Effect of ionic liquid on the enzymatic synthesis of cello-oligosaccharides and their assembly into cellulose materials

Imidazolium-based ionic liquids are important solvents for the processing of natural cellulose. Little is known about their use in synthesizing cellulose via bottom-up polymerization of β-1,4-D-glucosyl chains in solution. Here, we analyzed cellodextrin phosphorylase-catalyzed synthesis of cello-oligosaccharides, and the subsequent spontaneous self-assembly of the chains, in the presence of cellulose-dissolving ionic liquid, 1,3-dimethylimidazolium dimethyl phosphate ([Dmim]DMP) or 1-ethyl-3-methylimidazolium acetate ([Emim]OAc).

Carbohydrate Polymers 301 (2023) 120302 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Effect of ionic liquid on the enzymatic synthesis of cello-oligosaccharides and their assembly into cellulose materials Chao Zhong a, Krisztina Zajki-Zechmeister a, Bernd Nidetzky a, b, * a b Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, 8010 Graz, Austria Austrian Centre of Industrial Biotechnology (acib), 8010 Graz, Austria A R T I C L E I N F O A B S T R A C T Keywords: Synthetic cellulose Ionic liquid Chain self-assembly Hydrogel Phosphorylase bio-catalysis Imidazolium-based ionic liquids are important solvents for the processing of natural cellulose Little is known about their use in synthesizing cellulose via bottom-up polymerization of β-1,4-D-glucosyl chains in solution Here, we analyzed cellodextrin phosphorylase-catalyzed synthesis of cello-oligosaccharides, and the subsequent spontaneous self-assembly of the chains, in the presence of cellulose-dissolving ionic liquid, 1,3-dimethylimida­ zolium dimethyl phosphate ([Dmim]DMP) or 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) The average chain length dropped from ~7.4 in buffer to ~6.4 in ionic liquid (30 vol%) The synthetic cellulose exhibited allomorph II crystal structure and showed nanosheet morphology of 4–5 nm thickness and several μm length Its suspensions were hydrogels with viscoelastic properties dependent on solvent conditions used Reactions in 10 vol% [Dmim]DMP or [Emim]OAc gave a hydrogel with elastic modulus of ~13 kPa and loss factor of ~0.18 Collectively, interactions of the ionic liquid with enzyme and cello-oligosaccharides delimit the polymerization and tune the assembly into cellulose networks Introduction Cellulose is an abundant and eco-friendly natural polymer composed of β-1,4-linked D-glucose units The production of cellulosic materials is usually based on top-down processing of lignocellulosic biomasses (e.g., woody materials) (Abdul Khalil et al., 2014; Brinchi, Cotana, Fortunati, & Kenny, 2013) This often involves mechanical/physical disruption of the original chain structure in cellulose biomaterials (Abdul Khalil et al., 2014) as well as partial chemical depolymerization of the poly­ saccharide chains (Brinchi et al., 2013) In the extent that top-down processing alters the original cellulose structure physically and chemi­ cally (Abdul Khalil et al., 2014; Phanthong et al., 2018), bottom-up synthesis of cellulose chains can present a promising alternative of cel­ lulose material production The bottom-up concept involves synthetic build-up of cellulose chains, which then self-assemble into a hierar­ chically organized material Assembled structures of synthetic cellulose chains have been prepared by different strategies (Habibi, Lucia, & Rojas, 2010; Kontturi et al., 2018) The enzymatic approach of synthesis is gaining increased attentions since it offers simplicity and flexibility in controlling the properties of the resulting cello-oligomers and hence their self-assembly into cellulose materials Among the known options for the enzymatic synthesis of cellulose (Hiraishi et al., 2009; Petrovic, ănen et al., 2020; Serizawa, Kok, Woortman, Ciric, & Loos, 2015; Pylkka Kato, Okura, Sawada, & Wada, 2016), the approach using cellodextrin phosphorylase (CdP, EC 2.4.1.49) is promising, given the high chemical purity of the products, the simple substrates used, and the flexibility to prepare reducing end-functionalized cello-oligomers (Bulmer, de Andrade, Field, & van Munster, 2021; Nakai, Kitaoka, Svensson, & Ohtsubo, 2013) Cello-oligomers prepared by the CdP reaction selfassemble into different material structures in situ depending on the conditions used (Hata & Serizawa, 2021; Nidetzky & Zhong, 2020; Nigmatullin, de Andrade, Harniman, Field, & Eichhorn, 2021; Sugiura, Sawada, Tanaka, & Serizawa, 2021) Several studies of CdP-catalyzed synthesis of cello-oligosaccharides have shown that bulk parameters (e.g., pH, temperature) can affect the properties of the resulting cellulose (Hata, Kojima, Maeda, Sawada, & Serizawa, 2020; Hata, Sawada, Marubayashi, Nojima, & Serizawa, 2019) In addition, polymers (Hata et al., 2017) and colloidal particles (Hata, Sawada, Sakai, & Serizawa, 2018) that give a macromolecular crowding effect or cause a viscosity increase also affect the enzymatic synthesis and the subsequent self-assembly of the cello-oligomers Earlier works have placed a strong focus on the effect of * Corresponding author at: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, 8010 Graz, Austria E-mail addresses: czhong@tugraz.at (C Zhong), krisztina.zajki-zechmeister@tugraz.at (K Zajki-Zechmeister), bernd.nidetzky@tugraz.at (B Nidetzky) https://doi.org/10.1016/j.carbpol.2022.120302 Received 24 August 2022; Received in revised form 21 October 2022; Accepted 31 October 2022 Available online November 2022 0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) C Zhong et al Carbohydrate Polymers 301 (2023) 120302 macromolecules (i.e., molecular mass of about 20 to 150 kDa) on the enzymatic cellulose synthesis (Hata et al., 2017; Hata et al., 2018) However, effects of small-molecule additives (< kDa) have not been fully explored up to now A recent study reported the use of organic solvents (e.g., dimethyl sulfoxide, ethanol) to prevent the precipitation of the incipient cellulose oligomers during enzymatic synthesis (Hata, Fukaya, Sawada, Nishiura, & Serizawa, 2019) It was suggested that the cellulose chains might become more strongly solvated due to the in­ teractions (e.g., hydrogen bonding) with the solvents The exciting idea was put forward that small molecules could have a role in tuning the assembly of the synthesized cello-oligomers (Hata, Fukaya, et al., 2019) In the present study, therefore, the enzymatic synthesis of cellooligosaccharides was investigated using ionic liquids (ILs) as smallmolecule additives or co-solvents The ionic liquids might affect both the synthesis of the cello-oligosaccharides by the enzyme and the sub­ sequent self-assembly driven aggregation of the chains in bulk solution ILs are ion-containing organic salts that are known as one of the best solvents for carbohydrate polymers including cellulose ILs are effective because they contain loosely bound ions that can interact with polar groups (e.g., -OH) of oligo- and polysaccharides thus to solubilize them (Li, Wang, Liu, & Zhang, 2018; Morais et al., 2020; Verma et al., 2019; Wang, Gurau, & Rogers, 2012) To this end, 1,3-dimethylimidazolium dimethyl phosphate ([Dmim]DMP) and 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) were selected for their remarkable cellulose dissolution capacities (Koide, Urakawa, Kajiwara, Rosenau, & Wataoka, 2020; Li et al., 2018; Lopes, Bermejo, Martín, & Cocero, 2017; Zheng, Harris, Bhatia, & Thomas, 2019) We hypothesized that the ILs could interact with the synthesized cello-oligomers mainly through hydrogen bonds that might delimit the oligomerization and alter product prop­ erties accordingly The CdP-catalyzed reactions were carried out in the presence of IL (~30% by volume), and an increased soluble yield of cellulose (by ~40%) was observed when ILs were used Solid products were structurally analyzed (i.e., degree of polymerization (DP), morphology, crystallinity) to reveal the effect of ILs on the assynthesized cellulose In addition, hydrogels with viscoelastic proper­ ties were obtained depending on the conditions used It was hypothe­ sized that the interactions with IL facilitate the assembly of cellulose into highly-ordered material networks Overall, this study contributes to a better understanding of the role of small molecules in the enzymatic synthesis of oligo- and polysaccharides 2.3 Oligomerization reaction Reactions (in 0.5 mL volume) were performed at 45 ◦ C and 300 rpm through incubation on a ThermoMixer C (Eppendorf, Vienna, Austria) for 24 h α-D-Glucose 1-phosphate (αGlc1-P, 150 mM) and cellobiose (10 mM) were incubated with CcCdP (0.5, 1.0, and 2.0 U/mL, buffer ac­ tivity) in 50 mM MES buffer (pH 7.0) containing ionic liquid ([Dmim] DMP or [Emim]OAc) in a volume fraction of 10, 20, 30, and 40%, respectively The control reaction was performed under exactly the same conditions but without ionic liquid Insoluble materials were generated during the reactions To recover the solid materials, reaction mixtures were centrifuged at 21,130 ×g for at least (Centrifuge 5424/R, Eppendorf, Germany) until the su­ pernatant was clear After removal of the supernatant, the pelleted material was thoroughly resuspended in mL of distilled water and then centrifuged again at 21,130 ×g for (Centrifuge 5424/R, Eppen­ dorf) The washing step was repeated times In these steps, the su­ pernatant was carefully removed with a pipette, with the tips away from the pellet to avoid loss of the material The solid thus obtained was lyophilized and then weighed The insoluble ratio of the products was defined as the molar ratio of glucosyl units in the solid (estimated from the total amounts of insoluble products and the average DP of products calculated from mass spectrometry analysis) to the glucosyl units transferred from αGlc1-P during the reaction In addition, the supernatant of the reaction was heated (95 ◦ C, min) to inactivate the enzyme and then centrifuged The conversion of αGlc1P was determined by the phosphate released into the supernatant Phosphate was measured by a colorimetric assay (Saheki, Takeda, & Shimazu, 1985) Note that [Emim]OAc had no effect on the colorimetric assay and the influence of [Dmim]DMP (i.e., intrinsic phosphate con­ tent) was eliminated from the assay 2.4 Material characterization 2.4.1 Atomic force microscopy (AFM) The measurement was performed at room temperature using a Dimension FastScan Bio instrument (Bruker