# Improvement of the Transglycosylation Efficiency of a
## 3 Results and Discussion
### Screening of LnbB Variants
#### 3.1.2 Conserved Residues: W373F, W373H, W394F, W394H, W465F, W465H, and D467N  
LNT formation was not significantly improved for the D467N variant (Table 1; Figure S4), which is in line with the observations on the equivalent Bbhl variant D884N [26]. The total transglycosylation yield did improve compared to the WT, but regioselectivity was low  
Figure 4: Transglycosylation reactions using LNB-oxazoline as donor and lactose as acceptor at pH 7.4 and 37 \({}^{\circ}\)C for WT LnbB and the five best variants (W394F, W394H, Y419N, W465F, and W465H). Similar plots for the other variants are shown in Figure S4. The formation of transglycosylation products was followed over 24 h with LC-MS 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. (only half of the formed product was LNT; Table 1). Substitution of D467 in LnbB with Ala or Glu significantly reduced the hydrolytic activity, but did not improve transglycosylation dramatically [9]. D467 is conserved across GH20, and was selected for conservative mutation, i.e. substitution with structurally similar residues, to decrease transition state stabilization following the recently elaborated semi-generic sequence-based engineering approach [26]. Transglycosylation activity was significantly improved for the variants W394F, W394H, W465F, and W465H (Table 1). These Trp variants also exhibited preference for the formation of LNT (68-72% of the formed product was LNT). The variant W465F gave a maximum LNT yield of 1007 uM after 15 min of reaction. Unfortunately, the product was rapidly hydrolyzed, and only 7% of the formed LNT remained after 24 h of reaction (Table 1; Figure 4). The other variant of this position, W465H, showed a maximum LNT yield of 1235 uM after 3 h of reaction. Here, the secondary hydrolysis was statistically insignificant, and only 8% of the formed LNT was hydrolyzed after 24 h of reaction. The equivalent mutation in BbH, W882H, dramatically improved the transglycosylation yield to 66% (compared to 16% for the WT), yet the BbH W882H mutant suffered from a significant loss of activity and was dosed nine times higher than the WT in that study [26]. Recently, W394F was determined as the best among 23 LnbB variants with a maximum LNT yield of 32% using 2.5 mM pNP-LNB as donor and 200 mM lactose as acceptor [9]. Interestingly, the mutation of the equivalent position in BbH (W801H) did not affect the transglycosylation yield [26]. In the current work, the mutants W394F and W394H reached maximum LNT yields of 1279 and 1189 uM, respectively, both after 3 h of reaction. Considering both transglycosylation yield, secondary hydrolysis, and regioselectivity, W394H was the best-performing mutant in the variant screening on LNB-oxazoline and lactose (Table 1). Introducing conservative mutations of Trp residues involves a dilemma: should one aim to maintain the aromatic nature (mutation to Phe) or the hydrogen bonding capacity (mutation to His)? Some studies follow the Trp-to-Phe strategy [9,44], while others suggest Trp-to-His [26]. We included both substitutions. For both W394 and W465, the mutation of the Trp residue to a His appears to significantly reduce the LNT secondary hydrolysis (Table 1). For W465, the mutation to Phe retains the hydrophobic stacking with the GlcNAc residue, whereas the mutation to His enables hydrogen bonding to the hydroxyl groups on C4 and C6 or the ring oxygen. W394 interacts with the oxazoline ring, and the mutation in this position therefore impacts the interaction with this intermediate in the transition state. In Bbhl, W882H (corresponding to W465H in LnbB) gave the highest transglycosylation yield, but in this case, the secondary hydrolysis was still pronounced [26]. Bbhl W882F has not been investigated.