Single-batch, homogeneous phase depolymerization of cellulose catalyzed by a monocomponent endocellulase in ionic liquid [BMIM][Cl]

Article abstract of DOI:10.1016/j.molcatb.2014.04.021

The stability and enzymatic activity of an industrial, recombinant cellulase dissolved either in 1-butyl-3-methylimidazolium chloride [BMIM][Cl] or in mixtures of [BMIM][Cl]/aqueous buffer have been investigated and the results are here reported. The preparation used was a recombinant monocomponent endocellulase from Trichoderma reesei or EGIII (now renamed Cel12A), commercially referred to as IndiAge Super GX Plus. The key parameters studied were: the effect of temperature in the range between 75 and 90 °C, the enzyme stability at 75 °C and the ability of EGIII to hydrolyze cellulose in the presence of [BMIM][Cl]. This cellulase preparation turns out to be more stable and active in pure ionic liquid rather than in buffer. These results indicate that the recombinant, monocomponent endocellulase from T. reesei is a suitable biocatalyst for the depolymerization of cellulose in [BMIM][Cl] and it therefore opens the way to a possible one-pot process based on a homogeneous phase, enzyme-catalyzed depolymerization of cellulose.

Full text of DOI:10.1016/j.molcatb.2014.04.021

Accepted Manuscript
Title: Single-batch, homogeneous phase depolymerization of
cellulose catalyzed by a monocomponent endocellulase in
ionic liquid [BMIM][Cl]
Author: Paola D’Arrigo Chiara Allegretti Stefano Tamborini
Cristina Formantici Yves Galante Loredano Pollegioni Andrea
Mele
PII:
S1381-1177(14)00143-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.04.021
MOLCAB 2947
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
13-2-2014
18-4-2014
29-4-2014
Please cite this article as: P. D’Arrigo, C. Allegretti, S. Tamborini, C.
Formantici, Y. Galante, L. Pollegioni, A. Mele, Single-batch, homogeneous phase
depolymerization of cellulose catalyzed by a monocomponent endocellulase in
ionic liquid [BMIM][Cl], Journal of Molecular Catalysis B: Enzymatic (2014),
http://dx.doi.org/10.1016/j.molcatb.2014.04.021
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Single-batch, homogeneous phase depolymerization of cellulose catalyzed
by a monocomponent endocellulase in ionic liquid [BMIM][Cl]
Paola D’Arrigo,*a,b,d Chiara Allegretti,a Stefano Tamborini,b,e Cristina Formantici,c Yves
Galante,d Loredano Pollegioni,b,e Andrea Melea,b,d
aDepartment of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano,
Piazza L. Da Vinci 32, 20133 Milano, Italy.
bThe Protein Factory, Politecnico di Milano, ICRM CNR Milano, and Università degli Studi
dell’Insubria, via Mancinelli 7, 20131 Milano, Italy.
cLaboratory of Biotechnology, Lamberti S.p.A., via Piave 18, 21041 Albizzate (VA) Italy.
dICRM, CNR, Via Mario Bianco, Milano, Italy.
eDepartment of Biotechnology and Life Sciences, Università degli Studi dell’Insubria, via J.H. Dunant
3, 21100, Varese (VA) Italy.
corresponding author: paola.d’arrigo@polimi.it, Tel: +39 02 23993075 Fax: +39 02 23993180.
Abstract
The stability and enzymatic activity of an industrial, recombinant cellulase dissolved either in 1-butyl-
3-methylimidazolium chloride [BMIM][Cl] or in mixtures of [BMIM][Cl]/aqueous buffer have been
investigated and the results are here reported. The preparation used was a recombinant
monocomponent endocellulase from Trichoderma reesei or EGIII (now renamed Cel12A),
commercially refered to as IndiAge® Super GX Plus. The key parameters studied were: the effect of
temperature in the range between 75 and 90 °C, the enzyme stability at 75 °C and the ability of EGIII
to hydrolyze cellulose in the presence of [BMIM][Cl]. This cellulase preparation turns out to be more
stable and active in pure ionic liquid rather than in buffer. These results indicate that the recombinant,
monocomponent endocellulase from T. reseei is a suitable biocatalyst for the depolymerization of
cellulose in [BMIM][Cl] and it therefore opens the way to a possible one-pot process based on a
homogeneous phase, enzyme-catalyzed depolymerization of cellulose.
