# Improvement of the Transglycosylation Efficiency of a
###### Abstract  
The lacto-\(N\)-biosidase LnbB from _Bifidobacterium bifidum_ JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-\(N\)The ability of LNBases to release and transfer disaccharide moieties in a single reaction step is an attractive trait for targeted transglycosylation reactions, e.g., for the synthesis of HMOs, which are crucial for infant health and development, but absent from infant formula [12]. LNT is the most important HMO core structure as well as being among the most abundant HMOs in itself [13]. Two examples of the use of LNBases for transglycosylation exist in the literature, although only low transglycosylation yields were achieved: In the first example from 1999, a GH20 LNBase isolated from _Aureobacterium_ sp. L-101 was used for catalyzing the formation of LNT from _p_-nitrophenyl-\(\beta\)-lacto-\(N\)-bioside (pNP-LNB) and lactose [14] (Figure 1B). In the other example, the GH20 LNBase LnbB from _Bifidobacterium bifidum_ JCM 1254 was shown to catalyze transglycosylation with pNP-LNB and lactose to form LNT at low yields [3] (Figure 1B); LnbB also catalyzed the transfer of a lacto-\(N\)-bioside unit from LNT to 1-alkanols (C\({}_{1}\)-C\({}_{4}\)) as well as a condensation reaction with LNB and lactose to form LNT and an isomer [3]. Figure 1: Transglycosylation reactions catalyzed by LnbB in the current work with LNB-oxazoline (**A**) or pNP-LNB (**B**) as donor substrate and lactose as acceptor substrate. All reactions take place in competition with substrate and product hydrolysis (Figure S1). The terminal Gal residue (blue) was subsequently removed by the GH35 \(\beta\)1,3-galactosidase BgaC from _Bacillus circulans_ ATCC 31382 (Figure S2), and the resulting LNT2 (GlcNAc\(\beta\)1,3-Gal-\(\beta\)1,4-Glc) isomers were compared to their chemically synthesized counterparts (Schemes S1–S3 ). LNT (Gal-\(\beta\)1,3-GlcNAc\(\beta\)1,3-Gal-\(\beta\)1,4-Glc) isomers were also subjected to NaBH\({}_{4}\)-catalyzed reduction to confirm the presence of a LNB-1,1-Lac linkage. The fact that GH20 enzymes employ an oxazoline intermediate has recently been exploited for the synthesis of LNT [15] (Figure 1A) as well as for the synthesis of the HMO precursor lacto-N-triose II (LNT2; GlcNAc-\(\beta\)1,3-Gal-\(\beta\)1,4-Glc) [16; 17; 18]. To ensure oxazoline stability, these reactions require enzymes able to work at or preferably above neutral pH [8]. The ability to easily synthesize and use the reaction intermediate as donor substrate facilitates the use of glycosyl, i.e., GHs where the catalytic nucleophile is replaced by a nonfunctional residue to suppress the inherent hydrolytic activity of the enzyme and thus favor transglycosylation [8; 17; 19]. To construct glycosyntase variants of LnbB, two different substrate-interacting residues were targeted previously [15]. The polarizing Asp residue of the catalytic Asp-Glu pair (D320-E321), which apparently stabilizes the positive charge development in the oxazolinium ion transition state [7; 20], was substituted by Glu or Ala. Alternatively, a Tyr residue (Y419), which stabilizes the reaction intermediate and/or binds water or an alternative acceptor substrate through hydrogen bonding [7; 21; 22], was substituted by Phe to remove its hydrogen bonding capacity. This mutation was first shown to improve transglycosylation for chito-oligosaccharide synthesis in a GH20 \(\beta\)-\(N\)-acetylhexosaminidase from _Talaromyces flavus_; the substitution to Asn inspired by the closely related GH84 \(\beta\)-\(N\)-acetylglucosamine amidiases also improved transglycosylation performance [22]. In the reaction with pNP-LNB and lactose, all mutants of LnbB led to slightly increased LNT yields (10-13%) compared to the WT (8%). However, in the reaction with lacto-N-biose 1,2-oxazoline (LNB-oxazoline) and lactose, the highest LNT yields were obtained with the WT (67%) [15].