AXS, Karlsruhe, Germany) equipped with a NanoScope V controller in tapping mode Cellulose (washed pellets) dispersed in water (~2 mg/mL, 60 μL) was loaded onto a freshly cleaved mica surface and air dried A FastScan-A probe (Bruker AXS, Camarillo, USA) was used Analysis was performed using Gwyd­ dion 2.55 (http://gwyddion.net/download.php) Material and methods 2.1 Materials 2.4.2 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) Cellulose (washed pellets) suspended in water (~5 mg/mL) was prepared for measurement, which was performed according to the method described (Zhong, Zajki-Zechmeister, & Nidetzky, 2021) Mass spectra analysis was done using the mMass (http://www.mmass.org/) The number-average molecular weight (Mn) was calculated using the ∑ ∑ relationship, Mn = i (Ni × Mi)/ i Ni, where Ni is the peak intensity of the i-th cello-oligomer species and Mi is the molar mass of that species Here, sodium and potassium ion adducts of the oligomer in each DP (m/z +23 and +39) were included The average DP was calculated using the relationship, DP = (Mn − 18)/Mo, where Mo is the molecular mass of dehydrated glucose (in cellulose), 162.2 Da (Petrovic et al., 2015) Unless stated otherwise, the chemicals used were of highest purity available at Sigma-Aldrich (Vienna, Austria) or Carl Roth (Karlsruhe, Germany) Ionic liquids, [Dmim]DMP and [Emim]OAc, were from abcr GmbH (Karlsruhe, Germany) 2.2 Enzyme Cellodextrin phosphorylase from Clostridium cellulosi (CcCdP; Gen­ Bank identifier CDZ24361.1) was prepared according to the methods described (Zhong, Luley-Goedl, & Nidetzky, 2019) Briefly, enzyme was expressed in Escherichia coli BL21(DE3) and purified via its N-terminal His-tag Enzyme stock solutions (20 mg/mL) in 50 mM MES buffer (pH 7.0) were stored at − 20 ◦ C without appreciable loss of activity for at least one month The stock solutions were used as single-use aliquots and diluted to the desired working concentrations The enzyme showed a synthesis activity of 13.3 U/mg (on the acceptor substrate cellobiose) at 45 ◦ C in 50 mM MES buffer (pH 7.0) (Zhong et al., 2019; Zhong & Nidetzky, 2022) 2.4.3 Proton nuclear magnetic resonance (1H NMR) The 1H NMR spectra of the lyophilized material dissolved in 4% NaOD-D2O (10 mg/mL) were recorded on a Varian Inova-500 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) using a VNMRJ 2.2D software The chemical shifts were recorded relative to D2O (δH 4.8), and analyzed by MestReNova (https://mestrelab.com) The average DP of the product was calculated using the relationship, DP = (H1 + Hα + Hβ)/(Hα + Hβ), where the H1, Hα and Hβ present the C Zhong et al Carbohydrate Polymers 301 (2023) 120302 intensity of signals at 4.30 ppm (internal anomeric protons), 5.12 and 4.53 ppm (α- and β-anomeric proton), respectively (Hiraishi et al., 2009) synthesis of water-insoluble cellulose However, no turbidity was observed in the reactions with 40 vol% IL The reaction in buffer without IL gave a gel-like mixture that partially collapsed upon inversion (Fig 2a) The reactions containing 10–30 vol% IL also involved gel formation (Fig 2a), and a relatively stable gel was obtained in the re­ actions with 10 vol% IL (see later) The αGlc1-P conversion (after 24 h) in the reactions decreased with increasing IL concentration, from 56% in the control reaction to ~42% in the reaction with 30 vol% IL (Fig 3) A dramatic decline of αGlc1-P conversion (to just ≤12%) was further observed in the reactions con­ taining 40 vol% IL Time course analysis (Fig 3) also revealed that the αGlc1-P conversion was consistently lower when enzymatic reactions were performed in the presence of IL These results suggested that the ILs caused a lowering of the apparent activity of the CcCdP The inhibitory effect on activity (hence, the reaction rate) was dependent on the IL concentration used (Fig 3) The IL additionally caused a decrease in enzyme stability Fig 4a shows that incubation at IL concentrations of ≥20 vol% resulted in considerable loss of enzyme activity in 24 h Note that with the assay used, an irreversible process of enzyme inactivation was measured At 40 vol% of both [Dmim]DMP and [Emim]OAc, nearly all of the original enzyme activity was lost The two ILs used here are ăm, Rovio, & generally considered to be enzyme-friendly (Wahlstro Suurnă akki, 2012; H Zhao, Jackson, Song, & Olubajo, 2006) Nonethe­ less, tolerance of CcCdP to them as co-solvents was limited There can be different reasons for enzyme inhibition in the presence of IL (Zhao, 2005) One reason is a direct effect of the co-solvent on the enzyme structure The other is indirect and involves a co-solvent effect on substrate accessibility to the enzyme (Endo, Hosomi, Fujii, Ninomiya, & Takahashi, 2016; Li et al., 2018) The interaction of IL ions with the carbohydrate substrates may change the substrate partitioning between the solvent and the enzyme binding pocket, thus leading to a lowered apparent affinity for substrate binding and thus a decreased activity We cannot distinguish between these possibilities based on the evidence obtained Nevertheless, the selected ILs were usable as co-solvents for the purpose of current study when their concentrations were limited to 30 vol% Close