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Keywords: cellulase, ionic liquid, depolymerization, cellulose.
1. Introduction
Lignocellulosic biomass is an abundant, renewable, low cost non-food feedstock for biofuel production
and a source of C atoms for Chemistry alternative to fossile resources [1-4]. The major factor
hampering massive exploitation of cellulose (which is the main component of plants biomass), is its
well known low reactivity and recalcitrance to chemical processing, which is due to the high
crystallinity of cellulose fibrilles. Traditional chemical processes for cellulose modification or
derivatization are based on dissolution of cellulose under harsh and environmentally unfriendly
conditions, such as hot sodium hydroxyde, or those for the production of viscose, still largely applied
worldwide.
A possible greener alternative for the activation of cellulose fibrilles towards an easier hydrolysis
and/or chemical derivatization could imply the use of ionic liquids (ILs) [5-7]. Ionic liquids were
initially proposed as neoteric solvents for sustainable chemistry due to their unique properties, such as
negligible vapour pressure (thus eliminating VOC release in the environment), non-flamability, high
dissolving power, the possibility of modulating physico-chemical properties (e.g.,
hydrophilicity/hydrophobicity) and, in some cases, relatively low toxicity [8-12]. Although these
factors do not represent per se sufficient conditions for intrinsically green processes [13], they are
considered advantageous starting points for the design of environmentally friendly chemical
transformations. Along with their benign characteristics as solvents, ILs provide the chemist with new
and promising media for synthesis and catalysis on both a laboratory and industrial scale [14-18].
The pioneering work of Rogers and coworkers [19] demonstrated the ability of 1-butyl-3-
methylimidazolium chloride ([BMIM][Cl]) to dissolve up to 25 wt % cellulose. These authors showed
that the IL acts as a pure non-derivatizing solvent able to disrupt the tightly entangled structure of
cellulose by replacing the intermolecular hydrogen bonds, holding together the polysaccharide chains,
with IL/polysaccharide interactions. NMR studies confirmed the key role played by the interactions of
carbohydrates OH groups with Cl– ions of the IL in the dissolution process [20-22]. These findings
opened the way to the pretreatment of cellulose with [BMIM][Cl] or other ILs, followed by the
conversion of regenerated cellulose into fermentable sugars, possibly via enzymatic reactions, or into
new products by chemical derivatization. Indeed, the dissolution of cellulose in ILs and the
precipitation of “regenerated cellulose” in aqueous media causes the conversion of type I crystalline
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cellulose into type II amorphous cellulose [23-24]. From the biocatalytic reaction point of view, the
practical consequence of the pretreatment is the generation of a substrate that appears to have a greater
accessibility to cellulases for subsequent enzymatic depolymerization. In the last few years, many
contributions have highlighted the dramatic enhancement of hydrolysis rate of pretreated cellulose [25-
30] by cellulase, thus suggesting that a three-step process – cellulose dissolution, regeneration and
cellulase depolymerization – might be eligible for scale-up and potential industrial applications.
An obvious improvement of this strategy, in terms of time saving, energy optimization and
sustainability, would be the design of a single-batch process in IL, where the dissolution of cellulose
and the enzymatic degradation of the polysaccharidic chains take place in the same medium and
reactor. Such an approach would avoid the pretreatment - regeneration steps and the need to remove IL
traces before performing the enzymatic hydrolysis in an aqueous buffer. From the process point of
view, the one-batch approach would reduce the number of unitary operations, thus leading to saving of
time and energy in comparison to multi-batch processes. In principle, the reaction of controlled
depolymerization and eventual further chemical modification could be carried out in the same reactor,
with considerable advantages in an industrial setting. However, previous studies on biocatalytic
degradation of cellulose in [BMIM][Cl] by cellulases from Trichoderma reesei showed discouraging
results [31]: this work reported a complete loss of enzyme activity due to protein denaturation. Further
investigations confirmed the unfriendly character of [BMIM][Cl] towards “traditional” cellulases, as
loss of enzymatic activity was reported [32-33], although a significant retention of enzyme activity
was observed for methylimidazolium chloride [MIM][Cl] and other ILs with different anions. It is only
very recently that a commercial cellulase (Celluclast®) supported onto a polymeric support (Amberlite
XAD4) and “protected” by hydrophobic ILs coatings (e.g., 1-butyl-3-methylimidazolium-
bis(trifluoromethylsulfonyl)amide, [BMIM][NTf2]), showed efficient catalytic activity under
heterogeneous conditions in the saccharification of cellulose dissolved in [BMIM][Cl] [34].
