title: Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy.
abstract: The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.
authors.affiliation: Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Adams
authors.forename: Paul A
authors.initials: PA
authors.lastname: Kroon
authors.forename: Gary
authors.initials: G
authors.lastname: Williamson
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Morris
mesh_terms: D002240:Carbohydrate Sequence; D043364:Endo-1,4-beta Xylanases / Q000302:isolation & purification / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D015394:Molecular Structure; D014990:Xylans / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D014990:Xylans; C085118:arabinoxylan; D043364:Endo-1,4-beta Xylanases