Article
pubs.acs.org/JACS

Trade-oﬀ between Processivity and Hydrolytic Velocity of
Cellobiohydrolases at the Surface of Crystalline Cellulose
Akihiko Nakamura,†,‡ Hiroki Watanabe,§,‡ Takuya Ishida,†,∥ Takayuki Uchihashi,§,⊥,# Masahisa Wada,†,∇Toshio Ando,§,⊥,# Kiyohiko Igarashi,†,∥ and Masahiro Samejima*,††

Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan§
Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan∥
Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency, Goban-cho,
Chiyoda-ku, Tokyo 102-0076, Japan⊥
Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan#
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Sanbon-cho, Chiyoda-ku,
Tokyo102-0075, Japan∇
Department of Plant and Environmental New Resources, College of Life Sciences, Kyung Hee University, Seocheon-dong,Giheung-ku, Yongin-si, Gyeonggi-do 446-701, Republic of Korea
S Supporting Information*

ABSTRACT: Analysis of heterogeneous catalysis at an interface isdiﬃcult because of the variety of reaction sites and the diﬃculty ofobserving the reaction. Enzymatic hydrolysis of cellulose by cellulases is atypical heterogeneous reaction at a solid/liquid interface, and a key
parameter of such reactions on polymeric substrates is the processivity,
i.e., the number of catalytic cycles that can occur without detachment of
the enzyme from the substrate. In this study, we evaluated the reactionsof three closely related glycoside hydrolase family 7 cellobiohydrolases
from ﬁlamentous fungi at the molecular level by means of high-speed
atomic force microscopy to investigate the structure−function relationship of the cellobiohydrolases on crystalline cellulose. We found that highmoving velocity of enzyme molecules on the surface is associated with ahigh dissociation rate constant from the substrate, which means weakinteraction between enzyme and substrate. Moreover, higher values of processivity were associated with more loop regions
covering the subsite cleft, which may imply higher binding aﬃnity. Loop regions covering the subsites result in stronger
interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-oﬀ between
processivity and hydrolytic velocity among processive cellulases.

■

hydrolysis of crystalline cellulose.4 This technique enables
direct measurement of the processivity and catalytic velocity ofenzyme molecules without the need for enzyme labeling or
substrate modiﬁcation.
Cellulose, the main structural polysaccharide of plant cell
walls, is the most abundant biopolymer on Earth. In nature,
cellulose chains, linear polymers of β-1,4-linked D-glucose units,
are packed into ordered arrays to form crystalline cellulose I.5
Because cellulose chains have stable β-glycosidic bonds6 andeach chain is also stabilized by intra- and intermolecularhydrogen bonds in the crystal,7 cellulose I is quite resistant not
only to chemical hydrolysis but also to enzymatic degradation.
When cellulose I is treated by ammonia, however, the crystal

INTRODUCTION
Although homogeneous catalysis has been extensively studied,heterogeneous catalysis is less well understood because of thevariety of the reaction sites and the diﬃculty of observing thereaction.1 Among biochemical reactions, enzyme reactions at asolid/liquid interface with an insoluble substrate are particularly
intractable. The reasons for this include the complexity of solid
surfaces, which makes it diﬃcult to know how much of the
enzyme is catalytically active, and diﬀerences in the mobility ofthe enzyme molecules on the surface. In the case of polymeric
substrates, which are common in nature, a key parameter is theprocessivity, which is the number of catalytic cycles that canoccur without detachment of the enzyme from the substrate.2,3Processivity is diﬃcult to evaluate biochemically. However, werecently used high-speed atomic force microscopy (HS-AFM)
to visualize the behavior of single molecules of cellulase during© 2014 American Chemical Society

Received: November 24, 2013
Published: February 26, 20144584

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ArticleFigure 1. (A) Phylogenetic tree of the sequence-based alignment of Cel7s classiﬁed as “characterized” by CAZY (http://www.cazy.org/). Sequenceswere aligned and the tree was made with ClustalW2 server default settings. X-ray crystal structures of Cel7s and comparison of their overall
structures. Green, TrCel7A (8CEL); blue, PcCel7D (1GPI); yellow, TrCel7B (1EG1). Orange, Superimposed cellulose chain in enzyme subsite. (B)Diﬀerences of loop regions. TrCel7A has four loop regions (loop1 to loop4), and PcCel7D has loop2, loop4, and a short loop1 region. TrCel7B lacks
loop2 and loop3. Orange, superimposed cellulose chain in enzyme subsite. (C) Diﬀerent amino acids around the loop regions between PcCel7C and
PcCel7D. Y367 and H367 residues are located on loop4, and the others are located on loop2.

