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

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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 cellwall 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 3Hpolysaccharides 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)-b-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 3Holigosaccharides 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 reactionmixture 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-b-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 donorand acceptor-substrates.

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Ketersediaan

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Informasi Detail

Judul Seri

Integrative Plant Biology

No. Panggil

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Penerbit

: Journal of Integrative Plant Biology., 2015

Deskripsi Fisik

April 2015 | Volume 57 | Issue 4 | 411–428

Bahasa

English

ISBN/ISSN

doi: 10.1111/jipb.12

Klasifikasi

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Tipe Isi

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Tipe Media

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Tipe Pembawa

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Edisi

2015, Volume 57, Issue 4

Subjek

TANAMAN BIOLOGI

Info Detail Spesifik

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Pernyataan Tanggungjawab

deliza

Versi lain/terkait

Lampiran Berkas

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