## 3 Results and Discussion
### Evaluation of Transglycosylation Capacity with Natural Substrates  
#### 3.7.1 Comparison of Transglycosylation Properties with Raffinose and Locust Bean Gum  
Methanol was chosen as the acceptor for continued comparison of the transglycosylation properties of the two enzymes using HPLC, as both enzymes retained at least 30% of their initial activity during the first three hours under the used conditions and the fact that a product standard (methyl galactoside) was commercially available. Reactions were set up with raffinose or LBG as the donor and 10 _v/v_% methanol as the acceptor. First, a 24-h incubation time was chosen as quantitative product formation was confirmed at this time point with raffinose (Section 3.5). In addition, to quantify the transglycosylation product methyl galactoside, the hydrolysis product galactose was also quantified. Quantification of both products can be used to gain insight into the transglycosylation capacity of retaining GHs by calculating \(\mathrm{r_{S}/r_{H}}\), Equation (1) [10,25]. Theoretically, this ratio reflects the propensity for the glycosyl unit in the enzyme-glycosymetmediate to be released by a water molecule (hydrolysis) or an acceptor molecule (transglycosylation). The first comparison of transglycosylation capacity was made in reactions with 0.4 M raffinose as the donor and 10 _v/v_% methanol incubated with 12 nkat/mL of _B_oGal36A or Aga27A for 24 h at 37 \({}^{\circ}\)C. Both enzymes produced a substantial amount of both transglycosylation and hydrolysis products (Table 4). With these values, the fraction of transglycosylation products (methyl galactoside) of the total release of galactosyl units can be calculated and is presented as the \"Product yield\", Equation (2), in Table 4 (37% for _B_oGal36A and 39% for Aga27A). The product ratio [Me-Gal]/[Gal] is equal to the apparent ratio of the rate of synthesis over the rate of hydrolysis (\(\mathrm{r_{S}/r_{H}}\)) over the time period used (see Section 2.8). The product ratios of the 24h 400 mM raffinose incubations presented in Table 4 are informative for a first comparison of the transglycosylation propensity. _B_oGal36A had a  
Figure 5: Stability of BoGal36A (**left**) and Aga27A (**right**) in the presence of the different acceptor molecules when incubated at 37 \({}^{\circ}\)C over 24 h. The graphs show the remaining activity over a 24-h period correlated to a 0-h sample for each condition. Overall, Aga27A appears to be the more stable enzyme of the two in the presence of the various acceptors, with a noticeable exception for 10 _v/v_% methanol, where BoGal36A was the more stable enzyme. of 0.58 (\(\pm\)0.01) while Aga27A reached a r\({}_{\rm S}\)/r\({}_{\rm H}\) of 0.63 (\(\pm\)0.05) (Table 4). The values for transglycosylation yields are similar to those that were reported for other \(\alpha\)-galactosidases with other types of donors and acceptors [17; 23; 29], including Aga27A with LBG as the donor and allyl alcohol as the acceptor [25]. As can be concluded from the presented values, both enzymes appear to have more or less equivalent transglycosylation capacity under the tested conditions. In order to evaluate the transglycosylation capacity against polymeric substrates, reactions with 0.4% (_w/v_) LBG and 10% (_v/v_) methanol were set up as described in Section 3.4, with the exception of the concentration of _Bo_Gal36A which was increased to 33 nkat/mL to reach sufficient product for quantification. As was the case with raffinose, when using LBG, both enzymes produced similar amounts of methyl galactoside and had comparable yields to one another (Table 4). The data gives methyl galactoside product yields of 27% and 30% for _Bo_Gal36A and Aga27A, respectively. These product yields using LBG are lower than with raffinose but very similar to the product yield previously reported for Aga27A with 3% LBG using 10% allyl alcohol as the acceptor [25].