transforms into cellulose IIII,8 which is easily degraded bycellulase than cellulose I.9
Cellulolytic microorganisms secrete a set of cellulases,
cellobiohydrolases (CBHs: EC 3.2.1.91 and EC 3.2.1.176)
and endoglucanases (EGs: EC 3.2.1.4), to degrade cellulose.10
In the cellulose degradation system of the ascomycetes fungus

Trichoderma reesei, a dominant component of the secreted
proteins is glycoside hydrolase (GH) family 7 cellobiohydrolase(TrCel7A, formerly known as CBH I), which produces
cellobiose from the reducing end of cellulose.11−13 TrCel7A
has a two-domain structure,14 with a catalytic domain (CD)and a cellulose-binding domain (CBD) belonging to the4585

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ArticleThe reaction was stopped by the addition of 20 μL of 2.0 M Na2CO3,
and the absorbance of the reaction mixture was measured at 405 nm(Abs405 = 16.6 mM−1 cm−1). pNPL-hydrolyzing activity (units) was
calculated using the standard curve of p-nitrophenol. The purity ofeach fraction was checked by SDS-PAGE, and fractions containing
proteins of about 58 and 62 kDa proteins with pNPL-hydrolyzing
activity were collected. The 62 kDa protein in ﬂow-through fractions
was equilibrated with 20 mM Tris-HCl buﬀer, pH 8.0, and applied to a
SuperQ-Toyopearl 650S (TOSOH; CV = 100 mL) equilibrated withthe same buﬀer. Elution was done with a linear gradient of 0−0.1 M
NaCl in 800 mL of 20 mM Tris-HCl, pH 8.0. Next, ammonium sulfate
was added to the protein solution to a ﬁnal concentration of 1.0 M,
and fractionation was carried out on a Phenyl-Toyopearl 650S column(CV = 67 mL) equilibrated with 20 mM sodium acetate buﬀer (pH5.0) containing 1.0 M ammonium sulfate. Proteins were eluted with areverse linear gradient of 1.0 to 0 M ammonium sulfate in 134 mL.
Finally, the protein solution was equilibrated with 20 mM Tris-HCl
buﬀer (pH 8.0) and fractionated on a DEAE-Toyopearl 650S column(CV = 150 mL) equilibrated with the same buﬀer. Proteins were
eluted with a linear gradient of 0−50 mM NaCl in 1350 mL. The
fractions were assayed as described above, and fractions containing 62
kDa protein with pNPL-hydrolyzing activity were collected andequilibrated with 20 mM sodium acetate buﬀer (pH 5.0). The 58 kDa
protein solution was similarly fractionated on a Phenyl-Toyopearl
650S column (TOSOH; CV = 67 mL). Finally, the protein solution
was equilibrated with 20 mM potassium phosphate buﬀer (pH 7.0).
Elution was done with a linear gradient of 0−50 mM KCl in 1350 mL.
The fractions were assayed as described above, and 58 kDa proteincontaining fractions with pNPL-hydrolyzing activity but without pnitrophenylbitrophenyl-β-D-glucoside-hydrolyzing activity were collected and equilibrated with 20 mM sodium acetate buﬀer, pH 5.0, by
an ultraﬁltration.
TrCel7A was puriﬁed from Celluclast 1.5L (Novozymes, Denmark).
The crude enzyme was desalted by a gel ﬁltration column(TOYOPEARL HW-40S; TOSOH; CV = 45 mL), which was
equilibrated with 20 mM potassium phosphate buﬀer pH 7.0. The
protein concentrations of each fraction were measured by a Protein
Assay kit, and the fractions containing protein were mixed and injected
into an anion-exchange column (TOYOPEARL DEAE-650S, CV =150 mL), which was equilibrated with the same buﬀer. The proteins
were eluted by a linear gradient up to 300 mM KCl. After themeasurement of protein concentration, the pNPL-hydrolyzing activity
was measured, and the purity of each fraction was estimated by SDSPAGE. The fractions containing ∼60 kDa enzyme, which has activity
for pNPL, were collected and mixed with the same volume of 2.0 M
ammonium sulfate solution to a ﬁnal concentration of 1.0 M. The
enzyme solution was injected to a hydrophobic interaction column(TOYOPEARL Phenyl-650S; CV = 67 mL), which was equilibrated
with 20 mM of sodium acetate buﬀer, pH 5.0, containing 1.0 M
ammonium sulfate. The enzyme was eluted by a linear reverse gradientof ammonium sulfate to 0 M. The protein concentration, activity for
pNPL, and purity of each fraction were analyzed by the same methods,
and the enzyme was collected. The buﬀer solution of the enzyme waschanged to 20 mM of sodium acetate buﬀer, pH 5.0.
The enzyme solutions were ﬁltrated by a polyvinylidene ﬂuoride(PVDF) membrane with 0.1 μm pore size (Ultrafree Centrifugal
Filters, Merch-Millpore, Deutschland). The purity of the three
enzymes was checked by SDS-PAGE analysis on 12% polyacrylamidegel. The contamination of endoglucanase was estimated as <1% (mol/mol) from the viscometric measurement of carboxymethyl cellulose(CMC) degrading as shown in Figure S2 of the Supporting
Information.
Cellulose Preparation. Highly crystalline cellulose Iα and IIII wereprepared according to a previous report.33 Phosphoric acid-swollen
cellulose (PASC) was prepared from Avicel (Funakoshi, Tokyo,
Japan). Avicel was dissolved in 85% (w/w) phosphoric acid, and thesolution was made completely clear by vigorous agitation with a glass
stick. After overnight incubation at 4 °C, cellulose was regenerated inwater and homogenized in a high-speed blender. The cellulose
suspension was washed with water and stored at 4 °C.