Comparative evaluation of the cradle-to-gate Life Cycle Assessment (LCA) of [BMIM][Cl] versus the
established environmentally friendly N-methyl-morpholine-N-oxide (NMMO)/H2O in the industrial
dissolution of cellulose for the production of lyocell (which is a viscose-like, man-made fiber),
revealed that the IL has the potential for an environmental impact similar to the NMMO/H2O system,
thus renewing the interest in [BMIM][Cl] as a valuable and sustainable medium for industrial cellulose
processing [35].
In the present work, we describe a single-batch, homogeneous phase enzymatic depolymerization of
cellulose catalyzed by a commercial cellulase (EC 3.2.1.4) in the presence of varying volume ratios of
[BMIM][Cl] to buffer. There are several commercial preparations of cellulases available, which can be
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multi- or mono-component, native or recombinant, from T. reesei or Humicola insolens or other
microorganisms: most of them have been developed for textile and/or pulp & paper applications [36].
We used EGIII (Cel12A), a recombinant mono-component endocellulase from T. reesei (see Materials
for details), that showed good stability and activity towards carboxymethyl cellulose (CMC)
depolymerization in hydroalcoholic media [36, 37]. The IL was used both for cellulose dissolution and
cellulase-catalyzed depolymerization, without any pretreatment of cellulose. Our results indicate that a
higher specific activity and residual activity after 24 h of incubation is observed in IL than in buffer
and thus that a single-batch enzyme-catalyzed depolymerization of cellulose in pure, buffer-free
[BMIM][Cl] is actually feasible.
2. Experimental
2.1 Materials
Microcrystalline cellulose from cotton linters was obtained from Lamberti S.p.A. (Albizzate, Italy).
Carboxymethylcellulose sodium salt (CMC) was purchased from Fluka (Fluka 21900). Bicinchoninic
acid disodium salt hydrate (BCA) was purchased from Sigma-Aldrich (D8284).
The ionic liquid [BMIM][Cl] was purchased from Fluka and used without further purification. The
water content in the ionic liquid was less than 1% and this should account for the enzymatic hydrolysis
of cellulose that occurs in this medium (see below). The industrial enzyme preparation of mono-
component endoglucanase EGIII (Cel12A), in a concentrated granular form, from T. reesei used in this
study was IndiAge® Super GX Plus, referred as Super GX Plus, from Genencor Intl (now a subsidiary
of Dupont). The main characteristics of this enzyme are: 930 U/g, 248 mg protein/g commercial
cellulase product. The enzymatic activity was measured by the CMC method previously reported [36].
The enzyme shows an optimum activity at acidic pH, thus 50 mM sodium acetate buffer pH 4.8 was
used in all experiments.
All other reagents were of analytical grade and used without further purification.
2.2 Determination of reducing-ends by the BCA method
Quantification of the enzymatic depolymerisation of cellulose was based on the estimation of reducing
sugars produced during hydrolysis by the BCA method ([38] and references therein). Two stock
solutions were prepared and stored separately for one month at 4 °C. Solution A: 0.971 g of disodium
2,2-bicinchoninate, 27.14 g of Na2CO3 and 12.1 g of NaHCO3 were dissolved in 500 mL of distilled
water. Solution B: 0.624 g of CuSO4.5H2O and 0.631 g of L-serine were dissolved in 500 mL of
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distilled water. BCA working solution was freshly prepared before use by mixing equal volumes of
solution A and solution B.
In details: 1 mL of BCA working solution was added to 1 mL of sample in a vial and incubated at 70
°C for 30 min. The vials were cooled at room temperature, 1 mL of mixture was transferred to a
cuvette and the absorbance was determined at 560 nm.
A calibration curve was established by using a glucose solution as standard in the 0 to 82 µM
concentration range. In all cases, the contribution of the added protein to color development was
measured and subtracted from the final value.
