Solvent-Free Enzyme Activity: Quick, High-Yielding Mechanoenzymatic Hydrolysis of Cellulose into Glucose

Article abstract of DOI:10.1002/anie.201711643

Mechanochemistry enables enzymatic cleavage of cellulose into glucose without bulk solvents, acids, other aggressive reagents, or substrate pre-treatment. This clean mechanoenzymatic process (coined RAging) is also directly applicable to biomass, avoids m

Full text of DOI:10.1002/anie.201711643

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Accepted Article
Title: Solvent-free enzyme activity: quick, high-yielding mechano-
enzymatic hydrolysis of cellulose into glucose
Authors: Fabien Hammerer, Leigh Loots, Jean-Louis Do, J. P. Daniel
Therien, Christopher W. Nickels, Tomislav Friscic, and Karine
Auclair
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711643
Angew. Chem. 10.1002/ange.201711643
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10.1002/anie.201711643
Angewandte Chemie International Edition
COMMUNICATION
Solvent-free enzyme activity: quick, high-yielding mechano-
enzymatic hydrolysis of cellulose into glucose
Fabien Hammerer, Leigh Loots, Jean-Louis Do, J. P. Daniel Therien, Christopher W. Nickels, Tomislav
Friščić* and Karine Auclair*
Abstract: Mechanochemistry enables enzymatic cleavage of
cellulose into glucose without bulk solvents, acids, other aggressive
reagents or substrate pre-treatment. This clean mechano-enzymatic
process is also directly applicable to biomass, avoids many
limitations associated with the use of cellulases, and produces
glucose concentrations >3 times the highest obtained by
conventional methods.
processing.[15] Enzyme catalysis using mechanochemistry was
recognized only recently, by using lipase B from Candida
antarctica for kinetic resolution of secondary alcohols, or papain
for amide synthesis.[16] By enabling cellulase-catalyzed
hydrolysis of cellulose or biomass, without pre-treatment, bulk
solvent, acids or transition metals, the herein presented
mechano-enzymatic approach is a breakthrough in enzyme
catalysis and cellulose saccharification. It demonstrates
enzymatic turnover for a highly recalcitrant substrate, and a
route to overcome a persistent challenge with cellulase reactions.
We used inexpensive cellulase preparations from
Trichoderma reesei, Trichoderma longibrachiatum, and
Aspergillus niger, commercially available as lyophilized powders.
Microcrystalline cellulose (MCC) was used as a model substrate,
as it is more resistant to enzymatic action and chemically well-
defined. Cellulases are hydrolytic enzymes; the water content in
herein described reactions is given as η,[17] the ratio of water
volume to the weight of reactants, cellulose and cellulases. For
η=0.5-1 µL/mg, corresponding to mechanochemical liquid-
assisted grinding (LAG) conditions that enable reactivity of
poorly soluble substances,[16] the reactions were solid mixtures
which rapidly turned into thick paste. Cellulose hydrolysis was
monitored with 3,5-dinitrosalicylic acid (DNS), which reacts with
the reducing end of sugars to afford 3-amino-5-nitrosalicylic
acid,[18] allowing the non-discriminate detection of glucose and
oligosaccharides. It was used along with thin layer
chromatography (TLC) to estimate glucose formation. Percent
conversion of MCC was calculated as described in the SI.
Unless otherwise noted, all experiments were done in triplicate.
We first explored the activity of cellulases towards MCC
without mechanical agitation. Aging of a static mixture of MCC,
A. niger cellulases (0.5% w/w) and water (η=0.5 µL/mg) at 45°C
and 100% relative humidity (RH) for a week[19] gave modest
conversion (Fig. S1), improved at higher cellulase loading (Fig
S2; enzyme loading is defined in the SI). Highest conversions
were observed for η=1 µL/mg, with water or aqueous acetate
buffer as the liquid phase (50 mM, pH 5.0). As previously seen
in mechanochemical reactions,[16] η>1 µL/mg was detrimental for
reactivity (Fig S3). Analysis of soluble products by TLC revealed
mostly glucose, indicating that all three types of cellulase
activities: endoglucanase, exoglucanase and β-glucosidase, are
at work (Fig. S4). These results demonstrate that enzymatic
digestion of cellulose into glucose is possible without bulk
solvent and that excess water can harm the process.