carbohydrate-binding module (CBM) family 1. These domains
are connected by a highly O-glycosylated linker region. The CD
has a β-sandwich structure, formed by β-strands connected with
α-helices and loop regions stabilized by many disulﬁde bridges,
and contains substrate-binding sites for 10 glucose units(subsites −7 to +3).15 The CBD in TrCel7A consists of only
35 amino acids, being rather small compared with the CD,
which has a molecular weight of ∼50 kDa.16
The wood-rotting basidiomycete Phanerochaete chrysosporium
is one of the best-studied cellulolytic ﬁlamentous fungi,
together with T. reesei. The total genome of this fungus wassequenced by the Joint Genome Institute (http://genome.jgipsf.org/Phchr1/Phchr1.home.html),17−19 and the data showthat P. chrysosporium has seven genes coding Cel7 isozymes(Cel7A−Cel7F/G).19,20 Because the pattern of gene expression
varies depending on the conditions of cultivation, the geneproducts (Cel7s) are assumed to have distinct roles.21−25
Among them, PcCel7C and PcCel7D (formerly known as
CBH62 and CBH58, respectively26) are the major secretory
proteins in cellulolytic culture, accounting for up to 80% of
extracellular proteins. PcCel7D was ﬁrst identiﬁed as ahomologue of TrCel7A from P. chrysosporium.27,28 PcCel7C
has higher carboxymethyl cellulose-degrading activity than
PcCel7D.26 The X-ray structure of PcCel7D was solved, and ahomology model of PcCel7C was compared with PcCel7D
based on the high amino acid sequence identity of the catalytic
domains (81%).29 The comparison of the X-ray structure andthe homology model indicated that the overall fold is the same
between the two enzymes, but three amino acids in the catalytic
tunnel are diﬀerent in PcCel7C from those in PcCel7D (Figure
1).
On the basis of the X-ray structure of the catalytic core of
TrCel7A, which revealed a tunnel-like subsite structure
constructed from loop regions, the reaction of GH7 CBH
was suggested to be processive.30 TrCel7A and PcCel7D arelikely to show diﬀerent processivity because of the diﬀerence inthe number of loop regions covering the subsite.31,32 However,measurement of processivity is diﬃcult because of the variety ofenzyme-binding states on cellulose. In the present study, we
compared PcCel7C, PcCel7D, and TrCel7A in terms of moving
velocity on the substrate and half-life of the movement in orderto characterize the structure−function relationship GH family 7
CBHs.

