Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose
Surface
Kiyohiko Igarashi, et al.
Science 333, 1279 (2011);
DOI: 10.1126/science.1208386

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REPORTS
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Traffic Jams Reduce Hydrolytic
Efficiency of Cellulase on
Cellulose Surface
Kiyohiko Igarashi,1*† Takayuki Uchihashi,2,3,4* Anu Koivula,5 Masahisa Wada,1,6 Satoshi Kimura,1,6
Tetsuaki Okamoto,1,2 Merja Penttilä,5 Toshio Ando,2,3,4 Masahiro Samejima1
A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhancethe efficiency of biofuels development. We report here the real-time visualization of crystalline
cellulose degradation by individual cellulase enzymes through use of an advanced version ofhigh-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (Tr Cel7A) molecules
were observed to slide unidirectionally along the crystalline cellulose surface but at one point
exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic formof cellulose by means of an ammonia treatment increased the apparent number of accessible laneson the crystalline surface and consequently the number of moving cellulase molecules. Treatmentof this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase
II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on thesurface. Cellulose was completely degraded by the synergistic action between the two enzymes.
iorefining encompasses production offuels, power, heat, and value-added chemicals by appropriate conversion of lignocellulosic feedstocks. This prospect offers many
advantages, including diminished carbon dioxide
emission, productive use of renewable nonfood
crops or inedible waste products, and reductionof petroleum use. One of the bottlenecks to wide-B

1
Department of Biomaterial Sciences, Graduate School of
Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku,
Tokyo 113-8657, Japan. 2Department of Physics, Kanazawa
University, Kanazawa 920-1192, Japan. 3Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa
University, Kakuma-machi, Kanazawa 920-1192, Japan. 4Core
Research for Evolutional Science and Technology, Japan Scienceand Technology Agency, Sanbon-cho, Chiyoda-ku, Tokyo 1020075, Japan 5VTT Technical Research Centre of Finland, Post
Office Box 1000, FI-02044 VTT, Finland. 6Department of Plant
and Environmental New Resources, College of Life Sciences,
Kyung Hee University, 1, Seocheon-dong, Giheung-ku,
Yongin-si, Gyeonggi-do 446-701, Republic of Korea.

*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:aquarius@mail.ecc.u-tokyo.ac.jp (K.I.)

spread biorefining application is the enzymatichydrolysis of the cellulosic raw material into sugars. In attempts to maximize efficiency and reduce
costs, many studies on both individual cellulases
and enzyme cocktails have been carried out (1).
Various pretreatment methods have also been examined to increase the amount of sugar generated
from cellulosic biomass with reduced enzyme
loading and energy consumption, aiming at thedevelopment of commercially viable bioprocesses.
It is generally recognized that one of the problems
in cellulose hydrolysis is the slowdown of enzyme action with time and conversion (2, 3). Cellulose is a major component of plant cell walls,
accounting for almost half of their net weight.
Cell wall cellulose typically has ~70% crystallinity, suggesting that approximately one-thirdof net cellulosic biomass consists of natural crystalline cellulose, which is generally called cellulose I (4). Because cellulose chains have stableb-1,4-glucosidic bonds and each chain is alsostabilized by intra- and intermolecular hydrogenbonds, cellulose I is quite resistant not only towww.sciencemag.org

SCIENCEVOL 333

Acknowledgments: We thank T. Beatus, Y.-C. Lin, J. Brady,
L. Ristroph, and N. Wagner for useful discussions.
This research was supported by grants from NSF Civil,
Mechanical, and Manufacturing Innovation, Divisionof Materials Research (DMR), and DMR Materials
Research Science and Engineering Centers, and in part byaward KUS-C1-018-02 from King Abdullah University of
Science and Technology (KAUST). J.N.I. was supported by the
U.S. Department of Energy, Division of Materials Sciencesand Engineering under award DE-FG02-87ER-45331.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/333/6047/1276/DC1
Materials and Methods
SOM Text
Figs. S1 to S7
Movies S1 to S6
References (29–40)
15 April 2011; accepted 15 July 201110.1126/science.1207032

