Construction of an in vitro trans-sialylation system: surface display of Corynebacterium diphtheriae sialidase on Saccharomyces cerevisiae |
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Authors: | Seonghun Kim Doo-Byoung Oh Ohsuk Kwon Hyun Ah Kang |
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Institution: | (1) Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon, 305-333, South Korea;(2) Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heukseok-dong, Dongjak-gu, Seoul, 156-756, South Korea;(3) Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul, 156-756, South Korea;(4) Present address: Microbe-based Fusion Technology Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, 580-185, South Korea; |
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Abstract: | Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction
mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates
using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing
the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface
of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids
from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage.
The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis
of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling. |
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