■

EXPERIMENTAL PROCEDURES

Cultivation of Phanerochaete chrysosporium. P. chrysosporium
strain K-3 was grown on 2 L of Kremer and Wood medium containing
2% cellulose (CF11; Whatman) as the sole carbon source in a jar
fermenter with 5 L working volume (Takasugi Seisakusho, Tokyo,
Japan). The temperature was 37 °C, and the pH was maintained at 5.0
with phosphoric acid and potassium hydroxide. Stirring was done at
300 rpm, and air was supplied at 2.0 L/min. After cultivation for 5days, the culture supernatant was separated using a glass ﬁlter
membrane. Extracellular proteins were precipitated with ammonium
sulfate at 70% saturation and stored at 4 °C.
Puriﬁcation of Enzymes. Precipitated extracellular proteins weredissolved in 20 mM potassium phosphate buﬀer (pH 7.0) and applied
to a DEAE-Toyopearl 650S column (TOSOH, Tokyo, Japan; column
volume (CV) = 150 mL) equilibrated with 20 mM potassium
phosphate buﬀer (pH 7.0) after desalting. Proteins were eluted with alinear gradient of 0−300 mM KCl in 1050 mL. Protein concentrationof fractions was estimated with a Protein Assay kit (Bio-Rad, Hercules,
CA) according to the manufacturer’s instructions. Aliquots of 20 μL
were incubated with 20 μL of 10 mM p-nitrophenyl lactoside (pNPL),
20 μL of 1.0 M sodium acetate buﬀer (pH 5.0), and 140 μL of water.
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ArticleFigure 2. Time course of products formation (top) and ratio of cellobiose/glulose + cellotriose (bottom) formed from crystalline cellulose Iα (A, D),cellulose IIII (B, E), and PASC (C, F). Cellulose (0.1%) was incubated with 2.0 μM enzymes in 50 mM sodium acetate buﬀer, pH 5.0, at 30 °C.
Triangle, TrCel7A; square, PcCel7C; circle, PcCel7D. Error bars show standard deviation (SD) and are from the three independent measurements.

⎛ ln 2 ⎞f (t ) = a0 exp⎜−
·t ⎟
⎝
τ ⎠

Activity Measurement. Puriﬁed TrCel7A, PcCel7C, and PcCel7D(2.0 μM) were each incubated with 0.1% cellulose Iα, cellulose IIII, or
PASC in 50 mM sodium acetate buﬀer, pH 5.0, at 30 °C. After 15, 30,
60, 90, and 120 min incubation (additionally 5 and 10 min incubation
for cellulose Iα), the reaction mixture was ﬁltered using MultiScreen
0.22 μm ﬁlter plates (Millipore, Billerica, MA) and products wereseparated and quantiﬁed by high-performance liquid chromatography(HPLC; JASCO LC-2000) on a NH2-P column (Showa Denko K. K.,
Kanagawa, Japan) with a Corona-CAD detector (ESA Biosciences,
Chelmsford, MA). Concentrations of cellobiose in the reaction
mixtures, q(t), were ﬁtted to the equationsq(t ) = a ·(1 − exp(− b·t )) + c·(1 − exp(− d ·t ))where a0 is the intercept of the y-axis, τ is the half-life of the processive
movement, and t is duration time. In eq 5, (ln 2/τ) is the dissociation
constant of the moving-enzyme (koff) (the detail is available as
Supporting Information). For the HS-AFM experiments, highly
crystalline cellulose IIII was used as a substrate to obtain enoughnumbers of molecules for statistical analysis.

■

(1)

(2)

for cellulose IIII, where a, b, c, and d are constants and t is the
incubation time. Equation 1 and eq 2 were diﬀerentiated to eqs 3 and4, respectively, for the estimation of the velocity of cellobiose
production as follows:

ν(t ) = a · b exp(− b· t ) + c· d exp(− d· t )(3)

ν(t ) = a · b exp(− b· t ) + c(4)

RESULTS

General Characteristics of Cel7s. After ﬁve days ofcultivation, ∼2 g of crude enzymes were produced, andapproximately 300 and 600 mg of PcCel7C and PcCel7D,
respectively, were puriﬁed with suﬃcient purity for the present
experiments. After several steps of column chromatography,
PcCel7C, PcCel7D, and TrCel7A were each homogeneous on
SDS-PAGE as shown in Figure S2 of the Supporting
Information. The puriﬁed enzymes were incubated withcrystalline cellulose Iα, IIII, and PASC, and the amounts ofproducts were measured by HPLC. The progress curves areshown in Figure 2A−C. The major product was cellobiose,
whereas glucose was detected from all of the substrates andcellotriose was produced from PASC. TrCel7A produced
cellobiose from cellulose Iα with the highest velocity among
three CBHs (1.1 ± 0.1 μM/min at 60 min), whereas those of
PcCel7D and PcCel7C were 0.63 ± 0.01 and 0.35 ± 0.03 μM/
min at 60 min, respectively. When cellulose IIII was used, thevelocity of cellobiose production by TrCel7A was similar to thatof PcCel7D, whereas PcCel7C was less active toward cellulose
IIII, as shown in Figure 2B. In contrast, PcCel7C produced
cellobiose faster than PcCel7D and TrCel7A when PASC wasused as a substrate. In the degradation of PASC, the velocity ofcellobiose production by PcCel7C was 17.2 ± 1.1 μM/min at
30 min of incubation, which is 1.2 times faster than that offor cellulose Iα and PASC andq(t ) = a ·(1 − exp(− b·t )) + c·t(5)These functions are semiempirical and just used to calculate thevelocity of cellobiose production. The ratio of cellobiose/glulose +cellotriose production was estimated according to the previous
report.31
HS-AFM Observation. Movements of molecules of the threecellulases on the substrate surface were observed by HS-AFM based on
the previous reports.4,34,35 Histograms of averaged velocities ofindividual molecules during one processive reaction were drawn
using IGOR Pro 6 (WaveMetrics, Portland, OR) and ﬁtted to a
Gaussian distribution. Moving times of molecules were calculated fromthe number of frames in which the molecules were observed, and plotsof the number of molecules in each range of moving time were ﬁtted
to the equation
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ArticleTable 1. Summary of Biochemical Measurement and AFM Observations
biochemical measurementa
velocity of cellobiose production (μM/min)

