Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.

L.A.M. van den Broek, E.L. van Boxtel, R.P. Kievit, R.P. Verhoef, G. Beldman, A.G.J. Voragen

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Abstract

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards -glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while -glucose-1-phosphate served as donor. d- and l-arabinose, d- and l-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with d-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was -Glcp(11)-Araf.
Original languageEnglish
Pages (from-to)219-227
JournalApplied Microbiology and Biotechnology
Volume65
Issue number2
DOIs
Publication statusPublished - 2004

Keywords

  • leuconostoc-mesenteroides
  • enzymatic assay
  • alpha-galactosidase
  • purification
  • glucosides
  • cloning
  • gene
  • oligosaccharides
  • generation
  • phosphate

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