**1. Introduction**

Globally, soybean (*Glycine max* (L.) Merr.) is a large portion of major grain production, along with corn, wheat, and rice [1]. Isoflavones, a subgroup of flavonoids, are well-known plant secondary metabolites that are found mainly in the plant families *Fabaceae* or *Leguminosae*, including soybean [2,3]. Isoflavones in soybeans are present mainly in conjugated forms [4]. Soy isoflavones are classified as acetylisoflavones, malonylisoflavones, non-acylated isoflavone glucosides, and isoflavone aglycones (IFAs), depending on the side groups attached to the isoflavone skeleton [5]. IFAs have antioxidant properties, interact with epigenetic modifications, and exhibit estrogen-like activities [3]. Dietary IFAs can be metabolized to equol (4',7-isoflavandiol), which has significant estrogenic activity in the intestine [6]. Soy isoflavones exist naturally in conjugated forms rather than as aglycones; for example, daidzein [7-hydroxy-3-(4-hydroxyphenyl)chromen-4-one], glycitein [7-hydroxy-3-(4-hydroxyphenyl)-6-methoxychromen-4-one], and genistein [5,7-dihydroxy-3-(4 hydroxyphenyl)chromen-4-one] [5]. However, conjugated isoflavones are converted into aglycone

forms during processing or storage [4]. The bioaccessibility of IFAs is low due to their poor solubility in water [7]. Therefore, various methods have been developed to overcome these shortcomings of IFAs [8,9].

Glycosylation via chemical or non-chemical methods can change the physicochemical properties of compounds [10,11]. The use of enzymes, such as glycosyltransferases, is a natural way to improve the solubility of IFA [12]. Enzymatic glycosylation reactions are efficient and environmentally friendly [13]. Compared with harsh chemical methods, enzymatic methods typically generate five times less waste and have a 15-fold higher space–time yield [14]. Recently, biological glycosylation methods using a variety of enzymes to improve the water solubility of lipophilic compounds have been developed [11,15,16]. Transglycosylation improved genistein solubility by 3700–44,000 [17] and transglycosylated catechin, naringin, and rutin were found to have 100-, 1000-, and 30,000-fold higher water solubility than non-transglycosylated catechin, naringin, and rutin, respectively [18–20]. The use of glycosyltransferases from microbial sources for transglycosylation reactions is an efficient and eco-friendly biological alternative to chemical reactions [21]. Flavonoids can be glycosylated using enzymes from microbial sources such as cyclodextrin glycosyltransferase (CGTase) and uridine diphosphate glucose (UDP-glucose) [16,22–24]. CGTase is widely employed to increase the solubility and stability of polyphenols such as glycosylated isoflavones [23,25]. However, the CGTase-reacted complex solution is cloudy and contains high molecular weight compounds [26], while the UDP-glucose complex has low reaction yields and is expensive [16,27].

Amylosucrase (EC 2.4.1.4; AS) is a glycosyltransferase enzyme that belongs to the glucoside hydrolase family 13 [28]. One of the main characteristics of AS is that it catalyzes transglycosylation reactions to create α-1,4-glycosidic linkages given sucrose as a substrate [28,29]; AS promotes sucrose hydrolysis to release glucose and fructose, and α-1,4-oligosaccharides are formed using the released glucose as an acceptor [30]. *Deinococcus geothermalis* AS (DGAS) synthesizes α-1,4-glycosidic bonds using various acceptors such as flavonoids [31]. DGAS enhances the water solubility of flavonoid aglycones by transglycosylation [21,31,32]. Compared with glycosyltransferase enzymes such as CGTase, DGAS can react with high substrate specificity using relatively inexpensive donors [27]. In addition, the interpretation of experimental results is easier when using DGAS because of fewer impurities and the limited number of transglycosylated sugar moieties [27,32,33]. These features of DGAS can be exploited to increase the solubility and bioavailability of bioactive phenolic compounds in soybeans.

In this study, we investigated the physicochemical changes of major soybean IFAs in response to DGAS-mediated transglycosylation. To determine what reaction conditions resulted in high transglycosylated conversion yields of IFAs, factors such as the amount of donors, acceptors, and enzymes were evaluated. Optimal reaction conditions were applied to soybean isoflavone extract to confirm that the IFAs were efficiently transglycosylated. Based on our results, we propose an application of the DGAS enzyme process to enrich transglycosylated IFAs in soy-based foods such as soybeans, soybean extracts, and soy products in the industrial field.

## **2. Results and Discussion**