2.3 Measurements of cellulase activity
The enzyme activity was determined on a standard solution of 1% (w/v) CMC in distilled water [36].
500 μL of CMC solution were incubated in a 2 mL vial at 40 °C for 10 min. Then, 500 μL of cellulase,
previously diluted in buffer, were added; the mixture was vortexed and the vial incubated for 10 min at
40 °C. The reaction was stopped by adding 1 mL of the BCA working solution (the inhibition of the
enzymatic reaction is due to the high alkaline pH) and the determination of the reducing ends starts
simultaneously. The vial was vortexed again and incubated at 70 °C for 30 min. After cooling at room
temperature, 1 mL of mixture was transferred to a 3 mL cuvette. The absorbance at 560 nm was
determined versus a blank composed of 500 μL of buffer, 500 μL of 1% CMC solution or 500 μL of
cellulase and 1 mL of BCA working solution. Product concentration was determined based on a
calibration curve established with glucose as a standard.
One enzymatic unit (U) is defined as the amount of enzyme which liberates 1 μmol of glucose
equivalent from CMC in one min at 40 °C.
2.4 Measurements of cellulase stability in [BMIM][Cl] versus temperature and time of
incubation
For the evaluation of temperature stability of the enzyme preparation, 5 mg of cellulase Super GX Plus
was incubated in 250 µL of [BMIM][Cl] (or buffer for the control reaction) for 1 h under magnetic
stirring at different temperatures ranging from 75 to 90 °C. A 10 µL aliquot was withdrawn and diluted
to 1 mL with buffer: 500 µL of this solution were immediately used to assay the residual enzymatic
activity (see above).
The time stability of cellulase at a fixed temperature of 75 °C was assessed as follows: 5 mg of
cellulase were incubated in 250 µL of [BMIM][Cl] (or buffer for control reaction) under magnetic
stirring at 75 °C for up to 24 h. An aliquot (10 µL) was withdrawn and diluted to 1 mL with buffer;
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500 µL of this solution were used for cellulase activity determination on CMC (see above).
2.5 Enzymatic hydrolysis of cellulose
In a 1.5 mL screw-capped vial, 2.5 mg of cellulose were suspended in 1 mL of buffer/IL and allowed
to equilibrate at 75 °C. In different experiments, the volume ratio of IL to buffer was varied but
cellulose concentration (2.5 mg/mL) was kept constant. The reaction was started by adding 2.5 mg
equivalent to 2.33 U (or 25 mg equivalent to 23.3 U) of cellulase preparation and the mixture was
vigorously mixed by magnetic stirring. Samples (10 µL) were periodically withdrawn and reducing-
ends determined by the BCA method. Controls were prepared similarly but without cellulose. Assays
were performed at least three times each and mean values were calculated.
2.6 Fluorescence measurements
Protein fluorescence measurements were performed in a Jasco FP-750 instrument using a thermostated
cell holder at 25 °C. In order to measure fluorescence at this standard temperature on a liquid sample,
1% water was added to the pure ionic liquid. In details: 10 mg of enzyme sample were fully dissolved
by stirring for 2 hr at 75 °C in 1 mL of solvent (IL containing 1% of water or buffer), 20 μL of this
solution were diluted with the same solvent to a final volume of 800 μL (0.25 mg protein / mL).
Protein emission spectra were recorded from 300 to 400 nm with excitation at 280 nm (excitation slit
of 5 nm, emission slit of 10 nm) [39]. The emission of a blank control without enzyme was subtracted
from all measurements in order to correct for the contribution of the imidazolium ring to the overall
enzyme fluorescence spectra.
3. Results and Discussion
3.1 Effect of temperature
The effect of temperature on cellulase activity of Super GX Plus in [BMIM][Cl] was investigated and
compared to the value obtained in buffer as a control. Super GX Plus, being in granular form, was
assayed after 2 h at 75 °C in IL because of its slower dissolution at that temperature after 60 min
incubation.
