## 3 Results and Discussion
### Screening for the Ability to Utilze Raffinose as the Donor Substrate  
Having established t'e enzyme's ability to perform transglycosylation with pNP-Gal and various acceptors, their ability to utilize natural donor substrates was investigated. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Acceptor** & **_Bo_Gal36A** & **Aga27A** & **Product** \\ \hline pNP-Gal & N.D. & + & pNP-Gal-Gal \\ \hline
10\% Methanol & + & + & Methyl-Gal \\ \hline
5\% Propanol & + & + & Propyl-Gal \\ \hline
10\% Glycerol & + & + & Glycerol-Gal \\ \hline
5\% Allyl-OH & + & + & Allyl-Gal \\ \hline
2.5\% Propargyl-OH & + & + & Propargyl-Gal \\ \hline Enzyme, pNP-Gal and acceptor incubations and product analysis with MALDI TOF MS as presented in Figure 1, Figures S3 and S4. Enzyme concentration 8 nkat/mL, pNP-Gal concentration 40 mM, acceptor concentration (\(\
icefrac{{}}{{}^{\circ}}\)%): 10% methanol, 5% propanol, 10% glycerol, 5% allyl alcohol, 2.5% propargyl alcohol. Time 1 h, temperature 37 \({}^{\circ}\)C. & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Screening of transglycosylation products of _Bo_Gal36A and Aga27A with pNP-Gal as donor substrate and different acceptors. N.D., not detected. Figure 1: MALDI-TOF MS spectra of incubations with 4-nitrophenyl-\(\alpha\)-galactopyranoside (pNP-Gal). 8 nkat/mL. BoGal36A panel (**A**) or Aga27A panel (**B**) was incubated with 40 mM pNP-Gal alone (top) or with the addition of 5 \(\
icefrac{{}}{{}^{\circ}}\)/\(\
icefrac{{}}{{}^{\circ}}\) propanol (bottom) at 37 \({}^{\circ}\)C for 1 h. A peak corresponding to galactose (G1) (theoretical \(m/z\) for [m+Na\({}^{+}\)]: 203.05) was observed in all incubations. When incubated with pNP-Gal alone, transglycosylation products were observed in the form of pNP-galactobiose (pNP-G2, theoretical \(m/z\) for [m+Na\({}^{+}\)]: 486.12) and pNP-galactotriose (pNP-G3, theoretical \(m/z\) for [m+Na\({}^{+}\)]: 648.18) for Aga27A but not for BoGal36A. With propanol added, both enzymes generated transglycosylation products in the form of propyl-galactopyranoside (propyl-G1) (theoretical \(m/z\) for [m+Na\({}^{+}\)]: 245.10). Analytes and their \(m/z\) shown in parenthesis in the spectra were not detected. The trisaccharide raffinose (0.4 M) was investigated as a donor saccharide, with the same conditions as Section 3.2. The reactions were extended to 24 h since they were expected to be slower than for pNP-Gal [5] and analyzed with MALDI-TOF MS (Figures S5 and S6). The most striking difference compared to using pNP-Gal was that _Bo_Gal36A was able to catalyze self-condensation, likely producing a raffinose molecule with an extra galactose unit added (Figure 2). While Aga27A had the ability to utilize all the screened acceptors with raffinose as a donor, _Bo_Gal36A used methanol, propanol and glycerol but showed no signs of performing transglycosylation with allyl alcohol or propargyl alcohol under the tested conditions (Table 2, Figures 2 and S5). This is in contrast to when pNP-Gal was used as the donor substrate, where it was able to use both of these acceptors. The reason for this observation is not clear, but contributing factors may be comparably slower kinetics for raffinose [5], giving too little product formation to allow detection and/or occurrence of secondary hydrolysis [10]. In addition, enzyme active site architecture and substrate interactions may contribute, as discussed in Section 3.8. Figure 2: MALDI-TOF MS spectra of incubations with raffinose. In total, 12 nkat/mL BoGal36A panel (**A**) or 8 nkat/mL Aga27A panel (**B**) was incubated with 0.4 M raffinose alone (top) or with the addition of 5 %/% propanol (bottom) at 37 \({}^{\circ}\)C for 24 h. A peak corresponding to galactose (theoretical \(m/z\) for [m+Na\({}^{+}\)]: 203.05), marked G1 in the spectra, was observed in all incubations. When incubated with raffinose alone, transglycosylation products were observed in the form of a tetrasaccharide (theoretical \(m/z\) for [m+Na\({}^{+}\)]: 689.21) for Aga27A and BoGal36A.