AFM observation

ratio of cellobiose/glulose + cellotriose

enzymecellulose Iα

cellulose IIII

PASC

cellulose Iα

cellulose IIII

PASC

dissociation rate
constantb (s‑1)

PcCel7C
PcCel7D
TrCel7A

0.35 ± 0.03
0.63 ± 0.01
1.1 ± 0.1

6.6 ± 0.6
12.2 ± 0.3
12.7 ± 0.1

17.2 ± 1.1
14.6 ± 1.4
11.9 ± 2.2

14.5 ± 1.2
17.2 ± 0.8
20.3 ± 1.5

29.9 ± 0.1
33.3 ± 2.1
34.8 ± 0.9

14.7 ± 0.1
20.5 ± 0.6
15.5 ± 0.1

0.51 ± 0.01
0.32 ± 0.04
0.20 ± 0.01

half-life of
movementb(s)averagedvelocityc(nm/s)

half-life
processivityd

1.4 ± 0.0
2.2 ± 0.1
3.5 ± 0.2

14.7 ± 9.1
9.4 ± 3.7
6.8 ± 3.5

19.9
20.3
23.6

The values of cellulose Iα and cellulose IIII were at 60 min incubation, and the values of PASC were at 30 min incubation. The values are mean ±standard deviation. bThe mean ± SE of the ﬁtting with the exponential decay. cThe mean ± SD with the Gaussian. dHalf life processivity = half-life
period (s) × averaged velocity (nm/s)/cellobiose length (nm) = ln(2) × processivity.
aFigure 3. High-speed AFM images of the three CBHs. Positions of each enzyme molecule are marked by rectangles. Enzyme (0.55 μM) was
observed in 50 mM NaAc buﬀer, pH 5.0, at 25 °C. Frame rate was 0.2 frame/s.

Figure 4. Examples of movement characteristics and averaged velocity histograms of TrCel7A (A, D), PcCel7D (B, E), and PcCel7C (C, F).
Averaged velocities were calculated from the moving-distances and duration of each molecule. Peak values in each Gaussian distribution (D−F) areindicated by lines in the corresponding upper ﬁgures (A−C).

PcCel7D (14.6 ± 1.4 μM/min) and 1.4 times faster than that of
TrCel7A (11.9 ± 2.2 μM/min). The ratio of cellobiose/glucose+ cellotriose in cellulose Iα degradation was 20.3 ± 1.5 for

