## 3 Results and Discussion
### Evaluation of Transglycosylation Capacity Aga27A and BoGal36A  
In this paper, we presented a transglycosylation screening strategy for retaining \(\alpha\)-galactosidases, ultimately using natural donor saccharides (i.e., raffinose and galactomanan) and various acceptors. Mass spectrometry was used as the first rapid product screening method. The results with Aga27A show the strength of utilizing pNP-Gal as an initial screening substrate for evaluation of the transglycosylation capacity of enzymes that are known to act on polymeric substrates, as the acceptor utilization with pNP-Gal translated well to LBG. pNP-Gal has several advantages over LBG as an initial screening substrate, e.g., that \(\alpha\)-galactosidases often have a higher catalytic efficiency toward pNP-Gal [5,31,32]. This would allow for high throughput screening of a range of different conditions, e.g., varying enzyme, donor or acceptor concentrations. The procedure may be expanded to other acceptors, although several of the herein used acceptors generated potentially applicable glycosides. Allyl glycosides may effectively be used in polymerizations or thiol-ene glycosylation reactions [33]. Enzymatically synthesized alkyl glycosides  
Figure 6: The figure shows the time-resolved reactions with 40 mM raffinose as a glycosyl donor and 10 \(\rm\
icefrac{{v}}{{\rm{/}}}\)\(\rm\
icefrac{{{\rm{\%}}}}{{\rm{\%}}}\) methanol as an acceptor incubated with 33 nkat/mL BoGal36A in 50 mM sodium citrate pH 6 (**left** side) or 12 nkat/mL Aga27A in 50 mM sodium acetate pH 4.5 (**right** side). The concentration of methyl galactoside and galactose over the time course for BoGal36A and Aga27A is shown in panels (**A**,**B**), respectively, with galactose represented by the box while methyl galactoside is represented by the triangle. The change in apparent \(\rm r_{S}/r_{H}\) for the reactions is shown in panel (**C**) for BoGal36A and panel (**D**) for Aga27A. Error bars represent standard deviation for independent triplicate samples. gain interest as surfactants [34]. Furthermore, galactosylglycerol has shown prebiotic potential [22]. The lower stability for _Bo_Gal36A in the presence of some of the acceptors (Figure 5), in combination with slower kinetics with raffinose, and even more so with galactomanan, compared to pNP-Gal [5], likely negatively affected the product formation for these donors (Table 2, Table 3). These limitations can be overcome by molecular enzyme development [28] and choosing and optimizing reaction conditions (time, temperature) and usage of stabilizing components for a given application [1,2]. These possibilities also further motivate the use of pNP-Gal as a first screening substrate. _Bo_Gal36A was able to perform self-condensation with raffinose but not with pNP-Gal (Tables 1 and 2). The discrepancy in self-condensation behavior could have several explanations. The increase in substrate concentration is one possible explanation. It was shown that increased acceptor concentrations, both for saccharide and non-saccharide acceptors, can increase the yield in transglycosylation reactions with \(\alpha\)-galactosidasees [15,20,35,36]. Using longer saccharides as acceptors was shown to have a beneficial effect on the amount of transglycosylation products synthesized by a GH27 \(\alpha\)-galactosidasees [16]. In the paper, they suggested that the enzyme has several saccharide binding subsites and that these interactions would be beneficial for transglycosylation [16], which was also proposed for \(\beta\)-mannanases in regard to +2 subsites [37,38]. Subsite architecture may also play an important role for non-saccharide acceptors, highlighted in [PERSON] et al. where a single amino acid substitution in the acceptor region of a glucansucrase led to an 8-fold increase in transglycosylation towards a flavonoid [39]. Following the performed screening and evaluation, optimizing reaction conditions may increase the yield.