*3.2. Expression and Activity of DGAS*

The *dgas* gene has been described in previous studies [21,30]. This gene was identified in the genome of *D. geothermalis* DSM 11300. *E. coli* MC1061 was used to express *dgas*, whereas *E. coli* DH10B was employed for gene manipulation. DGAS was expressed by *E. coli* MC1061 harboring *dgas* in the pHCXHD vector system, as described previously [32,36]. *Escherichia coli* transformed with pHCXHD-DGAS were incubated for 18 h and then harvested by centrifugation (Hanil Combi 514R; Hanil Centrifuge Co., Gimpo, Korea) at 7000× *g* for 20 min. Then, the supernatant was discarded, and the pellet was resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole; pH 7.5) and disrupted using an iced ultrasonic bath (Sonifier 450; Branson Ultrasonics Corp., Danbury, CT, USA). Proteins extracted from the solution were collected by centrifugation (Hanil Combi 514R; Hanil Centrifuge Co.) at 7000× *g* at 4 ◦C for 20 min. Enzymes in the crude cell extract were purified with a nickel–nitrilotriacetic acid affinity column (Poly-Prep; Bio-Rad Laboratories, Inc., Hercules, CA, USA) filled with 500 μL nickel–nitrilotriacetic acid Superflow (Qiagen, Hilden, Germany). A purified enzyme was confirmed by performing sodium dodecyl sulfate–polyacrylamide gel electrophoresis

using 10% (*v*/*v*) acrylamide. Protein concentrations were measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Agawam, MA, USA) with bovine serum albumin as a standard.

The sucrose hydrolysis activity of DGAS was measured using 3,5-dinitrosalicylic acid solution, as described previously [32,36]. A reaction mixture comprising 50 μL 500 mM Tris-HCl (pH 8.0), 100 μL 25% (*w*/*v*) sucrose, and 300 μL deionized water was used. The enzymatic reaction was carried out by adding 50 μL of DGAS solution to the reaction mixture at 45 ◦C for 10 min. To stop the reaction, 500 μL of 3,5-dinitrosalicylic acid solution was added. After boiling for 5 min, the absorbance of the final reaction mixture was measured at 550 nm using a microplate reader (iMark™ Microplate Absorbance Reader; Bio-Rad Laboratories, Inc.). The reducing sugar concentration was calculated using a fructose standard curve. One unit of DGAS was defined as the amount of enzyme that produced one μmol of fructose per min under the assay conditions.

#### *3.3. Transglycosylation of IFAs*

Transglycosylation reactions with DGAS were performed using sucrose as the donor in sterilized water and IFA as an acceptor in DMSO. The reaction was performed in sodium phosphate buffer (pH 5.0 and 50 mM final concentration). Sucrose was used at concentrations of 10, 100, and 200 mM. Daidzein and genistein were used at concentrations of 20, 500, and 2000 μM, while glycitein was used at concentrations of 4, 100, and 400 μM. The amount of DGAS used was set to three enzyme units at 0.1, 1.0, and 5.0 U. The reaction temperature was 45 ◦C, and the reaction time was 24 h. These reaction conditions (temperature, buffer, and pH) for DGAS were based on those reported in previous studies [31,36]. The reaction was stopped by adding an equal amount of 10% (*v*/*v*) DMSO in methanol containing 0.1% (*v*/*v*) formic acid. The terminated reaction solution was subjected to vigorous mixing, sonicated for 10 min, and filtered through a 0.45 μm polyvinylidene fluoride syringe filter (GH Polypro; Pall Corp., Port Washington, NY, USA). Final samples were stored in the freezer at −20 ◦C prior to analysis.
