A unique Equisetum enzyme, hetero-trans-β-glucanase (HTG), enhances industrially and environmentally important cellulose-rich materials

Gigatons of cellulose are synthesised and degraded annually. Under-utilised cellulose, e.g. from sugar-beet pulp ('Curran') and recycled paper, could be valorised by Equisetum hetero-trans-β-glucanase (HTG), the only known enzyme modifying cellulose non-destructively. It catalyses hetero-transglycosylation, with cellulose as donor substrate and xyloglucan oligosaccharides (XGOs) as acceptor, creating cellulose-XGO covalent bonds. Interestingly, XGOs participate in this reaction even if linked to a biotechnologically-valuable 'cargo', e.g. a dye, fluorophore, cation, anion, hydrophobic compound or antibiotic, beneficially functionalising the cellulose. Here, we tested whether HTG acts on intransigent agricultural/industrial celluloses. Curran was found to be a pectin- and hemicellulose-poor, but cellulose-rich product, thus potentially enhanceable by HTG technology. To generate industrial quantities of HTG, we explored Equisetum extraction methods, comparing extractants (buffers versus tap-water), extractant ratios, homogenisation technique, work-up (centrifugation versus filtration) and salting out. Pleasingly, simplified 'industrial' methods were 75-85 % as effective as laboratory methods. Up-scaled heterologous expression in Pichia also yielded plentiful EfHTG. Thus diverse methods enabled bulk HTG production. All HTG preparations tested acted on pure cellulose (Whatman filter-paper; donor substrate) and radioactive or fluorescent cargo-bearing XGOs ([³H]XXXGol or XXXGol-sulphorhodamine respectively; model acceptor substrates). Diverse HTG preparations acted on potentially 'intractable' celluloses, e.g. Curran and crude recycled brown card, at rates comparable to those on pure celluloses (filter-paper and virgin white never-dried pulp). Where necessary, coarse industrial celluloses were H₂SO₄-hydrolysed to expose underestimated signal from incorporated but 'masked' radioactive substrate. Unlike surface-finish modifications, HTG-catalysed bonding of cargoes to cellulose is covalent (thus highly stable) and environmentally friendly (green sustainable technology).