Enzymically attaching oligosaccharide-linked ‘cargoes’ to cellulose and other commercial polysaccharides via stable covalent bonds

The Equisetum enzyme hetero-trans-β-glucanase (HTG) covalently grafts native plant cellulose (donor-substrate) to xyloglucan (acceptor-substrate), potentially offering a novel ‘green’ method of cellulose functionalisation. However, the range of cellulosic and non-cellulosic donor substrates that can be utilised by HTG is unknown, limiting our insight into its biotechnological potential. Here we show that HTG binds all celluloses tested (papers, tissues, hydrogels, bacterial cellulose) to radioactively- or fluorescently-labelled xyloglucan-heptasaccharide (XXXGol; acceptor-substrate). Glycol-chitin, glycol-chitosan and chitosan also acted as donor substrates but less effectively than cellulose. Cellulose-XXXGol conjugates were formed throughout the volume of a block of hydrogel, demonstrating penetration. Plant-derived celluloses (cellulose Iβ) became more effective donor-substrates after ‘mercerisation’ in ≥3 M NaOH; the opposite was true for bacterial cellulose Iα. Cellulose-XXXGol bonds resisted boiling 6 M NaOH, demonstrating strong glycosidic bonding. In conclusion, HTG stably grafts native and processed celluloses to xyloglucan-oligosaccharides, which may carry valuable ‘cargoes’, exemplified by sulphorhodamine. We thus demonstrate HTG's biotechnological potential to modify various cellulose-based substrates such as textiles, pulps, papers, packaging, sanitary products and hydrogels.
We thank Ms. Amy Wallace (The University of Edinburgh) for help in producing xyloglucan–[3H]XXXGol, MLG–[3H]XXXGol and cellulose–[3H]XXXGol, Dr. Tom Simmons (The University of Edinburgh) for heterologous production of EfHTG in Pichia, Ms. Lucile Druel (CEMEF, Mines ParisTech) for the preparation of cellulose hydrogels, and Ms. Anna Roig (ICMAB, Bellaterra) for help in preparing bacterial cellulose. We acknowledge The Royal Society, London, for organising a meeting (‘Cellulose: prospects and challenges’, 15–16 March 2017) which initiated part of this collaboration. This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/N002458/1]; and the BBSRC Impact Acceleration Account (IAA) project [grant number PIII057].