inspection of the different reactions in Fig revealed that the evolution of turbidity decreased with increasing concentration of each IL To quantitate the effect, the amount of insoluble product was measured from each reaction (0.5 mL volume; N = 4) The mass of insoluble product was 5.6 ± 0.2 mg from the buffer control reaction It was 4.8 ± 0.1 mg, 4.4 ± 0.4 mg and 3.2 ± 0.1 mg from the reaction in the presence of 10, 20 and 30 vol% [Dmim]DMP, respectively Using [Emim]OAc, it was 4.9 ± 0.1 mg, 3.3 ± 0.2 mg and 1.8 ± 0.1 mg from the reaction at 10, 20 and 30 vol%, respectively The decrease in the insoluble product formation dependent on the IL concentration used 2.4.4 X-ray diffraction (XRD) Measurement of the lyophilized cellulose material was done ac­ cording to the method described (Zhong et al., 2021) 2.5 Rheological measurement Dynamic rheological measurements were performed on a straincontrolled rheometer (MCR 502, Anton Paar, Austria) at 25 ◦ C, using a cone-and-plate measurement geometry (CP 50-1) with 50 mm diam­ eter and 1◦ cone For measurement, the sample (600 μL) was placed on the Peltier plate The linear viscoelastic range was measured with a strain sweep (0.01–100%) at a fixed frequency of 10 rad/s Frequency sweeps were performed over an angular frequency range of 1–100 rad/s with a constant strain amplitude of 0.1% (within the linear viscoelastic range) to record the storage modulus (G′ ) and loss modulus (G′′ ) of the mixtures Results and discussion 3.1 Enzymatic synthesis of cello-oligosaccharide chains in ILs Our previous study of the bottom-up synthesis of cellooligosaccharides exploited the CdP-catalyzed reaction using cellobiose as the “primer” substrate (Fig 1a) (Zhong et al., 2019) The synthesized cello-oligomers (with an average DP above 6) assembled into sheet-like nanocelluloses that aggregated/precipitated from buffer solution (Kli­ macek, Zhong, & Nidetzky, 2021) Here, we hypothesized that due to their known interaction with cellulose chains, ILs might tune the as­ sembly of the incipient cello-oligosaccharides and change the overall properties of the synthetic cellulose materials We here showed a CcCdP-catalyzed synthesis of cellooligosaccharides in the presence of [Dmim]DMP or [Emim]OAc (Fig 1b) The two imidazolium-based ILs are well-known for their cellulose-dissolving properties (Lopes et al., 2017; Wang et al., 2012) Cello-oligosaccharides were synthesized at a donor/acceptor molar ratio of 15:1 Earlier studies (Petrovic et al., 2015; Zhong et al., 2019) have shown that donor/acceptor ratios as high as this favor the chain elon­ gation and so the formation of insoluble cellulose as the product The conditions used were thus adjusted to investigate the chain assembly in the presence of IL Reactions involving 2.0 U/mL CcCdP (buffer activity) in the absence or presence of IL (10–40 vol%) were tested The mixtures with 0–30 vol% IL turned opaque after 24 h of reaction, indicating the Fig Bottom-up enzymatic synthesis of cello-oligosaccharides in the presence of imidazolium-based IL a) Reaction scheme of β-1,4-glycosylation of cellobiose using αGlc1-P as the donor catalyzed by cellodextrin phosphorylase; b) Chemical structure of the imidazolium-based ILs used in the current study: [Dmim]DMP, 1,3dimethylimidazolium dimethyl phosphate; [Emim]OAc, 1-ethyl-3-methylimidazolium acetate C Zhong et al Carbohydrate Polymers 301 (2023) 120302 Fig Photographs of the reaction mixtures (after 24 h) at different enzyme activities The activities here refer to the enzyme assay in buffer without IL The reactions were performed using 10 mM cellobiose, 150 mM αGlc1-P in 50 mM MES buffer (pH 7.0) containing IL concentrations of 0–30 vol% at 45 ◦ C, for 24 h To assess gelation, the tubes were inverted after the reactions Fig Time courses of αGlc1-P conversion from the enzymatic reactions with IL at various concentrations (0–30 vol%) a) [Dmim]DMP; b) [Emim]OAc Reactions using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL CcCdP (buffer activity) in 50 mM MES buffer (pH 7.0) containing IL at varied concentrations were performed at 45 ◦ C for 24 h might arise trivially from the fact that the conversion of the αGlc1-P substrate was also lowered when the IL concentration was increased However, it might also involve a shift in the ratio of insoluble and sol­ uble products released in the enzymatic reaction when IL was present Fig 4b shows that the portion of insoluble material in the total product mass decreased dramatically (by up to 40%) as the IL concentration increased In buffer without IL, all of the product (≥98%) accumulated in insoluble form Ability of the IL to interact with the incipient cellooligosaccharides can arguably be related to the Kamlet-Taft parameter of H-bond basicity (β) The β value of the two ILs used is ~1.0 while that of water is only 0.18 (Lopes et al., 2017) Solvent interactions of the cello-oligosaccharides that are stronger with the ILs than water could C Zhong et al Carbohydrate Polymers 301 (2023) 120302 vol%), and it was slightly lower than that of the oligosaccharides syn­ thesized in the buffer control reaction (Table 1) This result was consistent with the morphology analysis of the products (Fig 