TrCel7A, 17.2 ± 0.8 for PcCel7D, and 14.5 ± 1.2 for PcCel7C
at 60 min of incubation. These values were relatively higher
using cellulose IIII, and 34.8 ± 0.9 for TrCel7A, 33.3 ± 2.1 for
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ArticlePcCel7D, and 29.9 ± 0.1 for PcCel7C. In the case of PASC
degradation, PcCel7D showed the highest value (20.5 ± 0.6 at
30 min), and TrCel7A and PcCel7C showed the values of 14.7
± 0.1 and 15.5 ± 0.1, respectively. These values are
summarized in Table 1.
HS-AFM Observation for the Statistic Analysis ofProcessive Movement. TrCel7A, PcCel7C, and PcCel7D
molecules moving on the surface of crystalline cellulose wereobserved by HS-AFM (Figure 3A−C). Many moving molecules
were detected in all cases, although the velocity of the
movement and the duration varied, as shown in Movies S3−S5in Supporting Information and Figure 4A−C. The movements
were then analyzed statistically to quantify the diﬀerences. The
movement of TrCel7A molecules showed a Gaussian
distribution with mean ± SD of 6.8 ± 3.5 nm/s (n = 220,
Figure 4D), whereas those of PcCel7D and PcCel7C were 9.4 ±3.7 nm/s (n = 233, Figure 4E) and 14.7 ± 9.1 nm/s (n = 176,
Figure 4F), respectively. The longest movement was observed
for TrCel7A with ∼250 nm, whereas those of PcCel7C and
PcCel7D were ∼120 nm (Figure S6 in Supporting
Information). When the duration of the movements wasanalyzed, the graphs were ﬁtted to a single exponential decay
for all three CBHs. The values of half-life ± standard error (SE)(τ) of TrCel7A, PcCel7D, and PcCel7C were 3.5 ± 0.2, 2.2 ±
0.1, and 1.4 ± 0.0 s, respectively. In addition, the values ofdissociation rate constant ± SE (koff) of TrCel7A, PcCel7D, and
PcCel7C were 0.20 ± 0.01, 0.32 ± 0.04, and 0.51 ± 0.01 s−1,
respectively.

and HS-AFM observations in order to characterize thestructure−function relationship.
HS-AFM can observe single molecules moving on a cellulose
surface with catalysis.4,34,35 Here, we observed many moleculesmoving on the surface of crystalline cellulose for all three CBHs
(Figure 3), and the results indicated that PcCel7C and PcCel7D
are processive enzymes as well as TrCel7A, indicating that theregions of loop2 and loop4 are important but loop3 is notnecessary for processive reaction of CBH on crystalline
cellulose. Using cellulose IIII, we were able to observe enoughmolecules for statistical analysis and comparison of the three
CBHs. The moving velocity of TrCel7A on crystalline cellulose
IIII obtained in the present study (6.8 ± 3.5 nm/s) was almost
the same as that on crystalline cellulose Iα (7.1 ± 3.9 nm/s) as
reported previously,34 indicating that the conversion of
crystalline from Iα to IIII does not aﬀect the velocity of theprocessive movement. Therefore we compared the movementof the three CBHs on crystalline cellulose IIII. Among the three
CBHs, the values of averaged hydrolytic velocities of PcCel7C
and PcCel7D were higher than that of TrCel7A. In the present
work, we also analyzed the duration of processive movementsof CBH molecules on crystalline cellulose in order to calculate
the half-life of their movements. From the values of hydrolytic
velocities and the values of half-life of processive movements,
we calculated the half-life processivity of the three CBHs
(Table 1). The half-life processivity of TrCel7A was higher thanthe values of PcCel7C and PcCel7D. This result from thesingle-molecule observation well shows that the tunnel-likestructure of GH family 7 CBH is important for the processivity
as reported before.31 Among three enzymes, only TrCel7A
contains the loop3 region, which includes amino acids
interacting with the cellulose chain at subsites −2 (Y247), +1(T246, R251), and +2 (R251). This may imply that TrCel7A
has stronger aﬃnity for the cellulose chain, and this could be
the reason why the processivity of TrCel7A was higher thanthose of the other two CBHs. It was reported that the catalytic
domain of TrCel7A has more favorable binding free energy
than that of PcCel7D.37 Therefore, it is implied that the stabilityof the complex between CD and cellulose chain is important
for the processive reaction. The more loops covering the active
site causes higher processivity of cellobiohydrolase but lower
hydrolytic velocity.
Comparison of PcCel7C and PcCel7D showed no diﬀerencein the amino acids that are expected to interact directly with thecellulose chain in the subsites of the catalytic domains.29 The
CBDs of these enzymes are also very similar, and only one
amino acid, which is not located at the binding surface, isdiﬀerent. Thus, there is a high overall degree of similarity
between these two enzymes. However, three amino acids
constructing loop regions of tunnel-like structure are diﬀerent.
They are located in loop2 and loop4 covering the active-sitetunnel as shown in Figure 1C. Gly193, Thr196, and His367 in
PcCel7D are replaced by Ser, Ala, and Tyr, respectively, in
PcCel7C, and it has been predicted that the changes at loop2(G193S and T196A) make the loop more ﬂexible.29 Although
the structures of PcCel7C and PcCel7D only diﬀer aroundsubsites −4 and −3, the two enzymes exhibit diﬀerent velocities
of hydrolysis and diﬀerent aﬃnities for the cellulose chain. This
result suggests that the higher ﬂexibility of loop2 is related to
the faster velocity of the processive reaction.
When the parameters of the three CBHs obtained by HSAFM observations were compared, PcCel7D moved slower
than PcCel7C, but it had a longer moving-duration than■DISCUSSION
The structures of several GH family 7 cellulases (CBHs andEGs) have been solved, and the structure−function relationshipbetween CBH and EG can be understood in terms of thesestructures, i.e., CBHs contain rather closed, tunnel-shaped
active sites, whereas EGs lack the loops that cover the subsites
in CBHs and exhibit cleft-type active sites. This diﬀerence isclearly reﬂected in the phylogenetic tree (Figure 1A) of GH
family 7 cellulases, 35 CBHs and 17 EGs. At the sequence level,
GH family 7 cellulases are initially divided into two categories,
CBH and EG. The clade of CBH was further divided into two
nodes, representing ascomycete and basidiomycete CBHs.
Representative X-ray crystal structures of the three groups areshown in Figure 1A. Ascomycete CBHs have four loop regions,
two of which are absent in EGs, and the subsites of EGs are
more open than those of CBHs (Figure 1 B). Basidiomycete
CBHs lack one of the loops (loop3) covering the active site
compared with ascomycete CBHs. Therefore, the active-site
structure of basidiomycete CBHs is intermediate between thoseof ascomycete CBHs and EGs. Comparison of processivity
among these enzymes is expected to show how these structuralcharacteristics inﬂuence the processive reaction of GH7 CBHs.
It is well-known that ascomycete T. reesei produces CBH(TrCel7A) and EG (TrCel7B) in cellulose-grown culture
medium, whereas basidiomycete P. chrysosporium mainly
produces two similar CBHs, PcCel7C and PcCel7D. T. reesei
degrades cellulose by synergistic action between CBH-type andEG-type of GH7 enzyme (TrCel7A and TrCel7B).36 In
contrast, basidiomycetes such as P. chrysosporium have noapparent EG-type Cel7, indicating that basidiomycete employ adiﬀerent ratio of enzymes from ascomycete to degrade
cellulose. Therefore we compared the CBHs from P.
chrysosporium and T. reesei by the biochemical measurements
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ArticleFigure 5. Histograms of moving-durations. A, TrCel7A; B, PcCel7D; C, PcCel7C. Moving-durations were calculated from the number of frames inwhich each molecule was detected during tracking.

