## 3 Results and Discussion
### Evaluation of Transglycosylation Capacity with Natural Substrates
#### 3.7.1 Comparison of Transglycosylation Properties with Raffinose and Locust Bean Gum
Slower kinetics with LBG galactomann compared to oligomeric substrate, as observed for _Bo_Gal36A [5], could potentially contribute to a higher degree of secondary hydrolysis and lower yield. The r\({}_{\rm S}\)/r\({}_{\rm H}\) values were in the same range for both enzymes (0.42 for Aga27A 0.33 and for _Bo_Gal36A) but 33-44% lower than the values for raffinose (Table 4). 7.2 Determination of Initial r\({}_{\rm S}\)/r\({}_{\rm H}\) Ratios and Assessment of Secondary Hydrolysis in Raffinose and Methanol Reactions  
In order to further compare and evaluate the transglycosylation properties of the two enzymes, the product formation over the initial reaction time was analyzed. Reactions with 40 mM raffinose and 10 _v/v_% methanol using 33 nkat/mL _Bo_Gal36A or 12 nkat/mL Aga27A at 37 \({}^{\circ}\)C were followed by HILIC HPLC-analysis for up to 3 h.  
Both enzymes had continued production throughout the 3-h time period (Figure 6a,b), with the highest productivity during the first half-hour. Between the two- and three-hour timepoints, _Bo_Gal36A had a slight decrease in the methyl galactoside concentration (Figure 6a) while it increased slightly for Aga27A (Figure 6b). The decrease in methyl galactoside concentration is an indication of secondary hydrolysis in the _Bo_Gal36A reaction, which was further supported when the apparent r\({}_{\rm S}\)/r\({}_{\rm H}\) was plotted for both enzymes (Figure 6c,d). Both _Bo_Gal36A and Aga27A had the highest value of r\({}_{\rm S}\)/r\({}_{\rm H}\) at the 30-min timepoint with a ratio of 0.99 and 0.71, respectively. For _Bo_Gal36A, this value then decreased throughout the reaction time, going down to 0.64 after three hours (Figure 6c), while for Aga27A, the apparent r\({}_{\rm S}\)/r\({}_{\rm H}\) remained stable throughout the incubation time, with only a slight decrease to 0.63 after three hours (Figure 6d). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{1}{c}{**Donor**} & \multicolumn{1}{c}{**Enzyme**} & \multicolumn{1}{c}{**[Me-Gal] (mM)**} & \multicolumn{1}{c}{**[Gal] (mM)**} & \multicolumn{1}{c}{**Product Yield (\%)**} & \multicolumn{1}{c}{**r\({}_{\rm S}\)/r\({}_{\rm H}\)**} \\  & & & & **([Me-Gal]/** & **(Me-Gal]/[Gal])** \\ \hline \multirow{3}{*}{**0.4**_w/v_\% LBG**} & BoGal36A (24 h) & 0.7 (\(\pm\)0.1) & 2.1 (\(\pm\)0.4) & 27 & 0.33 (\(\pm\)0.06) \\ \cline{2-6}  & Aga27A (24 h) & 1.0 (\(\pm\)0.1) & 2.4 (\(\pm\)0.1) & 30 & 0.42 (\(\pm\)0.05) \\ \hline \multirow{3}{*}{**400 mM raffinose**} & BoGal36A (24 h) & 41 (\(\pm\)7) & 70 (\(\pm\)11) & 37 & 0.58 (\(\pm\)0.01) \\ \cline{2-6}  & Aga27A (24 h) & 35 (\(\pm\)11) & 55 (\(\pm\)13) & 39 & 0.63 (\(\pm\)0.05) \\ \hline \multirow{3}{*}{**40 mM raffinose**} & BoGal36A (30 min) & 1.3 (\(\pm\)0.1) & 1.4 (\(\pm\)0.2) & 48 & 0.99 (\(\pm\)0.06) \\ \cline{2-6}  & BoGal36A (2h) & 3.1 (\(\pm\)0.1) & 4.2 (\(\pm\)0.3) & 42 & 0.74 (\(\pm\)0.07) \\ \cline{2-6}  & Aga27A (30 min) & 2.3 (\(\pm\)0.3) & 3.3 (\(\pm\)0.4) & 41 & 0.71 (\(\pm\)0.03) \\ \cline{2-6}  & Aga27A (3 h) & 4.0 (\(\pm\)0.6) & 6.4 (\(\pm\)1.0) & 38 & 0.63 (\(\pm\)0.04) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Comparative table of transglycosylation capacity of _Bo_Gal36A and Aga27A with methanol as acceptor and raffinose or LBG as a donor. Based on the concentration of products at 30 min and the timepoint with the highest concentration of methyl galactoside for the respective enzyme (Table 4), product yields were calculated. For Aga27A, the product yield after 30 min was 41% and 38% after three hours. _Bo_Gal36A had product yields of 48% and 42% at 30 min and 2h, respectively. This is a sign of secondary hydrolysis for _Bo_Gal36A, as reflected by the decrease in \(\rm r_{S}/r_{H}\) over time (Table 4, Figure 6c). Thus, the two enzymes seem to be quite equal in transglycosylation capacity, even if _Bo_Gal36A displayed a 39% higher initial \(\rm r_{S}/r_{H}\) (Table 4). This advantage appears to be counteracted by secondary hydrolysis by _Bo_Gal36A. Similar findings were observed for \(\rm\beta\)-galactosidases and other GHs [10,28,30].