*2.3. E*ff*ects of Reaction Conditions on DGAS Transglycosylation*

Table 1 shows the conversion yields of IFAs to transglycosylated IFA glucosides according to the concentrations of donor and acceptors as well as the amount of DGAS. Glycitein reacted at lower concentrations than daidzein and genistein because of its lower solubility [38]. Regardless of IFA and sucrose concentrations, the conversion yield of each IFA used in this study increased as the amount of DGAS used increased, whereas the conversion yield of each IFA generally decreased with increasing IFA concentration. In transglycosylation reactions, a large amount of enzymes ensures not only a fast reaction rate, but also an increased reaction yield [33]. However, at a low donor concentration (0.1 M) and high acceptor concentration (20 mM), the conversion yields for daidzein and genistein were the lowest during transglycosylation for the highest amount of enzymes (5.0 U). The ratio of donor to acceptor suggests that the conversion yield is affected even if a sufficient amount of enzyme is supplied. Similar to the results of this study (Table 1), previous studies reported that the conversion efficiency increased when the concentration of the donor was higher than that of the acceptor [31,39,40]. The enzyme-modified glycosylation of lipophilic compounds is known to be complex because of the low solubility of lipophilic compounds in aqueous systems [41]. To increase the conversion yield despite low solubility, various solvents such as ionic liquids have been used in enzyme reactions for flavonoid glucoside synthesis [13,41]. In this study, we used dimethyl sulfoxide (DMSO) as the solvent to dissolve all IFAs (acceptors). However, cloudiness was observed during the enzyme reaction when the amount of acceptor was increased in an aqueous system containing a donor, acceptor, buffer, and enzyme (data not shown). Acceptor solubility was found to be the most important factor determining the lowest conversion yields at the highest acceptor conditions (glycitein, 4 mM; daidzein and genistein, 20 mM) (Table 1). Therefore, it is reasonable to assume that the reason glycitein had the lowest conversion yield was its relatively lower solubility than that of daidzein and genistein.


**Table 1.** Conversion yields of soy isoflavone aglycones after *Deinococcus geothermalis* amylosucrase (DGAS)-treated transglycosylation according to concentrations

 Data are expressed as means ± standard deviations (*<sup>n</sup>* = 3). Means with different superscripted letters indicate that for the same amount of DGAS, each isoflavonesignificantly by the Tukey–Kramer honestly significant difference test (*<sup>p</sup>*<0.05).

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The conversion yields of daidzein tended to be similar to those of genistein for the various reaction conditions used in this study. Glycitein had a lower conversion yield than genistein and daidzein (Table 1). Transglycosylation has been reported to occur at the hydroxyl group of the acceptor, resulting in the formation of an *O*-glycosidic bond between the acceptor and donor [35]. Likewise, a correlation between the number of hydroxyl groups and reactivity after the transglycosylation of hydroxyflavonoids with DGAS was observed [27]. The average conversion yields of daidzein and genistein were approximately 96.7% and 94.8%, respectively, at the highest amount of enzyme (5.0 U), lower acceptor concentrations (0.2–5.0 mM), and higher donor concentrations (1.0–2.0 M). In general, the highest conversion yield is achieved when sufficient enzymes and sucrose are provided for transglycosylation [33]. However, excess sucrose has been reported to reduce conversion yields [32]. Therefore, it is essential to have an adequate ratio of donor and acceptor as well as a sufficient amount of enzyme to attain higher conversion yields.