Figure 1
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As can be observed from Figure 1A, the specific activity in buffer of the cellulase used in this work
shows a significant decrease after 1 h incubation at temperatures ≥ 75 °C. Conversely, the activity of
the enzyme in [BMIM][Cl] increased up to 85 °C reaching a value 2-3 times higher than the
corresponding activity in buffer at 40 °C (Figure 1B). Thus, the ionic liquid appears to have the dual
effect of increasing the enzymatic activity and protecting the enzyme against thermal denaturation. As
a consequence, higher reaction temperatures than those used in an aqueous medium can be used for the
biocatalytic depolymerization of cellulose in IL. It is interesting to notice that at 90 °C Super GX Plus
in IL exhibits a marked decrease of stability (Fig. 1B), thus suggesting the 75-85 °C temperature range
as optimum for dissolution-hydrolysis of cellulose. The experimental values determined at 75 °C
deserve a specific comment. At that temperature, the enzyme in a patented granular form
(commercially refered to as Enzoguard®) dissolves more slowly in IL than in buffer, thus it expresses
less activity after the same time span. Super GX Plus needs about 2 h for full dissolution in
[BMIM][Cl] at 75 °C, while it dissolves instantly in buffer. Complete dissolution of the granules was
established by visual assessment and leveling off of cellulase activity. Also, the observed effect of
[BMIM][Cl] on Super GX Plus cellulase activity at high temperature (Figure 1) is probably related to
its ability to fully dissolve the enzyme preparation.
Protein fluorescence spectra of the cellulase after 1 or 2 h of incubation in buffer vs. [BMIM][Cl] was
compared (the reported fluorescence spectra have been corrected for contributions due to buffer or
ionic liquid): a dramatic quenching of fluorescence signal was revealed in IL, as shown in Figure 2.
Figure 2
This change is due to the significant alteration of the microenvironment surrounding the tryptophan
residues and, therefore, cannot be related to protein unfolding phenomena, since the enzyme is still
active in the ionic liquid. This result differs from what was observed by Turner et al. [31]: in that case
the change in protein fluorescence was directly proportional to the enzymatic activity loss. This
discrepancy is probably due to the different origin and preparation of the T. reesei cellulases used by
the two laboratories, i.e. a native enzyme in a lyophilized-KH2PO4 complexed physical form from
Sigma vs. a recombinant protein in a concentrated granules form from Danisco-Genencor.
The results obtained in the present work in [BMIM][Cl] and those reported in the literature [31-33]
indicate that the design of biocatalytic processes in ILs should carefully consider the type of cellulase
used, as a considerable variability can be observed in enzyme stability/activity, presumably related to
the microbial source of origin and/or to the introduction of genetic engineering manipulations.
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3.2 Enzyme stability at 75 °C in [BMIM][Cl]
The results of the previous section prompted us to investigate the stability time-course of the enzyme
in pure [BMIM][Cl] at 75 °C. This temperature was chosen because: i) it is the lowest at which
[BMIM][Cl] is in liquid state; ii) it efficiently solubilizes cellulose; iii) it seems to mantain the enzyme
active over extended periods of time. Results are shown in Figure 3.
Figure 3
The enzyme preparation showed a time-dependent inactivation when incubated in buffer while no
change in activity was apparent up to 6 h in ionic liquid (Figure 3). A significant decrease in activity
after 24 h in IL was observed (10% of the activity measured at 2 h, not shown). In any case, it should
be pointed out that the enzyme preparation showed higher residual activity at 75 °C in [BMIM][Cl]
compared to buffer even after 24 h of incubation (∼350 U/g in ionic liquid vs. 8-12 U/g in buffer
solution), further demonstrating that this highly viscous IL protects/preserves the protein activity
against inactivation more effectively than an aqueous medium. The stabilization of the protein in
[BMIM][Cl] at high temperature represents the prerequisite for the one-pot solubilization and
enzymatic cellulose hydrolysis process.
