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

Lookup NU author(s): Dr Lenka Frankova

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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 H-3-polysaccharides from unreacted H-3-oligosaccharidesthe former immobilized (on filter-paper, silica-gel or glass-fiber), the latter eluted. On filter-paper, certain polysaccharides [e.g. (13, 14)--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. [H-3]xylohexaitol, [H-3]mannohexaitol [H-3]cellohexaitol) remained immobile. On silica-gel, all H-3-oligosaccharides left an immobile ghost' spot (contaminating any H-3-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 H-3-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris trans--mannanase. In conclusion, our novel GF-blotting technique efficiently frees transglycanase-generated H-3-polysaccharides from unreacted H-3-oligosaccharides, enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donor- and acceptor-substrates.

Publication metadata

Author(s): Franková L, Fry SC

Publication type: Article

Publication status: Published

Journal: Journal of Integrative Plant Biology

Year: 2015

Volume: 57

Pages: 411-428

Print publication date: 04/04/2015

Online publication date: 31/01/2015

Acceptance date: 26/01/2015

ISSN (print): 1672-9072

ISSN (electronic): 1744-7909

Publisher: Wiley-Blackwell Publishing Asia

URL: https://doi.org/10.1111/jipb.12337

DOI: 10.1111/jipb.12337

PubMed id: 25641334

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