6) AFM observations of these sheet-like materials revealed an estimated thick­ ness of 4.9 ± 0.2 nm for the product from the control reaction The products from the [Dmim]DMP- and [Emim]OAc-containing reactions (20 vol%) exhibited a lower thickness of 4.2 ± 0.2 nm and 4.1 ± 0.2 nm, respectively It was shown in the earlier works (Hata, Sawada, et al., 2019; Serizawa, Fukaya, & Sawada, 2017) that the cellooligosaccharides are aligned perpendicular to the base plane of the cellulose nanosheets The nanosheet thickness is thus expected to reflect the average DP of the synthetic cello-oligosaccharides Detailed analysis as summarized in Table shows that the average DP of the cello-oligosaccharide products decreased with increasing IL concentration in the reactions The result can arguably be explained by the decrease of enzymatic reaction rate in the presence of the IL cosolvent As mentioned before, the decrease in rate may involve direct or indirect effect of the IL on the enzyme activity The rate of chain elongation in competition with the rate of chain aggregation delimits the average DP of cello-oligosaccharide present in the insoluble cellulose material The relative portion of longer-chain cello-oligosaccharides (DP ≥ 8) was dramatically reduced in the enzymatic reactions con­ taining IL (Fig 5b) In agreement with these findings, the research of Serizawa's group has shown that the average DP of cellulose chain can be modulated by changing the volumetric CdP activity in the reaction (Serizawa et al., 2017) or by using conditions (e.g., at lower temperature 20–30 ◦ C) that decrease the enzymatic rate (Hata, Fukaya, et al., 2019) The cellulose materials were also analyzed by XRD The XRD pat­ terns (Supplementary materials, Fig S1) were identical for all the cel­ lulose materials, irrespective of the type of IL and the IL concentration used in the synthetic reactions The XRD peaks at 2θ of 12.5◦ , 20.1◦ , and 22.1◦ indicated a highly ordered cellulose material of allomorph II crystal structure The cellulose II is the most stable crystalline form of cellulose (Moon, Martini, Nairn, Simonsen, & Youngblood, 2011) and is typical of the cellulose materials prepared by self-assembly driven as­ sociation of cello-oligosaccharides from aqueous solution (Hata, Sawada, et al., 2019; Hiraishi et al., 2009; Serizawa et al., 2017) Cel­ lulose II involves antiparallel organization of the cellulose chains The evidence from this study suggests that presence of the ILs used did not alter the fundamental characteristics of crystalline cellulose formation from the growing cello-oligosaccharide chains It would appear there­ fore, that the IL effect was mostly on the enzymatic process of synthesis, happening in solution In addition to the above features, no unassigned signal/peak was detected in the NMR and MS spectra of the products from the enzymatic reactions containing IL This result suggested the absence of chemical derivatization of the cello-oligosaccharides as-synthesized, consistent with the notion that ILs engage in non-covalent interactions, mainly involving hydrogen bonds and van der Waals forces, with oligo- and polysaccharides (Verma et al., 2019) Fig Effect of imidazolium-based IL on enzyme activity and insoluble product formation in reactions catalyzed by CcCdP a) Residual activity of CcCdP after 24 h incubation in the buffer solutions containing [Dmim]DMP or [Emim]OAc (0–40 vol%) The 50 mM MES buffer (pH 7.0) containing 2.0 U/mL CcCdP (buffer activity) and varied IL concentrations (0–40 vol%) were incu­ bated at 45 ◦ C, for 24 h The activity of CcCdP in solution after immediate preparation (0 h) and 24 h incubation was measured, and the residual activity was calculated; b) Insoluble ratio of cellulose products from the enzymatic re­ actions containing IL at varied concentrations Reactions were performed using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL of CcCdP (buffer activity) in 50 mM MES buffer (pH 7.0) containing IL at 45 ◦ C, for 24 h explain the largely decreased tendency to undergo self-assembly driven chain aggregation into solid material under conditions when the IL was present Addition of such IL can thus present a strategy to enhance the soluble product release from the CdP-catalyzed reaction 3.3 Gel-like properties of cellulose synthesized in the presence of IL As mentioned above (see 3.1 Enzymatic synthesis of cellooligosaccharide chains in ILs), strong gelation behavior was observed in reaction mixtures of enzymatic cellulose synthesis in the presence of IL While this behavior indicated the formation of network structures of solid material in suspension, the underlying mechanisms are not well understood from a number of earlier studies of gelation of cellulose in the presence of IL (Hopson et al., 2021) In hydrogels prepared from cellulose substrates that reflected different degrees of top-down pro­ cessing of natural raw materials, physical crosslinking by hydrogen bonding represented the principal force of stable network formation (Shen, Shamshina, Berton, Gurau, & Rogers, 2016) A series of experi­ ments were performed here to investigate the idea of a direct relation­ ship between the gel formation and the IL-mediated supramolecular 