP-Fuc and 584-mM D-lactose at pH 5 and 60 °C. The highest concentration in these conditions was 0.29 mM at 60 min [9], being less than that obtained in this study.

Estimation of a w in the Reaction System
Since water distribution in the reaction medium of enzymatic process tends to maintain balance with all the components of such system, the thermodynamic parameter a w is the best variable to determine water availability in mixed-solvents systems. The a w values of media containing test cosolvents were evaluated, according to a model previously described for enzymatic reactions in organic media [23,24] and reported in Fig. 5 and Table 1. As can be seen, the test solvents affected the a w of reaction system differently. Since the obtained isotherms followed the same pattern as those reported by Bell et al. [23] and Cruz-Guerrero et al. [20], the data were deemed valid.

Effect of a w on the Hydrolytic Activity and Stability of α-L-Fucosidase
By changing a w of the reaction medium, it is possible to increase production yield of fucosylated oligosaccharides. Prior to the experiment, enzyme stability was examined in a test medium with a variable a w value obtained by the addition of a water-miscible cosolvent. The three organic solvents and the corresponding a w of the reaction medium were acetone (a w 0.97), acetonitrile (a w 0.96), and DMSO (a w 0.99). As shown in Fig. 6a, no difference in enzyme activity was observed for the reaction systems tested (p < 0.05) during the reaction course, and the enzyme retained around 76% of its activity after 180 min in comparison to control. Pyeon et al. [36] reported that the recombinant β-glycosidase from Microbulbifer thermotolerans displayed 93, 79, and 20% of Fig. 4 Effect of pH (a), temperature (b), and enzyme concentration (c) on the initial velocity of transglycosylation activity of α-L-fucosidase from T. maritima. The effect of pH on the initial reaction velocity of the transglycosylation activity was performed at different pH and 60 °C by using pNP-Fuc 3.5 mM and lactose 438 mM. The effect of temperature was evaluated under the same conditions but at pH 8 and different temperatures. The effect of enzyme concentration was evaluated under the same conditions but at pH 8 and 90 °C. Error bars represent the standard deviation of the mean of triplicates its activity after incubation in acetone (a w 0.97), DMSO (a w 0.98), and acetonitrile (a w 0.96), respectively. In their previous study [37], a recombinant chitinase MtCh509 from M. thermotolerans was reported to retain up to 100% its activity in media with DMSO as an additive at a w of 0.99 and 0.96, whereas in the presence of acetone and acetonitrile, the enzyme activity decreased remarkably. As a general rule, it is assumed that thermophilic enzymes possess rigid structures that directly contribute to their apparent resistance to organic solvents [38]. In this context, the preserved enzymatic activity of α-L-fucosidase used in this work in the presence of the three cosolvents can be explained in a similar manner.
As can be seen in Fig. 6b, residual hydrolytic activity of α-L-fucosidase exceeded 100% in the presence of acetone (a w 0.97, 0.95, 0.93) and acetonitrile (a w 0.96, 0.93, 0.91). Nevertheless, at the a w values lower than 0.91, the enzyme activity declined. In case of DMSO as cosolvent in the reaction system at a w 0.99, no significant improvement of enzymatic activity was observed in comparison to the control assay (p < 0.05), but at lower a w , the α-L-fucosidase performance was negatively affected. Conversely, Mallek-Fakhfakh and Belghith [39] described no changes in the activity of β-glucosidase from Talaromyces thermophilus in the presence of DMSO at a w of 0.97, while further drop of a w to 0.91 caused remarkable enzyme deactivation. Furthermore, Jiang et al. [40] reported that the activity of xylanase B from T. maritima was unaffected by the addition of DMSO (a w 0.96). The solvent impact on enzyme activity is mostly correlated with Log P or another measure of its polarity rather than a combination of factors such as the dielectric constant, the dipole moment, the ability to form hydrogen  • acetonitrile, and ♦ DMSO. To observe the residual hydrolytic activity, reaction was performed at 60 °C and using either acetone, acetonitrile, or DMSO as a cosolvent, 3.5-mM pNP-Fuc and 0.0065 U/mL α-Lfucosidase from T. maritima. Control assay was performed in the absence of cosolvent under the same conditions and considered 100% of the residual hydrolytic activity. Error bars represent the standard deviation of the mean of triplicates bonds, as well as denaturation capability. Taken together, these properties determine how the solvent affects the way reactants interact with the active site of the enzyme [41]. The extremophile α-L-fucosidase from T. maritima employed in this work displayed higher resistance in the presence of different concentrations of organic solvents. This is an attractive feature for their usage in many industrial processes.