chemical hydrolysis but also to enzymatic degradation (5).
The industrially important cellulolytic ascomycete fungus Trichoderma reesei (anamorph of
Hypocrea jecorina) secretes two extracellularcellobiohydrolases (CBHs), which are cellulasesthat can hydrolyze glycosidic linkages particularly at a crystalline surface, and form cellobiose(b-1,4-glucosidic dimer) as a major product from
cellulose I (6). The two T. reesei CBHs hydrolyze
crystalline cellulose from the reducing and nonreducing ends, respectively (7). These enzymes
have a similar two-domain organization: The
cellulose-binding domain (CBD), which is categorized into carbohydrate-binding module (CBM)
family 1, contributes to adsorption on the insoluble
substrate, and the cellulose-hydrolyzing catalytic
domain (CD) catalyzes cleavage of glycosidic
bonds. The two T. reesei cellobiohydrolase CDs
have different types of folds. The CD of CBH I
belongs to the glycoside hydrolase (GH) family
7, and the CD of CBH II belongs to the GH
family 6, as listed on the Carbohydrate-Active
enZyme (CAZy) server (8), and thus, the two
enzymes are called TrCel7A and TrCel6A, respectively. In both cellobiohydrolases, the catalytic amino acids are located in a relatively longtunnel formed by surface loops extending fromthe central fold of the CD (9, 10). We previously
visualized the linear movement of wild-type andisolated CD on cellulose Ia using high-speed
atomic force microscopy (HS-AFM); the datasuggested that the sliding movement of wildtype TrCel7A reflects the processive degradationof the cellulose chain by catalysis at the CD andrequires initial recognition of the cellulose chain.
The chain recognition involves the tryptophan
residue W40 at the entrance of the TrCel7A active site tunnel (11).
In the present study, we succeeded in enhancing the temporal and spatial resolutions byusing a laboratory-built HS-AFM with extensive
improvements over the version reported previously (12), and we were able to visualize themovement of TrCel7A molecules on crystalline
cellulose in detail. Enzyme molecules sliding

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6. J. Vermant, M. J. Solomon, J. Phys. Condens. Matter
17, R187 (2005).
7. J. M. Brader, J. Phys. Condens. Matter 22, 363101(2010).
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961 (2004).
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Phys. Rev. Lett. 95, 268302 (2005).
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As already mentioned, cellulose I is quite resistant to enzymatic hydrolysis. We have shown
recently that crystalline polymorphic conversion
from cellulose I to cellulose IIII through supercritical ammonia treatment dramatically enhances
the hydrolysis of crystalline cellulose by TrCel7A(14). In order to investigate the reason for the enhanced hydrolysis of cellulose IIII, the degradationof the crystalline celluloses was visualized with HSAFM, and the results were compared with thoseof the conventional adsorption study. The binding isotherms—plots of free enzyme concentration
versus amount of adsorbed enzyme ([F ]-A)—
followed a Langmuir-type two-binding site model
on both crystalline celluloses (cellulose Ia andIIII) (Fig. 2A). These data show that TrCel7A has
higher binding capacity to cellulose IIII than to cellulose Ia. The simulated curves were divided into
high-affinity (solid line) and low-affinity (dashedline) components, as shown in Fig. 2B. There wasFig. 1. (A) Real-time observation of crystalline cellulose Ia incubated with TrCel7A by means of HS-AFM.
The time interval between images is 0.9 s. These images are taken from movie S1. (B) Time course of
distance from the initial position for TrCel7A molecules. The mobility of each molecule was analyzed byusing a routine developed in Igor Pro software, as described in the supporting online material. The
different symbols indicate different molecules observed in HS-AFM images. The black dotted line
represents the average velocity of TrCel7A (3.5 nm/s) estimated from previous HS-AFM observation (11).
(C) Velocity distribution of the linear movement of TrCel7A on crystalline cellulose Ia (n = 188 xxxxx). The
histogram was approximated by the combination of two Gaussian distribution curves (blue line) withmean T SD values of – 0.32 T 3.4 (red) and 7.1 T 3.9 (green).

