J Integr Plant Biol. ›› 2015, Vol. 57 ›› Issue (4): 411-428.DOI: 10.1111/jipb.12337

• Research Articles • Previous Articles     Next Articles

A general method for assaying homo- and hetero-transglycanase activities that act on plant cell-wall polysaccharides

Lenka Franková and Stephen C. Fry*   

  1. The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK
  • Received:2014-12-02 Accepted:2015-01-26 Published:2015-04-01
  • About author:*Correspondence: E-mail: s.fry@ed.ac.uk

Abstract:

Transglycanases (endotransglycosylases) cleave a polysaccharide (donor-substrate) in mid-chain, and then transfer a portion onto another poly- or oligosaccharide (acceptor-substrate). Such enzymes contribute to plant cell-wall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated 3H-polysaccharides from unreacted 3H-oligosaccharides—the former immobilized (on filter-paper, silica-gel or glass-fiber), the latter eluted. On filter-paper, certain polysaccharides [e.g. (1[RIGHTWARDS ARROW]3, 1[RIGHTWARDS ARROW]4)-β-d-glucans] remained satisfactorily adsorbed when water-washed; others (e.g. pectins) were partially lost. Many oligosaccharides (e.g. arabinan-, galactan-, xyloglucan-based) were successfully eluted in appropriate solvents, but others (e.g. [3H]xylohexaitol, [3H]mannohexaitol [3H]cellohexaitol) remained immobile. On silica-gel, all 3H-oligosaccharides left an immobile ‘ghost’ spot (contaminating any 3H-polysaccharides), which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glass-fiber (GF), onto which the reaction-mixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of 3H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris trans-β-mannanase. In conclusion, our novel GF-blotting technique efficiently frees transglycanase-generated 3H-polysaccharides from unreacted 3H-oligosaccharides, enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donor- and acceptor-substrates.

 

Franková L, Fry SC (2015) A general method for assaying homo- and hetero-transglycanase activities that act on plant cell-wall polysaccharides. J Integr Plant Biol 57: 411–428 doi: 10.1111/jipb.12337

Key words: Glass-fiber blotting, plant cell wall, polysaccharides (donor), radiolabeled oligosaccharides (acceptor), transglycanase

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