3.2 Structural characterization of the cello-oligosaccharides in insoluble cellulose material The chemical structure of the solid products was analyzed by 1H NMR and MALDI-TOF mass spectrometry The products, despite the different reaction conditions used for synthesis, exhibited similar and representative NMR signals (e.g., δH 4.3) assignable to the repeating β-glucosyl units of cellulose (Fig 5a) (Isogai, 1997) In addition, the mass spectra, with the peak-to-peak mass difference of 162.2 Da (one glucosyl unit), further confirmed the synthesis of cello-oligosaccharides under these conditions (Fig 5b) Here, the average DP of products calculated from both the NMR and MS spectra was 6–7 for the cellooligosaccharides synthesized from the IL-containing reactions (10–30 C Zhong et al Carbohydrate Polymers 301 (2023) 120302 Fig Structural characterization of synthetic cellulose a) 1H NMR and b) MALDI-TOF MS spectra of the solid products synthesized without and with IL at various concentrations (D indicates [Dmim]DMP; and E indicates [Emim]OAc) Reactions were performed using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL CcCdP (buffer activity) in 50 mM MES buffer (pH 7.0) containing IL at 45 ◦ C, for 24 h aforementioned reaction mixtures The G′ was nearly frequencyindependent, and the G′′ was weakly frequency-dependent, having a shallow minimum (0.5 U/mL reactions) or featuring decrease (1.0 and 2.0 U/mL reactions) at low frequency This rheological behavior, also observed in soft glassy materials (e.g., pastes and emulsions), might be related to structural relaxations that occur between low and high fre­ quencies (Mason et al., 1997; Mendoza et al., 2018) The G′ value is an indication of the hydrogel's ability to store deformation energy in an elastic manner It is correlated to the ability of the material to revert to a solid state and to retain its shape after cessation of shear (Ma et al., 2021) The G′ value normally increases with increasing fiber material concentration, within certain range, in the gel matrix, due to the stronger networks formed and the increased contribution to stiffness (Ma et al., 2021; Mihranyan, Edsman, & Strømme, 2007) This is suggested from the control reaction, where the G′ increased from 7500 to 9600 Pa as the enzyme activity increased from 0.5 to 2.0 U/mL and accordingly the αGlc1-P utilization increased by 15% (Fig 7b) Reactions at higher enzyme activity produced larger amounts of cello-oligosaccharides, in particular at an early stage of the conversion The increased molecular crowding thus generated may have promoted a stronger network of interactions between the initially formed nuclei of insoluble cellulose (Hata et al., 2017; Korhonen & Budtova, 2019) Interestingly, therefore, when IL co-solvent (10–20 vol %) was used, the G′ declined at high enzyme activity In reactions containing [Dmim]DMP (10–20 vol%), the G′ value dropped by almost 80% as the enzyme activity increased from 0.5 to 2.0 U/mL The effect of varied enzyme activity was by far more significant on the resulting gel properties than it was on the corresponding conversion of αGlc1-P, which was changed by just 12% (Fig 7b) Similarly, reactions in the presence of [Emim]OAc (10–20 vol%) involved a decrease in the G′ by almost 60% as the enzyme activity increased from 0.5 to 2.0 U/mL The corresponding change in αGlc1-P conversion was a mere 17% (Fig 7b) Moreover, reactions at 0.5 U/mL in the presence of 10 vol% [Dmim] DMP or [Emim]OAc yielded cellulose hydrogels with a G′ of ~10–13 kPa (Fig 7b) that was increased by up to 74% compared to the control (i e., hydrogel prepared in buffer lacking IL) Strikingly, these G′ values were even higher than the G′ recorded for the control at 2.0 U/mL, despite the fact that the control exhibited a 21% higher αGlc1-P con­ version than the reactions with 10 vol% IL Survey of all the reactions Table Characterization of the solid cellulose products synthesized by CcCdP in the presence of IL at varied concentrations Solvents β value Water/ buffer [Dmim] DMP 0.18 [Emim] OAc 0.95b 1.0–1.1 a Conc (vol%) DP_1H NMR DP_MALDITOF MS – 7.83 7.25 10 7.07 6.91 20 6.44 6.89 30 10 6.01 7.31 6.42 6.92 20 7.06 6.66 30 – 6.49 Allomorph Cellulose II Cellulose II Cellulose II – Cellulose II Cellulose II – a Ref (Brandt, Hallett, Leak, Murphy, & Welton, 2010; Fukaya, Hayashi, Wada, & Ohno, 2008) b Ref (Zhang et al., 2012) interactions between dispersed nanoscale nuclei of solid cellulose The volumetric enzyme activity was varied at three levels of 0.5, 1.0, and 2.0 U/mL (buffer activity), each at a variable IL content between and 30 vol% Gelation was observed in the majority of the reactions, except for those at 0.5–1.0 U/mL in the presence of 30 vol% IL, which yielded a suspension of the insoluble product (Fig 2) The gel-like properties of the reaction mixtures were further investigated by rheological means (Mendoza, Batchelor, Tabor, & Garnier, 2018; Roy, Budtova, & Navard, 2003) In this respect, the storage modulus G′ describes the solid-like or elastic behavior, and the loss modulus G′′ describes the liquid-like or viscous behavior of the materials The dependence of both G′ and G′′ on the angular frequency (1–100 rad/s) was assessed