*2.1. DGAS Expression and Activities*

DGAS was successfully expressed by *Escherichia coli* transformed with the shuttle vector pHCXHD-DGAS as confirmed by the detection of *dgas* expression in transformed cells (Figure 1a). Activity of AS was observed in cell extracts of pHCXHD-DGAS-transformed cells. DGAS fused with 6× histidine appeared as a single band with a molecular mass of about 73 kDa (Figure 1b), which is in good agreement with the estimated molecular mass of DGAS. This result confirmed that DGAS was successfully expressed as a functional protein in *E. coli*. DGAS was considered to be free of protein toxicity because it was not harmful to *E. coli*. Temperature and pH profiles of DGAS expressed in *E. coli* were investigated in the range of 30 to 55 ◦C and pH 4.0 to 9.0, respectively, based on a previous

report of DGAS expression in *E. coli* [30]. It was previously reported that isoflavone glucosides can be thermally transformed to their corresponding aglycone forms during processing [34]. In consideration of the stability of the IFAs, the temperature and pH for the DGAS-mediated transglycosylation of IFAs in this study were set at 45 ◦C and pH 5.0, respectively.

**Figure 1.** (**a**) Construction of expression vector of *Deinococcus geothermalis* amylosucrase (DGAS). (**b**) SDS-PAGE analysis of recombinant DGAS expressed in *Escherichia coli* and purified on a nickel– nitrilotriacetic acid affinity column. (Lane M, protein molecular standard marker; Lane 1, crude enzyme; Lane 2, crude passing through enzyme; Lane 3, inclusion body; Lane 4, purified enzyme).
