# Improvement of the Transglycosylation Efficiency of a
## 3 Results and Discussion
### Regioslectivity in Transglycosylation  
Previous studies on LnbB-catalyzed transglycosylation reported regioselective LNT formation detected by UV [15] or MS [9] using HILIC separation. However, this is quite in contrast to the data obtained in the current study: during the screening of LnbB variants, four different transglycosylation products were observed with masses corresponding to hex3 hexNAc1 (\(m/z\) 706 corresponding to the deprotonated [M\(-\)H]\({}^{-}\)) using a porous graphitized column and negative mode as detection (Figure 5). The same isomers were observed in the reactions with pNP-LNB as donor substrate. When the same sample containing four different LNT isomers was analyzed using a chromatographic setup as described by [PERSON] et al. [9] and detection in positive mode, we observed only one peak with an apparent LNT concentration of approximately the sum of the four isomers in our regular chromatography (Figure 6). The large irregular peak at 4 min in positive mode (Figure 6A) is likely due to the isomeric pattern of lactose in dimeric form (\(m/z\) 707) since it is also present in the blank sample (Figure 6C). As a control, the samples were analyzed using the same setup but in negative mode. This still resulted in a single peak yet without the noise observed in positive mode (Figure 6E). These results suggest that the previously reported regioselectivity of LnbB cannot necessarily be claimed. Low regioselectivity in transglycosylation was previously reported for GH20 \(\beta\)-\(N\)-acetylhexosaminidaseases from _Aspergillus oryzae_[49; 50], _Paraglaciecola hydrolytica_[18], _Talaromyces flavus_[51], _Nocardi orientalis_[52], the _Chamelea gallina_ mollusk [53], and soil metagenome HEX1 [28]. Observed products include $1,3-, $1,4-, $1,6-, and unidentified linkages [8] as well as $1,1-linked nonreducing oligosaccharides [18; 49; 51]. Numerous cases of regioselective transglycosylation with GH20 enzymes have also been reported [8], yet comparison of the current work with previous reports on LnbB transglycosylation suggests that great care must be taken in product analysis in order to confirm the true regioselectivity of an enzyme. Figure 5: Porous graphitized column chromatography of a representative sample in negative mode. Extracted ion chromatograms of LNT isomers (blue, \(m/z\) 706 [M\(-\)H]\({}^{-}\)) and lactose (red, \(m/z\) 341 [M\(-\)H]\({}^{-}+m/z\) 683 [2M\(-\)H]\({}^{-}\)) in a sample (**A**,**B**) and a blank reaction without enzyme (**C**,**D**). Reaction Product Analysis  
During the quantification of the reaction products formed by LnbB variants, four different products were observed with masses corresponding to hex\({}_{3}\)hexNAc\({}_{1}\) (\(m/z\) 706 and \(m/z\) 752, corresponding to the deprotonated [M\(-\)H]\({}^{-}\) and formic acid adduct [M\(+\)FA]\({}^{-}\), respectively; Figures 1 and 7). Based on retention time and fragmentation pattern, the dominant isomer was identified as LNT (LNB-\(\beta\)1,3-Lac). In order to compare the additional isomers to the synthesized GlcNAc-\(\beta\)1,2/3/4/6-Lac (LNT2 isomers) standards, the isomers were isolated by preparative HPLC and digested with the GH35 \(\beta\)1,3-specific \(\beta\)-galactosidase BgaC. The hydrolysis products of two isomers could be identified as 2\({}^{\prime}\)LNT2 (GlcNAc-\(\beta\)1,2-Lac) and 6\({}^{\prime}\)LNT2 (GlcNAc-\(\beta\)1,6-Lac) by the retention time (Figure 7E,I) and fragmentation pattern (Figure S5A-D), hence corresponding to LNB-\(\beta\)1,2-Lac and LNB-\(\beta\)1,6-Lac. However, the purified fraction of the identified LNB-\(\beta\)1,6-Lac (Figure 7H) appears to contain impurities in a separate peak. Since none of the compounds eluted at the same time as 4\({}^{\prime}\)LNT2, it can be concluded that no LNB-\(\beta\)1,4-Lac was formed by LnbB, and it can be assumed that the impurities could be LNB substitutions on the Glc moiety of lactose (Figure S2). Finally, the identification of LNT2 (GlcNAc-\(\beta\)1,3-Lac) verified the presence of LNT as a major product (Figures 7K and S5E,F).