Fig. 2. (A) Free TrCel7A concentration dependence of the amount of adsorbed enzyme on crystalline
cellulose Ia (red) and IIII (blue). These plots were fitted to curves (red and blue solid lines) simulated by useof Langmuir’s two-binding-site model, as described in (26); the high- (solid line) and low-affinity (dashedline) binding curves are individually drawn in (B). The binding parameters (A1, Kad1, A2, and Kad2) of
TrCel7A for cellulose Ia were 0.22 T 0.02, 8.4 T 1.4, 2.0 T 0.2, and 0.44 T 0.09, and those for cellulose IIII
were 1.9 T 0.1, 11 T 2, 1.8 T 0.3, and 0.31 T 0.06 (mean T SD), respectively. (C) HS-AFM images ofcrystalline cellulose IIII degradation by TrCel7A. The time interval between images is 30 s. These images
are taken from movie S2. (D) Schematic presentation of the increased number of observed lanes for
TrCel7A on cellulose Ia (top left) compared with cellulose IIII (top right) and comparison of sections ofcrystalline cellulose Ia (bottom left) and cellulose IIII (bottom right) according to previous reports (17, 18).

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unidirectionally on the cellulose Ia surface areshown in Fig. 1A and movie S1. This movement of Tr Cel7A was observed only at the topof the cellulose crystal, where the individual enzyme molecules were moving as one line. The
movement of the Tr Cel7A molecules was analyzed by using a custom software to track thelinear movement of the center of each molecule,and the time course of molecular movement isshown in Fig. 1B. Some molecules slid continuously without stopping (demonstrated by theorange plot in Fig. 1B), whereas the movementof other TrCel7A molecules was often disturbed
by halted and/or more slowly moving molecules(demonstrated by the open green and closed red
plots in Fig. 1B), resembling the movement oftraffic on a road. According to the histogram ofthe measured velocities (Fig. 1C), there would
seem to be two populations of TrCel7A molecules having average velocities of –0.32 T 3.4
and 7.1 T 3.9 nm/s. This is consistent with theidea that TrCel7A molecules intermittently repeatstop and go movements.
It is generally believed that CBHs have twomodes of adsorption on a cellulose surface:
the productive adsorption mode, in which both
the CD and CBD contribute to the binding, andthe nonproductive adsorption mode, in which
the enzyme molecules bind only via the CBD(13). In our previous HS-AFM study with TrCel7A
and its mutants, that variant that bound only
via the CBD (W40A without tryptophan residue at the active-site entrance) did not remain
long on the surface and therefore could not be
continuously visualized (11). In contrast, molecules bound to the substrate chain via the CD(the TrCel7Awild-type enzyme, isolated CD without CBD, and the inactive mutant E212Q) could
be continuously visualized during HS-AFM observation. Considering these results, it seems likely that the TrCel7A molecules visualized here as
stationary in the middle of cellulose fibrils havebeen stopped while holding a substrate chain in
the active site tunnel of the CD. Moreover, wepreviously reported an average velocity of 3.5 T
1.1 nm/s for the movement of TrCel7A (11), as
shown by a dotted line in Fig. 1B. The velocity ofmoving molecules obtained in the present study
was 7.1 T 3.9 nm/s—twice the velocity estimatedin the previous study—whereas the averaged velocity for all molecules (stopped and moving)was 5.3 T 4.9 nm/s. Therefore, it seems likelythat this earlier value (for average velocity) was
determined by the two populations of moving andstopped molecules. Although the time-resolutionof the HS-AFM observation used in this study
was up to 300 ms/frame, greater than threetimes faster than that in the previous work (up to
1 s/frame), this time-resolution is still not highenough to analyze each hydrolytic step of TrCel7A.
If one hydrolytic cycle produces one cellobiose
unit, which is approximately 1 nm in length, eachindividual hydrolysis-related movement could
occur within 140 ms, as calculated from thevelocity obtained in the present study.

