Exploiting cellulose:xyloglucan endotransglucosylase activity — exploring novel acceptor substrates carrying valuable ‘cargoes’

Hetero-trans-β-glucanase (HTG) is an Equisetum plant enzyme that covalently grafts cellulose (donor substrate) to xyloglucan heptasaccharide (XXXG; Xyl3.Glc4 as acceptor substrate). Commercial exploitation of this technology requires bulk XXXG production. Novel acceptor substrates bearing two or more XXXG moieties could potentially cross-link cellulose chains. We explored bulk XXXG production, the synthesis of novel (including multimeric) XXXG conjugates and novel coloured derivatives, and explored new activators of HTG action. Commercial tamarind gum (crude xyloglucan) was digested with xyloglucan endo-glucanase, producing mixed xyloglucan oligosaccharides, which were then enzymically de-galactosylated to XXXG. Sequential application of the two enzymes proved more reliable than simultaneous. XXXG was reductively aminated with 1,2-diaminoethane (DAE), generating conjugates with the constitution XXXGol₁_-₄-DAE. In some cases, [¹⁴C]DAE was used, generating radiolabelled conjugates. The products were characterised by ninhydrin staining, thin-layer chromatographic mobility and electrophoretically determined charge:mass ratio. All DAE conjugates of XXXGol proved satisfactory acceptor substrates for HTG. We also reductively aminated XXXG with toluidine blue O (TB), generating coloured conjugates (XXXGol₁_-₂-TB) that were likewise HTG acceptor substrates, allowing visualisation of this and other XXXG reactions on paper substrata. Furthermore, we discovered hydrophilic neutral polymers, e.g. polyvinylalcohol and polyethyleneglycol (but not a polyanion or polycation), that were highly effective activators of HTG action on cellulose - alternatives to the conventionally used bovine serum albumin. Casein and various food-grade plant protein preparations were also effective. In conclusion, this work opens the way to utilising Equisetum HTG to chemically modify cellulose, with exciting potential commercial benefits.