# Improvement of the Transglycosylation Efficiency of a
## 3 Results and Discussion
### Significance of the Donor Substrate
Figure 9: Transglycosylation reactions using pNP-LNB (2.5 mM) as donor and lactose (200 mM) as acceptor at pH 4.5 and 30 \({}^{\rm\circ}\)C for WT LnbB and the five best variants (W394F, W394H, Y419N, W465F, and W465H). The formation of trans-glycosylation products was followed over 24 h and quantified as LNT equivalents. Purple crosses represent the total formation of all LNT isomers, while pink triangles represent formation of LNT (LNB-\(\beta\)1,3-Lac), orange squares LNB-\(\beta\)1,2-Lac, green circles LNB-1,1-Lac, and blue triangles LNB-\(\beta\)1,6-Lac. In the previously presented reactions using LNB-oxazoline as donor, all five variants exhibited similar maximum LNT yields with no statistically significant differences between them--except for W465F, where the production of LNT was a slightly lower (Table 1). Interestingly, this is not the case when using pNP-LNB as donor, underlining the importance of the choice of donor substrate for transglycosylation reactions. With pNP-LNB as donor substrate, W394 of the subsite \(-1\) hydrophobic platform (Figure 3) was the best engineering target for improving transglycosylation. W394F showed the highest maximum LNT yield with 164 mM of LNT produced, whereas W394H showed a maximum LNT yield of 103 mM (after 2 h of reaction). Looking at the sum of LNT isomers, no difference was observed between the transglycosylation performances of W394F and W394H; both reached a maximum transglycosylation yield of 11% after 2 h of reaction. Thus, with pNP-LNB as donor substrate, regioselectivity was higher for W394F than for W394, but as also observed in the reactions with LNB-oxazoline as donor substrate, substituting Trp with His rather than Phe had a positive effect on the undesirable product hydrolysis (Figure 9). The same trend was observed for W465 (Figure 9), where the W465H did not reach the end of the reaction after 24 h (92 mM LNT formed). For Y419N, secondary hydrolysis was more severe with pNP-LNB as donor substrate compared to LNB-oxazoline (Figures 4 and 9). Recently, W394F was also determined as the best variant for improved transglycosylation with a maximum transglycosylation yield of 32% also using 2.5 mM pNP-LNB as donor and 200 mM lactose as acceptor; W394H was not included in that study, but several other variants were investigated, including the substitution of Trp with Ala, Lys, Glu, and Gln [9]. Furthermore, the substrate concentrations of pNP-LNB and lactose were optimized for LnbB W394H (Table 3, Figure 10), which was one of the best variants for both donor substrates, especially in terms of low secondary hydrolysis (Tables 1 and 2). Reaction conditions can be optimized to favor transglycosylation over hydrolysis, and it is generally observed that an increase in initial substrate concentrations and a high acceptor-to-donor (A/D) ratio tend to give improved transglycosylation yields [55]. In this study, the concentrations of pNP-LNB donor substrate ranged from 2.5 to 10 mM, whereas lactose acceptor concentrations were either 200 or 400 mM, with A/D ratio ranging from 20 to 160 at pH 4.5, 30 \({}^{\circ}\)C, and using 1 mM of LnbB W394H. As expected, the maximum LNT yield of 746 mM and a total LNT isomer yield of 1224 mM were reached when using the highest substrate initial concentrations of both donor (10 mM pNP-LNB) and acceptor (400 mM lactose). The best percentage yields on donor substrate of LNT (8%) and total LNT isomers (16%) were obtained in the reaction using the highest A/D ratio of 160 (Table 3; Figure 10). For the same initial donor concentration, maximum transglycosylation yields were significantly improved when doubling the acceptor substrate concentration, i.e., when doubling the acceptor-to-donor ratio (Table 3).