no apparent difference in the low-affinity component
between the two crystalline polymorphs, whereas
the high-affinity component in the case of crys-talline cellulose IIII was quite large comparedwith that of crystalline cellulose Ia, which is consistent with our previous study (14).

Fig. 3. (A) Occurrenceof molecular congestionof TrCel7A on cellulose
IIII observed by means of
HS-AFM. TrCel7A is movingfrom right to left. The timeinterval between images
is 0.3 s. These images aretaken from movie S3. (B)Fibrillation of crystalline
cellulose by TrCel7A after congestion was visualized by means of
HS-AFM. The time interval between images is
1.5 s. These images aretaken from movie S5. (C)Schematic presentation of possible mechanism of fibrillation after the appearance of a traffic jam of
TrCel7A molecules.

Fig. 4. Synergistic hydrolysis by TrCel6A and TrCel7A visualized by means of HS-AFM. (A) Microfibrils ofcellulose IIII observed on the highly oriented pyrolytic graphite surface. (B and C) Images after 3.0 min (B) and
8.0 min (C) from the addition of 2.0 mM TrCel6A. (D to F) Images after 0.5 min (D), 2.5 min (E), and 4.5 min(F) from the addition of TrCel7A after incubation with TrCel6A for 495 s. (A) to (C) and (D) to (F) are acquired
from movies S8 and S9, respectively. The numbers attached to the z axes, which correspond to the maximum
height in the lookup table (LUT), represent the maximum heights of the respective images. (G) Synergy
between TrCel6A and TrCel7A in cellobiose production from cellulose IIII. Green and red plots are the rates ofcellobiose production by TrCel6A and TrCel7A, respectively, and the blue plot shows the synergy between thetwo enzymes. The dotted line indicates the simple sum of cellobiose production calculated from the green andred plots. (H) Proposed mechanism for the exo-exo synergy between TrCel6A and TrCel7A.
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When the hydrolysis of crystalline cellulose
IIII by Tr Cel7A was observed with HS-AFM,
as shown in Fig. 2C and movie S2, differenceswere apparent in the number but not in the velocity of enzyme molecules moving on cellulose
IIII and cellulose Ia. In the case of crystalline
cellulose Ia, enzyme molecules slid only along
limited lanes (Fig. 1A and movie S1), whereas
on cellulose IIII, TrCel7A molecules moved overalmost the whole surface. This may reflect thedifference in the amount of high-affinity binding (Fig. 2, A and B). Lehtiö and co-workers reported recently that the isolated CBD of TrCel7A
binds to the 110 surface of Valonia cellulose(cellulose Ia), and they suggested that Tr Cel7A
degrades crystalline cellulose from this hydrophobic surface (15). The hypothesis was recently confirmed by means of AFM observation (16).
The characteristics of cellulose Ia and IIII differ(17, 18) in that cellulose Ia bears hydrophobic
110 surfaces between hydrophilic 100 and 010
surfaces, whereas cellulose IIII has similarly hydrophobic 110 and hydrophilic 010 surfaces but
moderately hydrophobic (and larger) 100 surfaces, as shown in Fig. 2D. Because we usedhydrophobic highly oriented pyrolytic graphite
as a grid in the experiments, the crystals are expected to be oriented with their hydrophobic surfaces in contact with it and by symmetry on the
opposite, top face as well. Therefore, it appears
that TrCel7A moved not only on the hydrophobic
110 surface but also on the (somewhat less) hydrophobic 100 surface of cellulose IIII. In contrast,
110 is the only surface sufficiently hydrophobic
for the enzyme to attach to in the case of cellulose
Ia. Increasing the number of suitable lanes forcellulase movement would be an effective way
to enhance crystalline cellulose hydrolysis, as
judged from the comparison between cellulose
Ia and cellulose IIII. In the case of TrCel7A action, moreover, when the movement of one
molecule was halted many following molecules
stacked behind it on the cellulose IIII surface,causing a traffic jam, as shown in Fig. 3A andmovie S3. In Fig. 3B and movies S4 and S5,
however, it becomes clear that after several additional molecules had also become blocked
the enzyme molecules started to move again on
the surface, and a cellulose bundle was peeled
off from the crystalline cellulose. This behavior
may reflect the presence of an obstruction on thesurface of crystalline cellulose, which a single
Tr Cel7A molecule is unable to climb over andis therefore halted. However, as schematically
represented in Fig. 3C the accumulation of subsequent molecules behind the blocked molecule
seems to lead to elimination of the obstacle (orobstacles). As a result, the blocked molecules
restart linear movement from the point where
the first molecule had stopped.
In the process of crystalline cellulose hydrolysis by T. reesei, two major cellobiohydrolases,
TrCel7A and TrCel6A, synergistically contributeas described above (7, 19). We imaged the hydrolytic processes with crystalline cellulose IIII