3.3 Effect of solvent on enzymatic cellulose hydrolysis
The effect of solvent composition (IL/water ratio) on the outcome of the enzymatic depolymerization
of cellulose was tested. Firstly, blank experiments without enzyme were carried out and demonstrated
that IL or mixture of IL/buffer did not cause cellulose hydrolysis in the absence of enzyme. To perform
enzymatic hydrolysis, cellulose was first dissolved in IL/buffer at 75 °C, followed by cellulase
addition. After 24 h, the reaction mixture was evaluated using the BCA assay for reducing-ends
determination. The data, reported after substraction of blank experiments without cellulose, are shown
in Figure 4, where the conversion of cellulose is expressed as reducing ends glucose equivalent
concentration (µM) per enzyme unit as a function of solvent composition. Setting 100% conversion for
Super GX Plus in buffer the enzyme is surprisingly active in pure IL showing 41.5% of its activity in
buffer. To the best of our knowledge, such experimental evidence of high enzymatic hydrolysis in pure
[BMIM][Cl] has not been previously reported. Intringuisly, the enzyme does not show any enzymatic
activity at IL/buffer 50/50 ratio.
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Figure 4
3.4 Effect of cellulose / enzyme ratio (w/w) on enzymatic hydrolysis
The work described in the previous sections was carried out by using a 1/10 (w/w)
cellulose/commercial enzyme preparation (S / E) ratio. In order to elucidate the influence of the
amount of enzyme on the reaction kinetics, the reducing sugars released during enzymatic hydrolysis
of cellulose at a different S / E ratio, namely 1:1 (w/w), were also evaluated. Along with the
predictable increase of conversion in the case of S / E = 1/10 (Figure 4) vs 1/1 (Figure 5), the data of
Figure 5 clearly indicate that the enzyme from T. reesei is very sensitive to the S / E ratio. At the S / E
= 1:1 ratio (Figure 5), the conversion yield is not significantly affected by the reaction medium, while
when an excess of cellulase is used (Figure 4), the highest conversion yields are observed in pure
solutions of IL or buffer. Indeed, the time course of cellulose hydrolysis in [BMIM][Cl] vs. buffer (not
shown) indicates that the cellulase conserves enzymatic activity during the entire course of the reaction
(24 h) at 75 °C.
Figure 5
4. Conclusions
This work demonstrates that [BMIM][Cl] might represent a suitable medium for the enzymatic
depolymerization of cellulose in a one-batch process. It suggests that a preliminary careful choice of
cellulase preparation and reaction conditions is required. Indeed, the recombinant, monocomponent
endocellulase EGIII (Cel12A) from Trichoderma reesei tested in this work (commercially known as
IndiAge Super GX Plus) was not denatured by the ionic liquid. Interestingly, this enzyme showed
exceedingly good performance for the depolymerization of dissolved cellulose in [BMIM][Cl] without
the usual pretreatment, known as dissolution-regeneration of cellulose in its amorphous form. Super
GX Plus is fully dissolved in [BMIM][Cl] and retains a great portion of its activity in this ionic liquid
for ≥ 6 h. This same mono-component endocellulase by recombinant DNA manipulation from the
natural cellulase complex of T. reesei was previously shown to be the most stable and active in
alcoholic reaction medium containing high percentage of 2-propanol or ethanol [36].
In conclusion, the approach outlined in the present work could be amenable of scaling-up and
innovative industrial applications for the efficient one-batch conversion of inexpensive cellulosic
materials into derivatives (biofuels, derivatized cellulose, monosaccharides for fine chemicals, etc.)
with high potential commercial interest and in the framework of environmentally “green”chemistry.
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The more friendly ILs based on 1-ethyl-3-methylimidazolium acetate diethylphosphate were also
studied and the manuscript is currently in preparation.
Acknowledgments:
The authors gratefully acknowledge PRIN 2010-2011 NANO Molecular Technologies for Drug
delivery - NANOMED prot. 2010 FPTBSH.
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[34] P. Lozano, B. Bernal, J.M. Bernal, M. Pucheault, M. Vaultier, Green Chem. 13 (2011) 1406.
[35] S. Righi, A. Morfino, P. Galletti, C. Samori, A. Tugnoli, C. Stramigioli, Green Chem. 13 (2011)
367.
[36] S. Cheroni, C. Formantici, Y.M. Galante, Enz. Microb. Technol. 47 (2010) 348.
[37] U. Arunachalam, JrJt Kellis, Biochemistry 35 (1996) 35, 11379-85.
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[39] L. Pollegioni, S. Iametti, D. Fessas, L. Caldinelli, L. Piubelli, A. Barbiroli, M.S. Pilone, F.
Bonomi, Protein Science 12 (2003) 1018.