Cellulose is the most abundant organic polymer on Earth,
generated at a rate of 1012 tons per year[1] and treated mostly as
a waste product. It is the strongest contender for replacing
petroleum, e.g. in the production of bioethanol or commodity
chemicals.[2] A necessary step for bioethanol production is
saccharification: breakdown of cellulose into glucose, used as a
substrate in microbial fermentation. However, cellulose is a
poorly soluble, recalcitrant material, requiring aggressive, toxic
reagents, e.g. transition metal ions,[3] strong acids,[4] or
hazardous solvents.[5] While such reagents are environmentally
taxing and hazardous, they also inhibit yeast fermentation.[6]
Cellulolytic enzymes (cellulases) offer
a
promising
approach to cellulose breakdown, but their performance is
limited by low conversion rates, and product or substrate
inhibition.[7] Current enzymatic processes require chemical
pretreatment of cellulose, large volumes of suspensions and
related waste management.[8] Attempts to increase substrate
loading and reach higher glucose concentrations[9] are
challenged by energy needed to stir viscous slurries and the
“solids effect”: loss of cellulase activity at higher substrate
content.[10] The solids effect is explained by mass transfer
limitations, and enzyme inhibition by hemicellulose, reaction
products, or other compounds.[10] From an evolutionary point of
view, the solids effect is unexpected, as most cellulases are
excreted by fungi which thrive on the surface of moist
lignocellulosic materials, e.g. wood or straw.[11]
We now present a route for efficient and selective
enzymatic cleavage of cellulose into glucose under
mechanochemical conditions,[12] without bulk solvent. Ball milling
is known to aid in cellulose amorphization, assisting in
subsequent hydrolysis,[13] and mechanochemistry[14] has
previously been used for rapid depolymerization of cellulose,
using processes relying on continuous mechanical impact and
acid impregnation, yielding complex products needing further
Next, we investigated cellulase activity upon ball milling.
After 1 hour of milling in a stainless steel milling jar mounted on
a Retsch MM400 mill operating at 30 Hz, a mixture of MCC, A.
niger cellulases and water (η=0.9 µL/mg) gave a ~3-4%
conversion (Table S1). Using a Teflon milling jar improved
conversion, likely due sample adhering less to jar walls.[20]
[a]
Dr. F. Hammerer, Dr. L. Loots, J.-L. Do, C. W. Nickels, J. P. D.
Therien, Prof. T. Friščić, Prof. Dr. K. Auclair
Department of Chemistry, McGill University
Montreal, Quebec, H3A 0B8 (Canada)
E-mail: tomislav.friscic@mcgill.ca, karine.auclair@mcgill.ca
Supporting information for this article is given via a link at the end of
the document.
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Maximum conversion was reached at η≈1 µL/mg, with water or
buffer (Fig. S5).
Milling was found to favor the formation of glucose and
cellobiose, while subsequent aging produced mostly glucose
(Fig. S8), suggesting preferential β-glucosidase activity under
static conditions.
Table 1. Performance of different enzyme preparations during milling, or upon
aging after milling. Conditions: 200 mg cellulose, 10 mg enzyme preparation
(5% w/w), 200 µL deionized water, milled 30 min at 30 Hz in a 10 mL Teflon
jar with 2×7 mm stainless steel balls, before aging for 1 week at 45 °C, 100%
RH.
Next, we explored a process based on alternating periods
of brief milling followed by aging (reactive aging, RAging). Thus
20% conversion of MCC was obtained after 20 days, by daily
milling a mixture containing 5% w/w of A. niger enzyme
preparation for 5 min, followed by ~24 h aging (Fig. 2). Exploring
the kinetics of milling and of aging (Figs. 1, S9) enabled the
technique optimization to 1-hour cycles consisting of 5 min
milling followed by 55 min aging. This was first explored using
the T. reesei enzyme preparation over 12 cycles, giving 20%
conversion in only 12 hours (Table 2). Increasing the aging
temperature to 55°C or increasing enzyme loading gave
moderate or no improvement.