Effect of Organic Media on the α-L-Fucosidase Transglycosylation Activity
Based on the results from previous experiments, α-L-fucosidase from T. maritima showed high stability and activity in the reaction media containing acetone (a w 0.97), acetonitrile (a w 0.96), and DMSO (a w 0.99) at reaction conditions of 60 °C and pH 8. Thus, these cosolvents were studied in the enzymatic synthesis of fucosylated oligosaccharides. Figure 7a demonstrates the reaction course of transglycosylation using different cosolvents in a reaction system. The highest productivity was achieved in the medium with the addition of acetone followed by DMSO and acetonitrile (acetone > DMSO > acetonitrile = 0.51 > 0.42 > 0.18 mM/h).
On the contrary, Trincone et al. [42] reported improved yields in the synthesis of β-Dglucopyranoside disaccharides and trisaccharides catalyzed by a mutant β-glycosidase from S. solfataricus (Ss-β-glyE387G) when acetonitrile was added to the reaction system. In other studies, a hyperthermophilic β-glycosidase was used in the synthesis of GOS in water-acetone system at a w of 0.49 and the highest conversion rate was 0.01 µmol/min mL [20]. In the case of porcine liver α-L-fucosidase employed to produce fucopyranosides in the reaction system with DMSO, the extended reaction time was needed, by approximately 9%, to afford the same yield as in conventional media [19]. The hydrolytic activity of α-Lfucosidase from T. maritima was quantified in the presence of different cosolvents. As it can be observed in Fig. 7b, similar patterns were obtained.
The effect of water activity and cosolvents on the r T/H ratio was also evaluated in the present study (Fig. 7c). In the presence of acetone, α-L-fucosidase showed higher transglycosylation than hydrolytic activity which corresponded to the r T/H value greater than 1, with a maximum recorded at 30 min (r T/H 1.43). In case of DMSO, the highest r T/H was obtained after 120 min and equaled to 1.21. Among the three cosolvents, acetonitrile presented the r T/H below 1, indicating on the predominant hydrolytic activity rather than transglycosylation. Overall, the addition of cosolvents, such as acetone and DMSO, to the reaction media improved kinetic equilibrium toward enzymatically catalyzed transglycosylation, as indicated by their maximal r T/H values when compared to the control (0.39). However, it was observed that by working on acetone and DMSO media, once that maximum concentration of fucosylated oligosaccharides was reached, they start to decrease. This decline could be due to the hydrolysis of the reaction product, which might be used as a precursor of longchain oligosaccharides as reported by Cruz-Guerrero et al. [20]. Similar results were reported by Li et al. [43], who observed a decrease in the transglycosylation activity of a commercially available collection of thermophilic glycosidases (CLONEZYME) in the reaction media containing acetonitrile at a w of 0.88. On the Fig. 7 Effect of the solvents on the a synthesis of fucosylated oligosaccharides, b hydrolytic activity, and c r S/H ratio catalyzed by α-L-fucosidase from T. maritima. Hydrolysis reaction was performed at 60 °C during 180 min either with acetone (a w 0.97), acetonitrile (a w 0.96), and DMSO (a w 0.99) as cosolvent in the reaction medium, 3.5-mM pNP-Fuc and 0.0065 U/mL of α-L-fucosidase from T. maritima. Transglycosylation reaction was performed at the same conditions but adding 438-mM D-lactose as the acceptor: □ acetone, • acetonitrile, and ▲ DMSO. Error bars represent the standard deviation of the mean of triplicate contrary, Li et al. [44] showed that addition of DMSO as a cosolvent at a w of 0.94 improved the yield of daidzein transglycosylation product by 14% (from 39 to 44.47%). The reaction was catalyzed by a recombinant maltosyl transferase from T. maritima expressed in E. coli. Additionally, Baek et al. [45] reported higher yield of transglycosylation products in a reaction catalyzed by maltogenic amylase from a Thermus strain when acetone was added as a cosolvent to the reaction media. The authors observed suppressed hydrolytic activity.

Conclusions
The gathered results indicate that α-L-fucosidase from T. maritima can catalyze the synthesis of fucosylated oligosaccharides in water-organic cosolvent media. The best results were observed for acetone (0.51 mM/h). Moreover, the addition of acetone or DMSO to the medium shifted the reaction equilibrium favoring transglycosylation over the enzymeassisted hydrolysis as the calculated r T/H values of 1.43 and 1.21, respectively, were higher compared to the value recorded for the control assay (0.39). Finally, it is possible that the fucosylated oligosaccharides synthesized in this study have biological functions, like human milk oligosaccharides, and can be used either for clinical applications or as an additive in infant formulas.