PcCel7C. In addition, TrCel7A moved much more slowly than
PcCel7D and had a longer moving-duration than PcCel7D(Figure 5). The movements in HS-AFM observation involve
catalysis, which has been conﬁrmed by the immobility of theinactive mutant of TrCel7A (E212Q) in our previous study,
indicating that the velocity of the movements is the velocity ofthe processive cycle when we analyze moving molecules on thesurface of the substrate. On the other hand, moving-duration isan indicator of the stability of the complex between CD andcellulose chain. The relationship between velocity and movingduration of the three CBHs suggests that higher stability of thecomplex hinders the moving on the cellulose. Recently it has
been discussed that the rate-limiting step of the degradation ofcellulose is a decrystallization process of the substrate.38,39
Allthough we are unable to distinguish decrystallization of thesubstrate and the sliding movement of CD, because the sliding
movement should promote decrystallization of cellulose, while
the decrystallization should cause movement vice versa, we
speculate that the rate-limiting step of crystalline cellulose
degradation is the process rather than the catalysis as discussed
from “bulk” experiments.38,39
Although a direct comparison between the results of singlemolecule observation and biochemical activity measurement isoften diﬃcult, the order of hydrolytic activity on PASC was thesame as the order of the velocity of processive movement andkoff. This result implies that the rate-limiting step of PASC
hydrolysis is the catalytic process or the oﬀ-rate from thesubstrate, but not decided by processivity. This is supported by
the report that a less processive mutant of chitinase hydrolyzes
chitosan faster than does wild-type chitinase.40 In addition,
because PASC contains large numbers of cellulose chain ends
and free chains for the initiation of endotype hydrolysis,32
capturing the substrate chain by CD or decrystallization of thesubstrate is not a rate-limiting step.41 In the hydrolysis of
crystalline cellulose Iα, the relationship among the three
enzymes was similar to that of processivity whereas thediﬀerence between PcCel7D and TrCel7A was smaller incellulose IIII hydrolysis. The big diﬀerence between the two
crystalline celluloses is the shape of the crystal. Cellulose IIII
has large hydrophobic surface and more numbers of accessible
chain ends for CBHs than cellulose Iα.34 These results may
imply that the bottleneck of crystalline cellulose degradation is
the process of picking up a chain-end. Processivity isdetermined as the balance between two kinetic parameters,
kcat and koff, which determines how many catalytic cycles occur
per binding episode of the catalytic domain. Therefore, it isreasonable that processivity is more important for the

