# Improvement of the Transglycosylation Efficiency of a
## 3 Results and Discussion
### Significance of the Donor Substrate  
The five best LnbB variants W394F, W394H, Y419N, W465F, and W465H were selected according to their higher LNT production and low secondary hydrolysis in the case of W394H, Y419N, and W465H. The variants were further characterized by monitoring transglycosylation reactions over 24 h using pNP-LNB as donor and lactose as acceptor and 1 \(\upmu\)M of enzyme at pH 4.5 and 30 \({}^{\circ}\)C (Table 2; Figure 9). Compared to the activity screening with LNB-oxazoline as donor substrate (Figure 4; Table 1), regooselectivity was lower when using pNP-LNB as donor substrate (Figure 9; Table 2). The percentage of LNT produced of the total transglycosylation products varied from 47% to 59%. As expected, all variants showed higher maximum LNT and total transglycosylation product yields compared to the WT. LnbB WT produced a maximum of 7 \(\upmu\)M LNT in the early stage of the reaction (15 min), representing 0.3% of the maximum theoretical molar yield based on the donor substrate. The five mutants showed maximum LNT yields ranging from 46 to 164 \(\upmu\)M representing 1.8% to 6.6% of the maximum theoretical LNT yield, whereas the maximum yield of total transglycosylation products (LNT and LNT isomers)  
Figure 8: LNT isomer reduction. Extracted ion chromatograms of native LNT isomers (blue \(m/z\) 706 and 752) and LNT alidtiols (green, \(m/z\) 708 and 754) from the native W394H 2 h sample (**A**,**B**) and from the reduced version of the same sample (**C**,**D**). ranged from 94 to 286 uM, corresponding to 3.7% to 11% of the maximum theoretical yield (Table 2; Figure 9). \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & & \multicolumn{3}{c}{**LNT Formation**} & \multicolumn{3}{c}{**Total LNT Isomer (TG) Formation**} \\ \hline \multirow{2}{*}{**Variant**} & **Time of Max.** & **Max. LNT** & **Max.** & **\% LNT** & **Max.** & **\% Total TG** \\  & **Yield [h]** & **[μM]** & **LNT Yield** & **Hydrolyzed after** & **[μM]** & **TG Yield** & **Hydrolyzed after** \\ \hline WT & 0.08 & 7 \(\pm\) 0.3 \({}^{\rm f}\) & 0.3\(\%\)\({}^{\rm f}\) & 55 \(\pm\) 8\% & 29 \(\pm\) 3 \({}^{\rm d}\) & 1.2\(\%\)\({}^{\rm d}\) & 52 \(\pm\) 7\% \\ W394F & 2 & **164 \(\pm\) 0.05\({}^{\rm a}\)** & **6.6\%**\({}^{\rm a}\) & 97 \(\pm\) 0.1\% & **285 \(\pm\) 5\({}^{\rm a}\)** & **11\%**\({}^{\rm a}\) & 91 \(\pm\) 0.3\% \\ W394H & 2 & \(\underline{103\pm 3}\)\({}^{\rm b}\) & 4.1\%\({}^{\rm b}\) & 6 \(\pm\) 18\%\({}^{\rm a}\) & **286 \(\pm\) 5\({}^{\rm a}\)** & **11\%**\({}^{\rm a}\) & 43 \(\pm\) 10\% \\ Y419N & 0.25 & 76 \(\pm\) 3 \({}^{\rm d}\) & 3.0\%\({}^{\rm d}\) & 95 \(\pm\) 1\% & 171 \(\pm\) 3\({}^{\rm b}\) & 6.8\%\({}^{\rm b}\) & 86 \(\pm\) 2\% \\ W465F & 0.08 & 46 \(\pm\) 0.04 \({}^{\rm e}\) & 1.8\%\({}^{\rm e}\) & 93 \(\pm\) 0.5\% & 94 \(\pm\) 0.7 \({}^{\rm c}\) & 3.7\%\({}^{\rm c}\) & 86 \(\pm\) 2\% \\ W465H & 24 & 92 \(\pm\) 0.7 \({}^{\rm c}\) & 3.7\%\({}^{\rm c}\) & - & 175 \(\pm\) 3\({}^{\rm b}\) & 7.0\%\({}^{\rm b}\) & - \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Maximum yields of LNT and of LNT plus isomers (total transglycosylation products; TG) from transglycosylation reactions by LnbB WT and variants, using 2.5 mM pNP-LNB as donor, 100 mM lactose as acceptor, and 1 uM of enzyme at pH 4.5 and 30 \({}^{\rm\circ}\)C. Superscript letters (\({}^{\rm a-f}\)) indicate statistically significant difference between the values within the column (\(p<\) 0.05). The time at which the maximum transglycosylation yields are reached is indicated (samples taken after 0.08, 0.25, 2, and 24 h of reaction). Secondary hydrolysis is indicated as the percentage of LNT, which has been hydrolyzed after 24 h of reaction (calculated as (max. LNT yield \(-\) LNT yield at 24 h)/[max. LNT yield]). An asterisk (*) indicates that the LNT concentration after 24 h is not significantly different from the maximum yield (secondary hydrolysis not statistically significant; \(p<\) 0.05). For each column, the best results are marked in bold, whereas the second-best results are underlined.