# Improvement of the Transglycosylation Efficiency of a
## 3 Results and Discussion
### Screening of LnbB Variants
#### 3.1.1 Glycoshrase Variants: D320E, D320T, and Y419N  
The synthesis of LNT was significantly reduced for the variants D320E and D320T, i.e., mutations targeting the assisting catalytic Asp residue (Figure 2). Both mutations thus also resulted in very low secondary hydrolysis of the formed LNT (Figure S4). D320T was designed based on recent work on the \(\beta\)-\(N\)-acetylhexosaminidase BBhI from _B. bifidium_ JCM 1254 (2018). In that work, BBhI was subjected to directed evolution, which revealed the polarizing Asp residue (D746 in BBhI) and W805 as important mutation targets to improve transglycosylation. Site-saturation mutagenesis of these two residues indicated D746T and W805R as the best variants with D746T exhibiting a transglycosylation yield of 85% vs. 45% for the WT and low secondary hydrolysis (2018). However, to reach this yield, the authors used high enzyme concentration of 41 \(\upmu\)M for the variant. Similarly, the mutation of the assisting Asp residue to Glu has previously had a large, positive effect on BBhI using LNB-oxazoline as donor substrate (2018), but this effect has not been transferable to LnbB due to both enzymatic and spontaneous LNB-oxazoline hydrolysis (2018). When using pNP-LNB as donor substrate, only the moderate improvement of transglycosylation was previously observed for D320 variants (2018; 2018). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Variant** & **Max. LNT [\(\mu\)M]** & **Max. TG [\(\mu\)M]** & **Time of Max. Yield [h]** & **\% LNT Hydrolyzed after 24 h** & **\% LNT of Total TG** \\ \hline WT & \(495\pm 14\)\({}^{\rm\,e}\) & \(854\pm 7\)\({}^{\rm\,g}\) & 0.25 & \(89\pm 0.2\)\% & \(58\pm 1\)\({}^{\rm\,e}\) \\ N259T & \(754\pm 32\)\({}^{\rm\,d}\) & \(1226\pm 26\)\({}^{\rm\,e}\) & 3 & \(66\pm 1\)\% & \(62\pm 1\)\% \({}^{\rm\,d}\) \\ D320E & \(185\pm 9\)\({}^{\rm\,f}\) & \(685\pm 51\)\({}^{\rm\,h}\) & 24 & - & \(27\pm 1\)\% \({}^{\rm\,i}\) \\ D320T & \(303\pm 5\)\({}^{\rm\,f}\) & \(798\pm 2\)\({}^{\rm\,h}\) & 24 & - & \(38\pm 1\)\% \({}^{\rm\,h}\) \\ W373F & \(839\pm 14\)\({}^{\rm\,c}\) & \(1192\pm 14\)\({}^{\rm\,e}\) & 0.25 & \(92\pm 2\)\% & \(70\pm 0.3\)\% \({}^{\rm\,ab}\) \\ W373H & \(696\pm 30\)\({}^{\rm\,d}\) & \(1020\pm 42\)\({}^{\rm\,f}\) & 0.25 & \(90\pm 2\)\% & \(68\pm 0.1\)\% \({}^{\rm\,b}\) \\ W394F & \(1279\pm 17\)\({}^{\rm\,a}\) & \(1865\pm 39\)\({}^{\rm\,b}\) & 3 & \(26\pm 2\)\% & \(69\pm 1\)\% \({}^{\rm\,b}\) \\ W394H & \(1189\pm 6\)\({}^{\rm\,a}\) & \(1655\pm 9\)\({}^{\rm\,c}\) & 3 & \(6\pm 6\)\% \({}^{\rm\,a}\) & \(72\pm 0.01\)\% \({}^{\rm\,a}\) \\ G398R & \(750\pm 106\)\({}^{\rm\,cd}\) & \(1183\pm 145\)\({}^{\rm\,e}\) & 0.25 & \(91\pm 3\)\% & \(63\pm 1\)\% \({}^{\rm\,cd}\) \\ \(\Delta\)G398 & \(720\pm 12\)\({}^{\rm\,d}\) & \(1169\pm 134\)\({}^{\rm\,ef}\) & 0.25 & \(91\pm 2\)\% & \(61\pm 3\)\% \({}^{\rm\,d}\) \\ Y419N & \(1285\pm 5\)\({}^{\rm\,a}\) & \(2388\pm 12\)\({}^{\rm\,a}\) & 3 & \(14\pm 6\)\% \({}^{\rm\,a}\) & \(54\pm 0.1\)\% \({}^{\rm\,f}\) \\ W465F & \(1007\pm 9\)\({}^{\rm\,b}\) & \(1467\pm 8\)\({}^{\rm\,d}\) & 0.25 & \(93\pm 2\)\% & \(69\pm 0.3\)\% \({}^{\rm\,b}\) \\ W465H & \(1235\pm 7\)\({}^{\rm\,a}\) & \(1930\pm 24\)\({}^{\rm\,b}\) & 3 & \(8\pm 4\)\% \({}^{\rm\,a}\) & \(64\pm 0.4\)\% \({}^{\rm\,c}\) \\ D467N & \(536\pm 3\)\({}^{\rm\,e}\) & \(1125\pm 1\)\({}^{\rm\,ef}\) & 3 & \(15\pm 0.4\)\% & \(48\pm 0.2\)\% \({}^{\rm\,g}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Maximum yields of LNT and of LNT plus isomers (listed as total transglycosylation products; TG) from transglycosylation reactions by LnbB WT and variants, using LNB-oxazoline as donor and lactose as acceptor and 1 \(\mu\)M of enzyme at pH 7.4 and 37 \({}^{\rm\,c}\)C. Superscript letters (\({}^{\rm\,a-i}\)) 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 0.25, 3, and 24 h of reaction). Secondary hydrolysis is indicated as the percentage of LNT, which was hydrolyzed after 24 h of reaction (calculated as (max.