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in the presence of both Tr Cel6A and Tr Cel7A.
The crystals were first observed without enzyme(Fig. 4A and movie S6) then incubated TrCel6A
for 495 s (Fig. 4, B and C, and movies S7 andS8), and the same crystalline cellulose was again
visualized after further addition of TrCel7A (Fig.
4, D to F, and movies S7 and S9). As shown in
Fig. 4, A to C, and movie S8, the appearance ofthe cellulose crystals did not change during incubation with TrCel6A, although many enzyme
molecules were observed on the surfaces. When
Tr Cel7A was added, enzyme molecules wereseen to start moving from many points on thesurface of the substrate, and the crystals apparently became thinner after the passage of the
enzyme molecules, as shown in Fig. 4, D to F,
and movie S9. This degradation of crystalline
cellulose was dramatically faster than was thecase with Tr Cel7A alone (movie S2).
We also examined this synergetic action between the two enzymes biochemically. Cellobiose
production from the reaction mixture containing
both TrCel6A and TrCel7A (Fig. 4G, blue line)
was greater than the sum (dotted line) of production by the individual enzymes separately (green
and red lines for Tr Cel6A and Tr Cel7A, respectively), confirming the occurrence of synergistichydrolysis of crystalline cellulose by these two
enzymes, which is in agreement with previous
findings. This has been called exo-exo synergy(20). These two enzymes both have active sites
located in a tunnel that passes through the whole
CD. The active site tunnel of TrCel6A is formedby two surface loops and is about 20 Å long,whereas that of TrCel7A is approximately 50 Ålong, comprising six loops (9, 10, 21, 22). Previoustransmission electron microscopic observationsof crystalline cellulose after hydrolysis by TrCel6A
and TrCel7A demonstrated that the two enzymes
degrade the substrate from different ends of thecrystal; TrCel7A degrades the substrate from thereducing end, leading to fibrillation, thinningof the crystal, or narrowing of the crystal end,