% Hydrolysis
Protein
content[a]
FPU/g
cellulose
milling and
aging [c]
Enzyme origin
milling[b]
A. niger
2
--[d]
16
5
3.2 ± 0.4
11 ± 1
11.7 ± 0.7
23.9 ± 0.6
18 ± 2
T. reesei
31
12
T. longibrachiatum
6.1 ± 0.6
[a] Measured using the Bradford assay, expressed in % mass. [b] After the
milling phase. [c] After the aging phase. [d] Below the detection limit.
Table 2. Selected RAging results: 200 mg MCC, enzyme (10 or 50 mg, 5 or
50% w/w, 16, 25 or 80 FPU/g cellulose) and deionized water. Results are
shown for 12 cycles of RAging. Milling in each cycle was done for 5 min at 30
Hz using a 10 mL Teflon jar with two stainless steel balls of 7 mm diameter.
Aging in each cycle was done for 55 min at 55 °C, unless otherwise noted.
Enzyme source
Enzyme
Loading
η (µL/mg)
Conversion (%)
T. reesei[a]
16[b] (5%)[c]
16 (5%)
0.5
0.5
0.4
0.4
0.8
20[d]
25±1
26[d]
T. reesei
T. reesei
80 (25%)
25 (25%)
25 (25%)
T. longibrachiatum
T. longibrachiatum
35[d]
49±2
[a] aging at 45oC; [b] expressed in FPU/g of MCC; [c] given in weight%;
[d] experiment not performed in duplicate.
Figure 1. Kinetics of milling reactions using 2 g MCC with 100 mg A niger
(red) or T. reesei (blue) cellulases and 2 mL deionized water (η=0.95 µL/mg).
Solid lines are reactions with milling and the broken line is a control reaction
without milling. Shown in green is a control experiment without enzyme. Milling
was done at 30 Hz in 20 mL stainless steel jars with 2×10 mm diameter
stainless steel balls.
Remarkably, T. longibrachiatum cellulases (25 FPU/g MCC or
25% w/w, η=0.8 µL/mg) gave 50% conversion in only 12 h
(Table 2). To the best of our knowledge, this is unprecedented
for untreated MCC at similar enzyme loadings. Increasing the
enzyme loading did not have a significant effect on the yield,
while decreasing it below 20 FPU/g MCC was detrimental (Fig.
S10). Importantly, reactivity did not depend on the jar being
closed or exposed to 100% RH while aging, suggesting that
water accumulation is not a critical factor. Powder X-ray
diffraction (PXRD, Fig. S11) on samples after RAging with T.
reesei enzymes revealed only broad Bragg reflections of
residual MCC, indicating that the glucose produced was not
crystalline and probably forms a highly concentrated aqueous
system. Under the best conditions identified so far, and taking
into account the water consumed in the reaction, the final
glucose concentration is ~580 g/L or 3.2 M. This is almost 4
times higher than the highest reported value for a cellulose
hydrolysis process (158 g/L or 0.9 M).[23] Analysis of the MCC
residue by atomic force microscopy (AFM, Fig. S12) revealed
nano-sized structures characteristic of cellulose nano-crystals
(CNCs), recently identified as a high value, renewable product
Investigating the conversion at milling times of up to 90
min revealed hyperbolic behavior, typical of many enzymatic
reactions, for both A. niger and T. reesei cellulases (Fig. 1). The
reduction of reaction rates upon increased milling time is not due
to enzyme denaturation, as subsequent aging for a week at
45°C led to a further 2- or 3-fold increase in conversion (Table 1)
even when milling was performed under harsher conditions, with
high-density tungsten carbide balls (Fig. S6).[21] Interestingly,
there was no obvious correlation between the in-solution activity
(in Filter Paper Units, FPU, defined in the SI) of the three
cellulase preparations and the reactivity observed under solvent-
free conditions (Table 1).
Consistent with the behavior of cellulases in solution,
increasing the enzyme loading beyond a certain point, for a
constant volume of water additive, did not improve the hydrolysis
yield (Fig. S7),
a
result usually attributed to enzyme
with
uses
overcrowding on cellulose surface.[22] Keeping a constant η=1
µL/mg however led to increased conversion even at high
enzyme loading (~46% after 30 min milling and 1 week aging).
