Lytic Polysaccharide Monooxygenase-Assisted Preparation of Oxidized-Cellulose Nanocrystals with a High Carboxyl Content from the Tunic of Marine Invertebrate Ciona intestinalis

Abstract

The tunicate species Ciona intestinalis is a fast-growing marine invertebrate animal that contains cellulose in its outer partthe tunic. The high crystallinity and microfibril aspect ratio of tunicate cellulose make it an excellent starting material for the isolation of nanocellulose. In the present work, tunic from C. intestinalis was subjected to organosolv pretreatment followed by bleaching and acid-hydrolysis steps for the isolation of nanocrystals. Applying an intermediate enzymatic treatment step with a lytic polysaccharide monooxygenase (LPMO) from the thermophilic fungus Thermothelomyces thermophila was proved to facilitate the isolation of nanocellulose and to improve the overall process yield, even when the bleaching step was omitted. LPMOs are able to oxidatively cleave the glycosidic bonds of a polysaccharide substrate, either at the C1 and/or C4 position, with the former leading to introduction of carboxylate moieties. X-ray photoelectron spectroscopy analysis showed a significant increase in the atomic percentage of the CO/O−C−O and O−CO bonds upon the addition of LPMO, while the obtained nanocrystals exhibited higher thermal stability compared to the untreated ones. Moreover, an enzymatic post-treatment with LPMOs was performed to additionally functionalize the cellulose nanocrystals. Our results demonstrate that LPMOs are promising candidates for the enzymatic modification of cellulose fibers, including the preparation of oxidized-nanocellulose, and offer great perspectives for the production of novel biobased nanomaterials.

Structure and Self-Assembly of Lytic Polysaccharide Monooxygenase-Oxidized Cellulose Nanocrystals

“…Such green reaction that requires only molecular oxygen and an electron donor for conversion of the crystalline celluloses has an enormous potential for biorefinery , and environmentally friendly nanomaterial production. LPMOs have been studied for enhancing cellulose nanofibrillation − and more recently for CNC production, although in the latter case, the LPMO oxidation was combined with harsh sulfuric acid hydrolysis. The C1 carboxyl group deriving from the LPMO oxidation has previously been shown to stabilize cellulose nanofiber suspensions in a much similar manner to the C6 carboxyl group derived from TEMPO-mediated oxidation. , Thus, the aqueous suspensions of LPMO-oxidized CNCs could be expected to self-assemble into a liquid crystalline phase.…”

High resolution product profiling of lytic polysaccharide monooxygenases

“…The first structures of an AA9 (PDB ID: 2VTC) and an AA10 LPMO (PDB ID: 2BEM) have been elucidated in 2008 and 2005, respectively (142,152). Subsequently, more structures of AA9 and AA10, as well as of AA11, AA13, AA14 and AA15 LPMOs, have been elucidated as extensively reviewed previously (13,146,149,150,153,154). Overall, though generally LPMOs exhibit a low amino acid sequence similarity, both in between and within families, they display a common fibronectin-/immunoglobulin-like β-sandwich core structure (Fig.…”

“…Overall, though generally LPMOs exhibit a low amino acid sequence similarity, both in between and within families, they display a common fibronectin-/immunoglobulin-like β-sandwich core structure (Fig. 6) (153,154). Different from GH enzymes, which has 1 a surface cleft or groove, the LPMO active site is located at a relatively flat, solvent (and substrate) exposed, surface (Fig.…”

“…In AA9, AA11, AA13 and AA14 LPMOs, the third amino acid is a tyrosine, while a phenylalanine is found in AA10 (90% conserved) and chitin-specific AA15 LPMOs (149,150,154). This structural coordination is recognized as a histidine-brace motif, which is conserved in all LPMOs to date (145,154,158,159). The histidine-brace motif has been shown to be key for the LPMO catalytic reaction.…”

“…In addition to lignocellulose degradation, LPMOs have been used to assist the production of functional materials and compounds. Several studies have investigated the LPMO-assisted production of nanocellulose (152)(153)(154)(155)(156)(157)(158)(159)(160)(161), which is a valuable material used as emulsion stabilizer and gas barrier, also even in food industry (162-165).…”