Structure and assembly of epiglucan, the extracellular (1!3;1!6)-b-glucan produced by the fungus Epicoccum nigrum strain F19 Frank Schmid, a Bruce A. Stone, b Robert T. C. Brownlee, c Barbara M. McDougall d and Robert J. Seviour d, * a School of Agriculture and Wine, The University of Adelaide, PMB1, Glen Osmond, SA 5064, Australia b School of Biochemistry, La Trobe University, Bundoora, Vic. 3083, Australia c Department of Chemistry, La Trobe University, Bundoora, Vic. 3083, Australia d Biotechnology Research Centre, La Trobe University, Bendigo, Vic. 3550, Australia Received 14 July 2005; accepted 30 October 2005 Available online 15 December 2005 Abstract—In a previous article [Carbohydr. Res. 2001, 331, 163–171] two different structures for the possible modular repeating unit of the extracellular b-glucan, epiglucan produced by the fungus Epicoccum nigrum strain F19 were proposed. Clarifying which was the more likely one was considered essential before attempts were made to understand how epiglucan was assembled by this fungus. Data from Smith degradation analyses of epiglucan were consistent with the repeating unit of structure I, where single glucosyl res- idues are attached by (1!6)-b-linkages to two out of every three glucosyl residues in the (1!3)-b-linked glucan backbone. Repeated Smith degradations of 14 C-glucose labelled epiglucan showed that chain elongation occurred from its non-reducing end. Side chain insertion into the growing glucan was followed by analysis of real time incorporation of 13 C-glucose into epiglucan by 13 C NMR, and 14 C-glucose by enzymic digestion of the synthesised 14 C-epiglucan. All data obtained were consistent with the view that single (1!6)-b-linked glucosyl side residues are inserted simultaneously as the glucan backbone elongates. →3)-β-D-Glc p-(1→3)-β-D-Glc p-(1→3)-β-D-Glcp-(1→ β-D-Glc p 1 ↓ 6 β-D-Glc p 1 ↓ 6 Ó 2005 Elsevier Ltd. All rights reserved. Keywords: b-Glucan assembly; Epiglucan elongation; Side chain insertion; Extracellular polysaccharide; NMR spectroscopy; Biosynthesis; Radio- isotope labelling 1. Introduction Previous investigations into the assembly of linear cell wall and mucilage fungal glucans have concentrated mainly on understanding their direction of elongation, and the purification and characterisation of the glucan synthases involved. 1,2 Many fungal extracellular glucans are branched, which adds another level of complexity to such studies, as both the timing and enzymology of side branch insertion into the backbone glucan need to be considered. Batra et al. 3 studied the direction of elonga- tion and timing of side branch insertion of the extra- cellular (1!3;1!6)-b-glucan from Sclerotium rolfsii. Cultures of S. rolfsii were pulsed with 14 C-glucose, the glucan isolated and the relative proportions of 14 C-glu- cose incorporated into reducing and non-reducing ends 0008-6215/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2005.10.013 * Corresponding author. Tel.: +61 3 54447459; fax: +61 3 54447476; e-mail: r.seviour@latrobe.edu.au Carbohydrate RESEARCH Carbohydrate Research 341 (2006) 365–373