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Figure 2. Extent of MCC hydrolysis upon different combinations of milling followed by aging (RAging). Milling was performed: (green) only once; (red) once a
week, , (blue) once a day; (black) every hour. Milling was done at 30 Hz for 5 min, using a stainless steel jar, 2×10 mm diameter stainless steel balls. Aging was
done at 45°C and 100% RH. Reaction mixtures (blue, red, green) were composed of 2 g MCC, 100 mg (5% by weight) of A. niger cellulase preparation and 2 mL
deionized water (η=1 µL/mg) or (black) 200 mg MCC, 50 mg (25% w/w) of T. longibrachiatum cellulase preparation and 200 µL deionized water (η=0.8 µL/mg).
ranging from liquid crystals to therapeutics.[24]
within 12 h. Avoiding the use of bulk solvent enables this
process to selectively yield glucose at concentrations >3 times
larger than those seen in conventional processes, while also
producing CNCs, a value-added form of cellulose. This is also a
significant improvement compared to enzymatic hydrolysis by
milling of cellulose suspensions.[25] While the calculated enzyme
productivity of our process (0.022 g of glucose/FPU) compares
well with that of others (Table S2), the benefits are even more
obvious when considering the space-time yield; at 20 g of
glucose/L/h, this process is at least 20-fold superior to other
reported enzymatic digestions of MCC. Importantly, the process
can be applied directly to untreated biomass. Other groups have
shown that cellulases can be recycled,[26] and our own
preliminary studies indicate that simple centrifugation of the
reaction mixture allows direct isolation of >30% of the glucose
produced, and further RAging of the pellet, without adding more
enzyme, leads to an additional 20% conversion of the MCC (Fig.
S14). By enabling a high, selective conversion of cellulose
directly to glucose, in a surprisingly short time for a cellulase-
catalyzed process, and without solvents, acids or transition
metals, this approach provides significant advantages over
known enzymatic or chemical processes for cellulose
breakdown. We are confident that this approach can be further
A preliminary study of scalability was done on a 5 g model
reaction using a planetary mill with 25 FPU of enzyme per gram
of MCC (25% w/w) and η=0.75 µL/mg (using water), 10 stainless
steel balls of various sizes, in a stainless steel jar (100 mL).
Without optimization, 43% of the MCC was transformed to
glucose over 36 cycles using the T. longibrachiatum preparation.
Finally, we evaluated reactivity of two locally available raw
biomass samples, hay and cedar wood saw dust, which were
either used untreated or cryo-milled before enzymatic cleavage
(Figs 3, S13). At equal enzyme loadings (1% solid loading w/v in
acetate buffer or water, solid loading is defined in the SI),
RAging gave higher yields compared to analogous aqueous
suspension. This also shows that RAging circumvents the need
for biomass pre-treatment, enabling the release of ~1.5 mmol of
glucose per gram of biomass without any optimization, a ~3-fold
increase compared to reactivity in aqueous suspension.
optimized, leading to
a
paradigm shift in enzymatic
transformation of biomass and other recalcitrant substrates.
Acknowledgements
This work was funded by NSERC DG, CREATE and I2I grants.
FRQNT Center in Green Chemistry & Catalysis is acknowledged
for support, CSACS McGill and M. Ramkaran for access to AFM,
and Dr. A. Tomberg for cedar wood samples.
Keywords: biocatalysis • cellulase • cellulose • glucose •
Figure 3. Enzymatic hydrolysis of native or cryo-milled cedar wood saw dust
by RAging (blue, η=0.8 μL/mg) or in 1% w/v (η=100 μL/mg) suspension in
aqueous buffer (red) or water (green). All reactions lasted 12 h at enzyme
loading of 25 FPU/g of MCC (25% w/w).
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hydrolysis of cellulose into glucose, by switching from
conventional solution media to mechanochemistry without bulk
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Entry for the Table of Contents (Please choose one layout)
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We report a solvent-free
Fabien Hammerer, Leigh Loots,
Jean-Louis Do, J. P. Daniel
Therien, Christopher W. Nickels,
Tomislav Friščić* and Karine
Auclair*
enzymatic approach combining
mechanochemistry and aging to
achieve efficient saccharification
of cellulose, reaching molar
glucose concentrations.
Page No. – Page No.
Solvent-free enzyme activity:
quick, high-yielding
mechanoenzymatic hydrolysis
of cellulose into glucose
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