The behavior of the mixtures was found to be predominantly elastic, indicated by the evi­ dence that G′ was larger than the corresponding G′′ over the entire fre­ quency range Fig 7a depicts this behavior for mixtures obtained from the 0.5 U/mL reaction Similar profiles of storage/loss modulus versus angular frequency were obtained for the 1.0 and 2.0 U/mL reactions, as shown in Figs S2–3 The results confirmed the gel property of the C Zhong et al Carbohydrate Polymers 301 (2023) 120302 Fig Atomic force microscopy (AFM) images of the synthesized cellulose: a) control; b) synthesized at 20 vol% [Dmim]DMP; c) synthesized at 20 vol% [Emim] OAc Materials were prepared from the reaction using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL CcCdP (buffer activity) in 50 mM MES buffer (pH 7.0) with/ without IL at 45 ◦ C, for 24 h The nanosheet crystalline material is shown, and its thickness (in single layer) was measured through cross-sectional analysis performed (Fig 7b) suggested a minimum degree of αGlc1-P conversion of ~42% required for gel formation Reactions that yielded only sus­ pensions of insoluble cellulose (with G′ ≤ kPa) exhibited low αGlc1-P conversion of 27–36% (Figs and 7b) In summary, therefore, two factors appear to be critical in order to promote cellulose gel formation efficiently A sufficient amount (concentration) of nanoscale cellulose nuclei (supposedly in nanosheet form or smaller structure) must accu­ mulate as gel precursors from the synthesis reaction Stiffness of the resulting gel is affected by the precursor concentration Physical cross­ linking of the gel precursors, and probably their further growth into higher-order structures (e.g., nanoribbons; Hata, Fukaya, et al., 2019; Hata, Sawada, et al., 2019), is then necessary to establish the final gel network structure The IL co-solvents appear to facilitate these processes in particular and so generate a gel reinforcement effect Fig illustrates the proposed mode of cellulose gel formation under the assistance of IL Previous studies have demonstrated the transition into gels of cel­ lulose solutions in IL upon the addition of water It was hypothesized that the added water breaks some of the original cellulose-IL interactions and thus promotes the restoration of cellulose-internal hydrogen bonding interactions that lead to the stabilization of supramolecular cellulose chain assemblies (Lee et al., 2017) With the formation of such cellulose assemblies, able to interact with IL ions and serving as nuclei for gelation via physical cross-linking, chain entanglement and forma­ tion of self-supporting gel networks could follow (Lee et al., 2017; Zhao et al., 2020) Evidence of the current study emphasizes in particular the prominent role of cellulose-IL interactions in the process of stable gel formation To allow for the relevant interactions with IL ions to be developed in the pre-gelation state, a moderate synthetic rate that en­ ables self-assembly of cello-oligosaccharides of DP ≥ is required An excessive synthetic rate can lead to a supramolecular aggregation of the cellulose nuclei (chain assemblies) that may be too fast for IL ions to intervene Using an IL concentration (e.g., 10 vol%) suitably combined with enzyme-catalyzed synthetic rate, the incipient cellooligosaccharides would self-assemble and generate cellulose nuclei sufficiently stabilized/solvated by the IL ions in suspension (for a rele­ vant discussion of related effects of small molecules on cellulose as­ sembly, see Hata, Fukaya, et al., 2019) With IL ions mediating the interactions among cellulose nuclei, they might be further entangled and assembled, thus promoting the growth into a highly ordered matrix (Fig 8) This IL-mediated assembly route would impart a stronger network of intermolecular interactions between the cellulose and confer a higher elasticity to the resulting gel as compared to a gelation that merely involves an all-cellulose network of interaction The viscoelastic properties of the gels were further evaluated on the basis of the so-called loss factor (tan δ), defined as tan δ = G′′ /G′ The tan δ indicates how well the material performs in absorbing and dissipating energy Its value was found to increase with increasing IL concentration used in the reactions (Fig 9), suggesting a trend towards liquid-like behavior of the semi-solid system (i.e., the viscous response is larger than the elastic contribution) at the higher IL concentrations The observable trend (Fig 9) might reflect the evidence discussed above that the total content of insoluble cellulose in the suspension decreased as the IL concentration was increased A lowered concentration of cellulose nuclei as gel precursors might affect the further crosslinking and so the C Zhong et al Carbohydrate Polymers 301 (2023) 120302 Fig Rheological characterization of the synthetic cellulose prepared in the presence of IL a) Dynamic viscoelastic properties of the cellulose product mix­ tures obtained from enzymatic reactions in the pres­ ence of IL (left panel, [Dmim]DMP; right panel, [Emim]OAc) Reactions were done with 0.5 U/mL (buffer activity) Storage modulus G′ and loss modulus G′′ are shown with filled and open symbols, respectively