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).

Available online 3 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/)

Effect of ionic liquid on the enzymatic synthesis of cello-oligosaccharides

and their assembly into cellulose materials

Chao Zhonga, Krisztina Zajki-Zechmeistera, Bernd Nidetzkya,b,*

aInstitute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, 8010 Graz, Austria

bAustrian Centre of Industrial Biotechnology (acib), 8010 Graz, Austria

A R T I C L E I N F O

Keywords:

Synthetic cellulose

Ionic liquid

Chain self-assembly

Hydrogel

Phosphorylase bio-catalysis

A B S T R A C T 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-methylimida1,3-dimethylimida-zolium 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

1 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,

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, &

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, Kok, Woortman, Ciric, & Loos, 2015; Pylkk¨anen et al., 2020; Serizawa,

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, &

assemble into different material structures in situ depending on the conditions used (Hata & Serizawa, 2021; Nidetzky & Zhong, 2020;

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,

2019) In addition, polymers (Hata et al., 2017) and colloidal particles

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)

Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2022.120302

Received 24 August 2022; Received in revised form 21 October 2022; Accepted 31 October 2022

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 (< 1 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,

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 cello-

oligosaccharides was investigated using ionic liquids (ILs) as small-

molecule 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;

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,

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 as-

synthesized 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 Material and methods

2.1 Materials

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 &

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 5 min (Centrifuge 5424/R, Eppendorf, Germany) until the su-pernatant was clear After removal of the susu-pernatant, the pelleted material was thoroughly resuspended in 1 mL of distilled water and then

centrifuged again at 21,130 ×g for 5 min (Centrifuge 5424/R,

Eppen-dorf) The washing step was repeated 3 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, 5 min)

to inactivate the enzyme and then centrifuged The conversion of αGlc1-

P was determined by the phosphate released into the supernatant Phosphate was measured by a colorimetric assay (Saheki, Takeda, &

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)

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 (N i ×M i)/∑i N i , where N i is the peak intensity of

the i-th cello-oligomer species and M i 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)

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

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)

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 strain-

controlled 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

3 Results and discussion

3.1 Enzymatic synthesis of cello-oligosaccharide chains in ILs

Our previous study of the bottom-up synthesis of cello-

oligosaccharides 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 (

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 cello-

oligosaccharides in the presence of [Dmim]DMP or [Emim]OAc

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

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 generally considered to be “enzyme-friendly” (Wahlstr¨om, Rovio, &

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 2 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

Fig 1 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, 1,3- dimethylimidazolium dimethyl phosphate; [Emim]OAc, 1-ethyl-3-methylimidazolium acetate

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

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 cello- oligosaccharides 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

Fig 2 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 3 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

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

3.2 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 cello-

oligosaccharides synthesized from the IL-containing reactions (10–30

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.,

oligosaccharides 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 1 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 co- solvent 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

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 celCel-lulose 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)

3.3 Gel-like properties of cellulose synthesized in the presence of IL

As mentioned above (see 3.1 Enzymatic synthesis of cello-

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

experi-ments were performed here to investigate the idea of a direct relation-ship between the gel formation and the IL-mediated supramolecular

Fig 4 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 Rere-actions 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

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 0 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

aforementioned reaction mixtures The G′ was nearly frequency-

independent, 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, &

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 &

%) 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

Fig 5 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

Table 1

Characterization of the solid cellulose products synthesized by CcCdP in the

presence of IL at varied concentrations

Solvents β value Conc

(vol%) DP_

1 H NMR DTOF MS P_MALDI- Allomorph Water/

buffer 0.18 – 7.83 7.25 Cellulose II

[Dmim]

DMP 1.0–1.1

II

II

[Emim]

II

II

aRef (Brandt, Hallett, Leak, Murphy, & Welton, 2010; Fukaya, Hayashi,

Wada, & Ohno, 2008)

b Ref (Zhang et al., 2012)

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′≤1 kPa) exhibited low αGlc1-P

conversion of 27–36% (Figs 2 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;

network structure The IL co-solvents appear to facilitate these processes

in particular and so generate a gel reinforcement effect Fig 8 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

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 ≥ 6 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 cello- oligosaccharides 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

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

Fig 6 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

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;

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

Fig 7 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 8 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 solid- in-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);

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)

4 Conclusions

Using purified preparation of the CcCdP, bottom-up enzymatic

syn-thesis of cellulose material composed of short cello-oligosaccharide

chains (DP 6 - 7) was possible in the presence of water-miscible,

cellu-lose-dissolving types of IL ([Dmim]DMP, [Emim]OAc) up to a co-solvent

concentration of 30 vol% Reversible and irreversible processes of

enzyme inactivation caused by the IL prohibited the use of co-solvent

concentrations of 40 vol% or higher The IL co-solvent affected the

cellulose synthesis in two distinct ways First, it slowed down the

iter-ative polymerization process catalyzed by the enzyme, presumably due

to a direct co-solvent effect on the CcCdP to decrease its activity An

indirect effect on the enzyme activity, resulting from a lowered

avail-ability of the growing oligomer chain to the enzyme due to cello-

oligosaccharide interaction with the IL ions in solution, might be an

additionally contributing factor The cellulose chains self-assembled

into highly ordered nanosheets of cellulose II crystal structure and

un-derwent further physical cross-linking aggregation/growth into polymer

networks that had gel-like characteristics Whereas the self-assembly

process appeared to be unaffected by the presence of IL in terms of

chain organization into crystalline material, the higher-order network

structure formation during gelation was decisively influenced by the IL,

probably by IL ions participating in the physical cross-linking The

viscoelastic properties of the resulting gels were tunable by the IL

con-tent used in the synthesis Enzymatic reaction in 10 vol% [Dmim]DMP

or [Emim]OAc gave a robust material that showed elasticity and

stiff-ness desirable in hydrogel 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 self-

assembly processes

CRediT authorship contribution statement

Chao Zhong: Conceptualization, Methodology, Formal analysis,

Investigation, Writing – original draft, Visualization Krisztina Zajki-

Zechmeister: Methodology, Investigation, Software Bernd Nidetzky:

Conceptualization, Writing – review & editing, Resources, Funding

acquisition

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

761030 (CARBAFIN) The authors acknowledge Prof Hansj¨org Weber 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

Appendix A Supplementary data

Supplementary data to this article can be found online at https://doi

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