## 2 Results and Discussion  
In this experiment, LgtB was expressed as an N-terminal His\({}_{6}\)-tagged protein heterogeneously in _E. coli_ BW25113 (DE3). The expression of the soluble protein was best when 0.5 mmol isopropyl-\(\beta\)-D-thiogalactopyranoside (IPTG) was used for the induction of bacterial culture. Figure 1: Chemical structures of the protopanaxadiol and ginsengide derivatives of protopanaxadiol. The ginseng CK was modified by using purified LgtB protein and attaching galactopyranosyl moiety at the 4-OH position of glucose in CK. By using a \(\beta\)-1,4 galactosyltransferase, the glycosylation of CK was quantitatively analyzed by an ultrahigh-performance liquid-chromatography photo-diode array (UHPLC-PDA) analysis. The reaction mixture yielded a peak (P) which appeared before a retention time (\(t_{\rm R}\)) of a substrate peak (S). The control reaction performed with a soluble lysate from _E. coli_ Bw25113 (DE3) expressing LgtB in pET24 vector, which was not induced, did not show any new peak. The peaks of the substrate and product were observed at \(t_{\rm R}\) of \(\sim\) 38.1 and 36.2 min, respectively (Figure 2a). A total of 0.5 mmol CK was transformed completely into the product within 3 h. The UV spectra of the substrate and that of the product, as shown in Figure 2b, were alike. We further elucidated the chemical structure of the metabolite by its accurate mass and mass-spectral fragmentation pattern and by 1D and 2D NMR analyses. The exact calculated mass  
Figure 2: HPLC of the glycosylation reaction of compound K (CK) by purified recombinant LgtB. (**a**) HPLC analysis of the reaction of CK; Control (i), Reaction at 0.5 h (ii), reaction at 1 h (iii), and reaction at 3 h (iv). (**b**) UV maxima of (i) CK (S), and (ii) CKGal (P). S refers to substrate and P refers to product. of [CK + galactose + H]\({}^{+}\) was 785.5051 Da, and the product peak displayed a similar mass fragment \(m/\)z\({}^{+}\) of 785.5067 (Figure 3). To further confirm the structure of the product peak, it was purified by preparative-HPLC and prepared for NMR analysis as described in the materials and methods section. The \({}^{1}\)H-NMR spectrum of the reaction product showed two proton signals representing beta (\(\beta\)) configurations for H-1 of the glucopyranosyl and galactopyranosyl moieties, which appeared at \(\delta\) 5.12 ppm (1H, \(d\), \(J\) = 7.8 Hz) and \(\delta\) 5.04 ppm (1H, \(d\), \(J\) = 7.8 Hz), respectively. Other proton signals of sugar moieties were seen in the region from \(\delta\) (3.0-4.0) ppm (Supplementary Figure S1A). When the \({}^{13}\)C-NMR spectrum of the reaction product was compared with that of CK, six new signals for C-1\({}^{\prime\prime}\) to C-6\({}^{\prime\prime}\) of galactopyranoside were present between \(\delta\) 60 ppm and \(\delta\) 105.57 ppm. Other signals of product exactly matched with that of CK (Supplementary Figure S2). The signal for the anomeric carbons of glucopyranosyl and galactopyranosyl moieties appeared at \(\delta\) 97.65 ppm and \(\delta\) 105.57 ppm (Supplementary Figure S1B), respectively. Their correlations were measured in heteronuclear single-quantum correlation (HSQC) for approving the position at which sugar is conjugated (Supplementary Figure S1C). A downfield glycosylation shift of \({}^{13}\)C (\(\sim\)11 ppm) of the C4\({}^{\prime}\) carbon of glucose was observed, which indicated the attachment of a galactosyl moiety to the C4\({}^{\prime}\)-OH of CK (Table 1). As seen in Supplementary Figure S1D, in the heteronuclear multiple-bond correlation (HMDC), a long-range correlation between C4\({}^{\prime}\) of the CK backbone (C4 of glucosyl moiety of CK, \(\delta\) 81.96) and anomeric proton of the galactosyl moiety (H1\({}^{\prime\prime}\), \(\delta\) 5.04) further confirmed that the product was 20-O-\(\beta\)-D-lactoside of PD.