Please wait a minute...
J Integr Plant Biol, 2015, 57 (4): 411-428, Research Article
A general method for assaying homo- and hetero-transglycanase activities that act on plant cell-wall polysaccharides
Lenka Franková and Stephen C. Fry*
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK
*Correspondence: E-mail: s.fry@ed.ac.uk
doi: 10.1111/jipb.12337
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[RIGHTWARDS ARROW]3, 1[RIGHTWARDS ARROW]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.

 

Franková L, Fry SC (2015) A general method for assaying homo- and hetero-transglycanase activities that act on plant cell-wall polysaccharides. J Integr Plant Biol 57: 411–428 doi: 10.1111/jipb.12337

--------------------------------------------------------------------------------------------------------------------
Received: 02 December 2014      Accepted:    Online on:
This Article
   Full Text  
   Full Text (PDF) 
   Supporting information
Services
   E-mail Link to this Article
   Export Citation for this Article
Citing Articles
   Cited By
Google Scholar
   Articles by Lenka FrankovÁStephen C. Fry
PubMed
   Articles by Lenka FrankovÁStephen C. Fry
Editorial Office, Journal of Integrative Plant Biology, Institute of Botany, CAS
No. 20 Nanxincun, Xiangshan, Beijing 100093, China
Tel: +86 10 6283 6133 Fax: +86 10 8259 2636 E-mail: jipb@ibcas.ac.cn
Copyright © 2020 by the Institute of Botany, the Chinese Academy of Sciences
Online ISSN: 1744-7909 Print ISSN: 1672-9072 CN: 11-5067/Q
网站备案号:京ICP备16067583号-22