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

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→3, 1→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.