TY - JOUR
T1 - Transglycosylation activity of engineered bifidobacterium lacto-n-biosidase mutants at donor subsites for lacto-n-tetraose synthesis
AU - Castejón-vilatersana, Mireia
AU - Faijes, Magda
AU - Planas, Antoni
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/3/2
Y1 - 2021/3/2
N2 - 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 Bifidobacte-rium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hy-drolyzes 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 fur-ther engineering for practical enzymatic synthesis of LNT.
AB - 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 Bifidobacte-rium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hy-drolyzes 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 fur-ther engineering for practical enzymatic synthesis of LNT.
KW - Bifidobacterium
KW - Biocatalysis
KW - Human milk oligosaccharides
KW - Lacto-N-biosidase
KW - Protein engineering
KW - Transglycosylation
UR - http://www.scopus.com/inward/record.url?scp=85102727712&partnerID=8YFLogxK
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=pure_univeritat_ramon_llull&SrcAuth=WosAPI&KeyUT=WOS:000645741500001&DestLinkType=FullRecord&DestApp=WOS_CPL
UR - http://hdl.handle.net/20.500.14342/3922
U2 - 10.3390/ijms22063230
DO - 10.3390/ijms22063230
M3 - Article
C2 - 33810098
AN - SCOPUS:85102727712
SN - 1661-6596
VL - 22
SP - 1
EP - 15
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 6
M1 - 3230
ER -