11
Page 11 of 20

Figure captions
Figure 1: Effect of temperature on cellulase residual activity in buffer (Panel A) and in [BMIM][Cl]
(Panel B). The cellulase activities were measured after incubation of 5 mg of enzyme preparation in
250 μL of [BMIM][Cl] (or 50 mM sodium acetate buffer pH 4.8 for control reaction) for 1 h at
different temperatures. A 10 μL aliquot of this solution was withdrawn, diluted with suitable buffer
and used for cellulase activity assay on CMC as indicated in Section 2.3. For Super GX Plus in
[BMIM][Cl] at 75 °C the reported data have been recorded after 2 h of incubation because the
commercial preparation takes this long time to be fully dissolved in IL (see text). Error bars are smaller
than the symbols used (standard deviation is < 5%).
Figure 2: Fluorescence spectra of Super GX Plus in buffer (50 mM sodium acetate pH 4.8, continuous
line) and in [BMIM][Cl] (dotted line), following subtraction of the pure solvent contribution.
Conditions: 0.25 mg protein/mL, 25 °C. The IL contains 1% of water to allow solubilisation at the
assay temperature.
Figure 3: Time course of cellulase stability at 75 °C in 50 mM sodium acetate pH 4.8 buffer (Panel A)
and in [BMIM][Cl] (Panel B) for Super GX Plus (■). The reactions were performed as indicated in
Section 2. The standard deviation (<5%) is such a small value that the error bars are smaller than the
symbols used.
Figure 4: Effect of the concentration of ionic liquid -expressed as volume ratios of IL/buffer (50 mM
sodium acetate pH 4.8)- for Super GX Plus on the conversion of cellulose after 24 h reaction. The
conversion yield after 24 h reaction at 75 °C is expressed as micromoles reducing end glucose
equivalent/ mg of starting cellulose (100% of conversion is the equivalent of 0.667 micromoles of
reducing end glucose equivalent/enzyme unit in buffer). The reactions were performed at 75 °C using
2.5 mg of cellulose, 25 mg of enzyme preparation and 1 mL of solvent as indicated in Section 2.5. The
standard deviation was <5%.
Figure 5: Effect of cellulose/enzyme ratio (w/w) on cellulose saccharification versus solvent
composition expressed as volume ratios of IL/buffer (50 mM sodium acetate pH 4.8) for Super GX
Plus. The conversion yield after 24 h reaction at 75 °C is expressed as micromoles reducing end
glucose equivalent/ mg of starting cellulose. The reactions were performed using 2.5 mg of cellulose,
12
Page 12 of 20

2.5 mg of cellulase (ratio (w/w) 1:1 substrate / enzyme preparation) and 1 mL of solvent (IL/water) as
indicated in Section 2. The standard deviation was <5%.
13
Page 13 of 20

Graphical abstract
Cellulose dissolution and enzyme catalyzed depolymerization is achieved one-pot and in homogeneous
liquid phase by using an industrial preparation of cellulase from T. resei in ionic liquid 1-butyl-3-
methylimidazolium chloride [BMIM][Cl].
14
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Highlights
The enzymatic depolimerization of cellulose has been performed in [BMIM][Cl].
The recombinant monocomponent endocellulase from Trichoderma reesei is active in [BMIM][Cl].
Cel12A is not denaturated by the ionic liquid [BMIM][Cl].
Page 15 of 20

Figure 1
Figure 1
Panel A
50
45
40
35
30
25
20
15
10
5
0
75
80
85
90
Panel B
2500
2000
1500
1000
500
0
75
80
85
Temperature ( °C)
90
Page 16 of 20

Figure 2
Page 17 of 20

Figure 3
Figure 3
Panel A
35
30
25
20
15
10
5
0
0
2
4
6
time (h)
Panel B
2500
2000
1500
1000
500
0
0
1
2
3
4
5
6
time (h)
Page 18 of 20

Figure 4
Figure 4
4
3,5
3
2,5
2
1,5
1
0,5
0
100:0
99:1
95:5
90:10
50:50
0:100
IL/water (v:v)
Page 19 of 20

Figure 5
Figure 5
0,45
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
100:0
99:1
95:5
90:10
50:50
0:100
IL/water (v:v)
Page 20 of 20