Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis
| Autor: | Mireia Castejón-Vilatersana, Antoni Planas, Magda Faijes |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Models Molecular Glycosylation Protein Conformation ved/biology.organism_classification_rank.species Oligosaccharides 01 natural sciences Mass Spectrometry Substrate Specificity lcsh:Chemistry chemistry.chemical_compound Glycoside hydrolase Lactose lcsh:QH301-705.5 Spectroscopy Chromatography High Pressure Liquid Bifidobacterium chemistry.chemical_classification lacto-N-biosidase biology Molecular Structure Hydrolysis General Medicine Computer Science Applications Biochemistry biocatalysis Glycoside Hydrolases Catalysis Article Inorganic Chemistry 03 medical and health sciences Structure-Activity Relationship Humans Physical and Theoretical Chemistry Molecular Biology Bifidobacterium bifidum Milk Human 010405 organic chemistry ved/biology Organic Chemistry Substrate (chemistry) protein engineering Protein engineering biology.organism_classification 0104 chemical sciences Enzyme Activation Kinetics 030104 developmental biology Enzyme chemistry lcsh:Biology (General) lcsh:QD1-999 Mutation human milk oligosaccharides transglycosylation |
| Zdroj: | International Journal of Molecular Sciences Volume 22 Issue 6 International Journal of Molecular Sciences, Vol 22, Iss 3230, p 3230 (2021) |
| ISSN: | 1422-0067 |
| Popis: | The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT. |
| Databáze: | OpenAIRE |
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