Recently, β-glucan transferases of family GH17 have been characterized for proteobacteria. They cleave (β1→3) glucan chains internally and transfer the newly generated reducing end to the non-reducing end of another (β1→3)-glucan molecule through (β1→3), (β1→4), and/or (β1→6) linkages [1].
A gene from Bradyrhizobium japonicum, encoding a β-glucosyltransferase domain of family GH17, was cloned and expressed in E. coli, and the recombinant enzyme was isolated and purified. To investigate the activity of the enzyme, we characterized transfer products obtained by incubating linear (β1→3)-linked gluco-oligosaccharides (Lam-Glc2-10) and their corresponding alditols (Lam-Glc2-10-ol) with the recombinant enzyme. Detailed analyses by thin-layer chromatography (TLC), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 1D/2D 1H and 13C nuclear magnetic resonance (NMR) spectroscopy showed that the enzyme has a non-Leloir trans-β-glucosylation activity, forming to a substantial extent (β1→6) linkages at branching points. The enzyme seems to cleave laminaribiose units from the reducing end of the laminari-substrate (DP>4) donors and adds the remaining oligosaccharide part to another laminari-substrate molecule or already formed product. That the activity of the enzyme started from the reducing end of the substrate, was confirmed by the fact that the enzyme was not active on oligosaccharide-alditols.
The potential uses of the unique activity of this novel recombinant enzyme will be further discussed.
[1] G.O. Hreggvidsson, J.M. Dobruchowska, O.H. Fridjonsson, J.O. Jonsson, G.J. Gerwig, A. Aevarsson, J.K. Kristjansson, D. Curti, R.R. Redgwell, C.-E. Hansen, J.P. Kamerling, T. Debeche-Boukhit, Glycobiology 21 (2011) 304-328/664-688.
Acknowledgements
This work was financially supported by grants from the European Union (the European Regional Development Fund), the Dutch Ministry of Economic Affairs, Agriculture and Innovation, the Northern Netherlands collaboration initiative (SNN EZ/KOMPAS RM119), and the 7th Framework EU project Amylomics (FP7 KBBE.2010.3.3-01).