whereas TrCel6A hydrolyzes the cellulose chain
from the nonreducing end less processively
than TrCel7A, sharpening the crystal tip (23, 24).
It is somewhat difficult to find an explanation forthis type of synergistic action on highly crystalline cellulose. One possibility is that the twoloops forming the active site tunnel of Tr Cel6A
can open and allow generation of nicks in the
middle of crystalline cellulose and that thesenicks become starting and ending points for
Tr Cel7A activity. This mode of synergistic
action (schematically shown in Fig. 4H) could
also be called endo-exo synergy (25), although
as explained above, TrCel6A is generally defined
as an exo-glucanase (cellobiohydrolase). The
synergistic hydrolysis was also observed when
Tr Cel7A and Tr Cel6A were incubated at thesame time with cellulose IIII.
The present results suggest that the roughness of the crystalline cellulose surface leads
to the formation of traffic jams of productively
bound cellulases. Thus, flattening the surface,removing obstacles, and/or increasing the number of lanes, entrances, and exits by means ofpretreatment or combined use of synergisticallyacting enzymes should reduce the molecularcongestion, improving the mobility of the cellulase molecules and increasing the efficiency ofhydrolysis.
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Acknowledgments: The authors are grateful to K. Tokuyasu
of the National Food Research Institute, J. Ståhlbergof the Swedish University of Agriculture Sciences, and
A. Isogai of the University of Tokyo for their criticalsuggestions during the preparation of this paper. We
thank T. Tsukada for his help in checking the activity
and purity of TrCel6A. This research was supported by
Grants-in-Aid for Scientific Research to K.I. (19688016
and 21688023), T.U. (21023010 and 21681017), andT.A. 20221006) from the Japanese Ministry of Education,
Culture, Sports, and Technology; by a grant of theKnowledge Cluster Initiative to T.A.; by a Grant forDevelopment of Technology for High Efficiency Bioenergy
Conversion Project to M.S. (07003004-0) from the
New Energy and Industrial Technology Development
Organization; and by a Grant for Development of
Biomass Utilization Technologies for Revitalizing Rural
Areas to M.S. from the Japanese Ministry of Agriculture,
Forestry and Fisheries.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/333/6047/1279/DC1
Materials and Methods
References (27–33)
Movies S1 to S9
16 May 2011; accepted 29 July 201110.1126/science.1208386

variable, and their mechanisms are
Isotopic Signature of N2O Produced by temporally
not fully understood. Marine N O sources to theatmosphere are estimated to represent ~30% of
total “natural” inputs, or ~4 Tg N O-N per year
Marine Ammonia-Oxidizing Archaea (2).
Changes in the magnitudes of these sources22

Alyson E. Santoro,1*† Carolyn Buchwald,2 Matthew R. McIlvin,1 Karen L. Casciotti1*‡
The ocean is an important global source of nitrous oxide (N2O), a greenhouse gas that contributes
to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be
the primary sources of marine N2O, but the isotopic signatures of N2O produced by these processes
are not consistent with the marine contribution to the global N2O budget. Based on enrichment
cultures, we report that archaeal ammonia oxidation also produces N2O. Natural-abundance
stable isotope measurements indicate that the produced N2O had bulk d15N and d18O values
higher than observed for ammonia-oxidizing bacteria but similar to the d15N and d18O values
attributed to the oceanic N2O source to the atmosphere. Our results suggest that ammonia-oxidizing
archaea may be largely responsible for the oceanic N2O source.
ropospheric concentrations of nitrous oxide (N2O) are currently 322 parts per billion and rising at a rate of ~0.25% per yearT1282(1). Future projections for this radiatively active
trace gas are uncertain, primarily because biological sources and sinks of N2O are spatially and2 SEPTEMBER 2011

VOL 333SCIENCEdepend on the mechanisms and controls of microbial N2O production.
Marine N2O production is thought to be carried out by ammonia-oxidizing bacteria (AOB)
1
Department of Marine Chemistry and Geochemistry, Woods
Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
2
Joint Program in Chemical Oceanography, Massachusetts
Institute of Technology–Woods Hole Oceanographic Institution,
Woods Hole, MA 02543, USA.

*To whom correspondence should be addressed. E-mail:asantoro@umces.edu (A.E.S.); kcasciotti@stanford.edu (K.L.C.)†Present address: Horn Point Laboratory, University of Maryland
Center for Environmental Science, Cambridge, MD 21613, USA.
‡Present address: Department of Environmental Earth System
Science, Stanford University, Stanford, CA 94305, USA.

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