degradation of crystalline cellulose, which has less accessible
chain ends.
The velocity of processive movement estimated from the HSAFM observation and the velocities of cellobiose production
determined by biochemical experiments were very diﬀerent asreported previously4,34 (Table 1). Considering only reactive(working) enzyme molecules are visualized in the HS-AFM
observation and biochemical bulk experiments cause underestimation of the speciﬁc activity of the CBHs, the number ofactive enzymes was quite limited (<1% of added enzyme) at thesurface of the crystalline cellulose. In contrast, the processivity
obtained from the ratio of cellobiose/glulose + cellotriose in
the biochemical experiment reﬂects the processivity estimatedfrom the HS-AFM observation more than we expected,
suggesting that the biochemical estimation is eﬀective toknow the rough value of processivity for CBHs. The similarity
between the values of processivity estimated from the HS-AFM
observation and the ratio of cellobiose/glulose + cellotriose
indicates the ratio cancels the amount of nonworking enzyme.
The processivity of TrCel7A was previously reported as ∼60 forthe degradation of bacterial cellulose,32,42 which is about twice
the value determined by HS-AFM observation in this study(kcat/koff = 34). We think the major reason of the diﬀerence isthe diﬀerence of the substrate and/or diﬀerent techniques toestimate the value. We are now determining the processivity
and koff by the diﬀerent single molecular methods, such as a
ﬂuorescence microscope in addition to recently developed HSAFM combined with single-molecule ﬂuorescence microscope.43 Although the absolute value of processivity is still
open for discussion, we may have to use the same substrateprepared by the same protocol to compare the processivity.
In applications of cellulases for degradation of biomass, thesubstrate will contain both crystalline and amorphous regions.
From the comparison among TrCel7A, PcCel7C, and PcCel7D
in this study, higher processivity is better for the degradation of
the crystalline region of the cellulose, whereas higher hydrolytic
activity by higher dissociation rate constant is better for the
degradation of the amorphous region. Therefore, the suitable
enzyme may diﬀer depending upon the crystallinity of cellulose
in biomass. Moreover, the contribution of the processivity
becomes smaller when the substrate is treated chemically,
because decrease of degree of polymerization causes an increaseof accessible chain-ends of the substrate, which also occurred by
the reaction of other enzymes in the enzyme cocktail such as
EGs and redox enzymes. We believe that the present ﬁndings
will be helpful to select CBHs for industrial cellulosic biomass
conversion.
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CONCLUSION
We used HS-AFM to evaluate the kinetic parameters forprocessive reaction of three cellobiohydrolases, as well as therelationship between processivity and the structure of the
enzymes. The correlation between catalytic rate constant anddissociation rate constant indicated that higher aﬃnity for thesubstrate causes a lower catalytic rate, which is a trade-oﬀ
between processivity and hydrolytic velocity in cellobiohydrolases. Considering the higher processivity has the advantage
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cocktails for better hydrolysis of a cellulosic biomass.

■

ASSOCIATED CONTENT

S Supporting Information*

Additional results and details of methods described within the
manuscript. Supplementary Movie S1, crystalline cellulose
degradation movie of TrCel7A. Supplementary Movie S2,
crystalline cellulose degradation movie of PcCel7D. Supplementary Movie S3, crystalline cellulose degradation movie of
PcCel7C. This material is available free of charge via theInternet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

E-mail: amsam@mail.ecc.u-tokyo.ac.jp.
Author Contributions‡

These authors contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
This research was supported by a grant for Development of an
Innovative and Comprehensive Production System for
Cellulosic Bioethanol to M.S. (09000833-0) from the New
Energy and Industrial Technology Development Organization,
by a grant from the Advanced Low Carbon Technology
Research and Development Program (ALCA) of the Japan
Science and Technology Agency (JST) to K.I., by Grants-in-Aid
for Scientiﬁc Research for Young Scientists (A: No. 19688016
and 21688023) and (B: No. 17780245), by a Grant-in-Aid for
Scientiﬁc Research (B: No. 24380089), and by a Grant-in-Aid
for Innovative Areas (No. 24114008) to K.I. from the Japanese
Ministry of Education, Culture, Sports, and Technology(MEXT) and Grant-in-Aid for JSPS Fellows to A.N.
(25•7574) from Japan Society for the Promotion of Science(JSPS).

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