## 3 Results and Discussion
### Stability in the Presence of Acceptor Molecules  
In the previous sections, we presented a rapid screening methodology using MALDI-ToF MS, which was followed by a screening of detectable transglycosylation products using HPLC. While some of the screening reactions displayed the absence of transglycosylation products, this does not exclude that products would be formed under other conditions using the same enzyme and reactants. There is a possibility that factors such as reduced activity towards the more complex substrates raffinose and LBG [5], secondary hydrolysis of transglycosylation products and enzyme instability in the presence of acceptors affected product formation. Previous studies of \(\alpha\)-galactosidase transglycosylation reactions with alcohols showed an enzyme-destabilizing effect by alcohol acceptors [17; 29]. In order to address this destabilization effect and its potential influence on the results observed in the previous sections, the stability of both _BoGal36A_ and Aga27A in the acceptors was investigated. Both enzymes displayed high stability when incubated at 37 \({}^{\circ}\)C for 24 h in the buffer. _BoGal36A_ retained 80\(\%\) and Aga27A 56\(\%\) of their respective initial activity (Figure 5). Very similar values were obtained in the presence of 10 \(\
icefrac{{v}}{{v}}\)\(\%\) glycerol (data not shown). When incubating in the presence of the other acceptors, both enzymes were more unstable, but no immediate decrease in activity was observed upon the addition of any of the acceptors. Propargyl alcohol (2.5 \(\
icefrac{{v}}{{v}}\)\(\%\)) had the most destabilizing effect, _BoGal36A_ had no detectable activity after one hour and Aga27A had \(<\)5\(\%\) remaining activity after six hours. While _Bo_Gal36A_ had almost no activity left in the presence of 5 \(\
icefrac{{v}}{{v}}\)\(\%\) allyl alcohol or propanol after three hours, Aga27A had 33\(\%\) and 19\(\%\) remaining activity, respectively. Overall, Aga27A was the more stable enzyme of the two under the tested conditions, with the exception of 10 \(\
icefrac{{v}}{{v}}\)\(\%\) methanol. In this condition, _Bo_Gal36A had higher stability compared to Aga27A, retaining 42\(\%\) of the initial activity after 24 h, while Aga27A had no detectable activity at this timepoint but had 28\(\%\) residual activity at 3 h. \begin{table}
\begin{tabular}{c c c c} \hline
**Acceptor** & _BoGal36A_ & **Aga27A** & **Product** \\ \hline
10\(\%\) Methanol & + & + & Methyl-Gal \\ \hline
5\(\%\) Propanol & N.D. & + & Propyyl-Gal \\ \hline
10\(\%\) Glycerol & N.D. & + & Glycerol-Gal \\ \hline
5\(\%\) Allyl-OH & N.D. & + & Allyl-Gal \\ \hline
2.5\(\%\) Propargyl-OH & N.D. & N.D. & Propargyl-Gal \\ \hline Enzyme, LBG and acceptor incubations and product analysis with MALDI-TOF MS are presented in Figure 3 and Figure 5. Enzyme concentration 8 nkat/mL, LBG concentration 0.4 \(\
icefrac{{v}}{{v}}\)\(\%\), acceptor concentration (\(\
icefrac{{v}}{{v}}\)\(\%\)): 10\(\%\) methanol, 5\(\%\) propanol, 10\(\%\) glycerol, 5\(\%\) allyl alcohol, 2.5\(\%\) propargyl alcohol. Time 24 h, temperature 37 \({}^{\circ}\)C. \\ \end{tabular}
\end{table} 
Table 3: Screening of transglycosylation products with _Bo_Gal36A and Aga27A using LBG as a donor and different acceptors. N.D., not detected. Figure 4: Chromatograms for evaluation of transglycosylation capacity with raffinose as donor substrate. Panel (**A**,**B**) show chromatograms of 0.4 M raffinose incubated with 12 nkat/mL of BoGal36A (**A**) and Aga27A (**B**) in the presence of various acceptors. (1): incubation with raffinose, without additional acceptor, (2): 10% MeOH, (3): 5% PrOH, (4): 10% glycerol. Incubations with methanol, propanol or glycerol had additional peaks in the chromatograms at 3.8, 3.4 and 4.4 min, respectively, not present in the hydrolysis control. Controls with BoGal36A or Aga27A had no peaks, and a control sample with raffinose had a single peak with an elution time of 5.8 min (data not shown).