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

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 3 H‐polysaccharides from unreacted 3 H‐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. [ 3 H]xylohexaitol, [ 3 H]mannohexaitol [ 3 H]cellohexaitol) remained immobile. On silica‐gel, all 3 H‐oligosaccharides left an immobile ‘ghost’ spot (contaminating any 3 H‐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 3 H‐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 3 H‐polysaccharides from unreacted 3 H‐oligosaccharides, enabling high‐throughput screening of multiple postulated transglycanase activities utilising chemically diverse donor‐ and acceptor‐substrates.