Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering

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-tetraose (LNT; Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide in itself and constitutes the structural core of a range of more complex human milk oligosaccharides as well. Thirteen different LnbB variants were expressed and screened for transglycosylation activity by monitoring transglycosylation product formation using lacto-N-biose 1,2-oxazoline as donor substrate and lactose as acceptor substrate. LNT was the major reaction product, yet careful reaction analysis revealed the formation of three additional LNT isomers, which we identified to have a β1,2-linkage, a β1,6-linkage, and a 1,1-linkage, respectively, between lacto-N-biose (Gal-β1,3-GlcNAc) and lactose. Considering both maximal transglycosylation yield and regioselectivity as well as minimal product hydrolysis, the best variant was LnbB W394H, closely followed by W465H and Y419N. A high transglycosylation yield was also obtained with W394F, yet the substitution of W394 and W465 of the subsite −1 hydrophobic platform in the enzyme with His dramatically impaired the undesirable product hydrolysis as compared to substitution with Phe; the effect was most pronounced for W465. Using p-nitrophenyl-β-lacto-N-bioside as donor substrate manifested W394 as an important target position. The optimization of the substrate concentrations confirmed that high initial substrate concentration and high acceptor-to-donor ratio both favor transglycosylation.

Enzymatic Hydrolysis of Human Milk Oligosaccharides. The Molecular Mechanism of Bifidobacterium Bifidum Lacto-N-biosidase

“…In addition, we provide the substrate conformational itinerary and the structure of the transition states along the reaction coordinate, which will be useful for the engineering of LnbB and related β-hexosaminidases for biocatalytic applications. These results increase our understanding of GHs following substrate-assisted mechanisms and can aid the engineering of the enzyme for the synthesis of HMOs for infant formula milk. ,, …”

“…These results increase our understanding of GHs following substrate-assisted mechanisms and can aid the engineering of the enzyme for the synthesis of HMOs for infant formula milk. 12,45,46 ■ ASSOCIATED CONTENT * sı Supporting Information…”

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases

“…Identification of transglycosylation product regioisomers and quantitation of potentially regioisomeric mixtures were done with liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS) using samples from the reaction conditions and time giving rise to the highest product yield for each enzyme, namely, UB pH 7.2 for CloFuc, UB pH 8.3 for CpAfc2, UB pH 4.5 for BbAfcB, SB pH 5.9 for SpGH29 C , and SB pH 7.9 for BiAfcB. The LC-ESI-MS analysis was performed on an amaZon SL ion trap (Bruker Daltonics, Bremen, Germany) coupled to an UltiMate 3000 UHPLC from Dionex (Sunnyvale, California, USA) equipped with a porous graphitized carbon column (Hypercarb PGC, 150 mm × 2.1 mm, 3 μm; Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described previously, allowing separation of regioisomers, , using a target mass of 850 m / z . Quantitation was performed in Compass TASQ 2.2 (Bruker Daltonics) by using LNFP II and LNFP V as external calibration standards.…”

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome

“…For fractionation of individual isomers, 100 μL of 4 h transfucosylation reaction sample was injected and collected by hand based on retention time, dried in a speed vacuum concentrator, and resolubilized in miliQ water, as previously described (Vuillemin et al 2021 ). For structural elucidation of the products, selected reaction samples were reduced according to Vuillemin et al with some modifications: selected transfucosylation fractions were mixed with freshly made aqueous NaBH 4 to a final concentration of 0.125 M and incubated for 15 h at room temperature (Vuillemin et al 2021 ). Reactions were stopped by adding 0.085 M of acetic acid.…”