Symbols: square (□) for control reac­ tion; circle (○) and triangle (△) for the reaction containing 10 vol% and 20 vol% IL, respectively; b) Heatmap of storage modulus G′ (upper panel) and αGlc1-P donor conversion (lower panel) in the re­ actions with various enzyme loadings (0.5–2.0 U/mL) and IL concentrations (0–30 vol%) Reactions with enzyme loading (0.5, 1.0 and 2.0 U/mL of CcCdP, buffer activity) were performed using 10 mM cello­ biose and 150 mM αGlc1-P in 50 mM MES buffer (pH 7.0) containing IL concentrations 0–30 vol% at 45 ◦ C, for 24 h The G′ values at angular frequency of 20 rad/s (middle range) were selected Fig Proposed mode of synthetic cellulose gel formation in the presence of IL co-solvent Incipient cello-oligosaccharide chains self-assemble into nuclei of insoluble cellulose, supposedly in nanosheet form Direct intervention of the IL at this stage cannot be excluded but seems to be of minor importance Under involvement of IL, however, the cellulose nuclei are further assembled and grown into organized solid networks that confer gel-like properties to the resulting solidin-liquid dispersion (Hata, Fukaya, et al., 2019) Crosslinking of the cellulose nuclei involves participation from the IL ions via non-covalent hydrogen bonding Besides hydrogen bonds, the IL ions can interact via van der Waals and CH-π bonds (Liu, Sale, Holmes, Simmons, & Singh, 2010; Zhang et al., 2017); resulting stiffness of material A tan δ value of ≤1 is typical of concen­ trated polymer gels and one of ≤0.18 (phase angle δ ≤ 10◦ ) indicates a strong gel (Mihranyan et al., 2007; Naji-Tabasi & Razavi, 2017) Mate­ rials from reactions at ≥20 vol% IL exhibited tan δ values of 0.24 or greater (Fig 9) These values are too high to be practically suitable for hydrogel development However, by controlling the IL content to ≤10 vol%, materials showing tan δ values of ≤0.18 were obtained Such materials can be classified as a truly elastic gel (Hopson et al., 2022; Naji-Tabasi & Razavi, 2017) Taken together, the materials prepared at 10 vol% IL showed desirable rheological properties (i.e., a high G′ value ≥ 10 kPa; relatively low tan δ value) for hydrogel applications in gen­ eral Such properties can be useful in further micro-structured fabrica­ tion by 3D printing (Ma et al., 2021) Also, the presence of ions in the material (cellulose ionogel) can be important in electrochemical C Zhong et al Carbohydrate Polymers 301 (2023) 120302 Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Data availability Data will be made available on request Acknowledgement This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No ărg Weber 761030 (CARBAFIN) The authors acknowledge Prof Hansjo and Prof Brigitte Bitschnau from Graz University of Technology (TUG) for the 1H NMR and XRD support, respectively The authors also thank Prof Iain B.H Wilson and Dr Jorick Vanbeselaere from University of Natural Resources and Life Sciences (Vienna) for MALDI-TOF MS sup­ port Addition thanks to Prof Michaela Flock and Dr Angela Chemelli from TUG for rheology measurement support Fig Viscoelastic properties of enzymatically synthesized cellulose gels Loss factor (tan δ) of the reaction mixtures as reflected by varied enzyme loadings (0.5, 1.0, 2.0 U/mL, buffer activity) and IL concentrations (0–30 vol%): a) [Dmim]DMP; b) [Emim]OAc Symbols: square (□), diamond (⋄) and circle (○) presents the reaction with enzyme loading of 0.5, 1.0 and 2.0 U/mL, respec­ tively The loss factor was calculated using the values of G′ and G′′ at angular frequency 20 rad/s (middle range) Appendix A Supplementary data applications (Ge et al., 2021; Liu et al., 2020) Lastly, the hydrogels with tunable properties have significant application potential in medical treatment (Du et al., 2019; Shen et al., 2016) Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2022.120302 References Conclusions Abdul Khalil, H P S., Davoudpour, Y., Islam, M N., Mustapha, A., Sudesh, K., Dungani, R., & Jawaid, M (2014) Production and modification of nanofibrillated cellulose using various mechanical processes: A review Carbohydrate Polymers, 99, 649–665 Brandt, A., Hallett, J P., Leak, D J., Murphy, R J., & Welton, T (2010) The effect of the ionic liquid anion in the pretreatment of pine wood chips Green Chemistry, 12(4), 672–679 Brinchi, L., Cotana, F., Fortunati, E., & Kenny, J M (2013) 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Zhong, C (2020) Phosphorylase-catalyzed bottom-up synthesis of shortchain soluble cello-oligosaccharides and property-tunable cellulosic materials Biotechnology Advances, 51, Article 107633 10 ... applications Overall, the study expands the scope of phosphorylase-catalyzed synthesis of cellulose materials and advances the understanding of the role of IL co-solvent in cellulose selfassembly... of cel­ lulose solutions in IL upon the addition of water It was hypothesized that the added water breaks some of the original cellulose- IL interactions and thus promotes the restoration of cellulose- internal... indirect effect of the IL on the enzyme activity The rate of chain elongation in competition with the rate of chain aggregation delimits the average DP of cello-oligosaccharide present in the insoluble