A general method for assaying homo‐ and hetero‐transglycanase activities that act on plant cell‐wall polysaccharides. Issue 4 (April 2015)

• Record Type: Journal Article
• Authors: Franková, Lenka; Fry, Stephen C.
• Is Part Of: Journal of integrative plant biology. Volume 57:Issue 4(2015)
• Page Start: 411
• Page End: 428
• Publication Date: 2015-04
• Subjects: Plants -- Periodicals; Plants -- China -- Periodicals; Electronic journals; 580.5
• DOI: 10.1111/jipb.12337
• Languages: English
• ISSNs: 1672-9072

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <sec id="jipb12337-sec-0001" sec-type="section"> <p>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 <sup>3</sup>H‐polysaccharides from unreacted <sup>3</sup>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‐<sc>d</sc>‐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. [<sup>3</sup>H]xylohexaitol, [<sup>3</sup>H]mannohexaitol [<sup>3</sup>H]cellohexaitol) remained immobile. On silica‐gel, all <sup>3</sup>H‐oligosaccharides left an immobile 'ghost' spot (contaminating any <sup>3</sup>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 <sup>3</sup>H‐polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF‐blotting was well demonstrated for <italic>Chara vulgaris</italic> trans‐β‐mannanase. In conclusion, our novel GF‐blotting technique efficiently frees transglycanase‐generated <sup>3</sup>H‐polysaccharides from unreacted <sup>3</sup>H‐oligosaccharides, enabling high‐throughput screening of multiple postulated transglycanase activities utilising chemically diverse donor‐ and acceptor‐substrates.</p> </sec> </abstract>