www.sciencemag.org/cgi/content/full/333/6047/1279/DC1

Supporting Online Material for

Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose
Surface
Kiyohiko Igarashi,* Takayuki Uchihashi, Anu Koivula, Masahisa Wada, Satoshi Kimura,
Tetsuaki Okamoto, Merja Penttilä, Toshio Ando, Masahiro Samejima*To whom correspondence should be addressed. E-mail: aquarius@mail.ecc.u-tokyo.ac.jp (K.I.)

Published 2 September 2011, Science 333, 1279 (2010)
DOI: 10.1126/science.1208386
This PDF file includes:
Materials and Methods
References (27–33)
Other Supporting Online Material for this manuscript includes the following:(available at www.sciencemag.org/cgi/content/full/333/6047/1279/DC1)
Movies S1 to S9

1

Supporting Online Materials

2
3

Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

4

Kiyohiko Igarashi, Takayuki Uchihashi, Anu Koivula, Masahisa Wada, Satoshi Kimura,

5

Tetsuaki Okamoto, Merja Penttilä, Toshio Ando, and Masahiro Samejima

6
7

Materials and Methods

8

Cellulose and enzyme preparations

9High-speed AFM observations

10Adsorption of TrCel7A on crystalline celluloses

11Synergistic hydrolysis of crystalline cellulose

12
13

Captions for Supporting Online Movies

1415

Supporting Online Movies S1 to S9161

1Materials and Methods

2Cellulose and enzyme preparations- Cellulose from green alga Cladophora sp., having more

3

than 95% crystallinity, was used in this study (14, 26). Cel7A from T. reesei was purified from4

a commercial cellulase mixture, Celluclast® 1.5L (Novozyme, available from Sigma-Aldrich)5

as described previously (26), and hydrolysis of highly crystalline celluloses from Cladophora

6was performed as described previously (26).

7T. reesei Cel6A wild-type was purified from a T. reesei strain lacking the endogenous genes

8

for the major endoglucanases Cel5A and Cel7B. The presence of contaminating cellulolytic

9activities was ruled out by measuring the activities towards MeUmb(Glc)1, MeUmb(Glc)2,10

and hydroxyethyl cellulose (HEC), as described earlier (27). Concentrations of purified

11wild-type protein were determined from UV absorbance at 280 nm using the molar extinction

12coefficient ε = 104,000 M-1 cm-1, based on the result of total amino acid analysis (28).

13
14High-speed AFM observations- High-speed atomic force microscopic observations were15

carried out using a laboratory-built HS-AFM apparatus and a BL-AC10EGS-A2 small

16

cantilever (Olympus Corporation), based on previous reports (12, 29-31). Two microliters of17

crystalline cellulose suspension in water (0.2 %) was dropped on a graphite sample disc,18

which was rinsed 3 times with 18 µl of 20 mM sodium acetate buffer, pH 5.0, after incubation

19for 5 minutes at 25˚C. Crystalline cellulose on the sample disc was initially observed without

20

enzyme in 70 µl of the same buffer, followed by the addition of 20 µM enzyme solution with21

final concentration of 2.0 μM. The AFM images were taken at 1-4 frames sec-1 at 25˚C.

22Digital movies were constructed using Adobe ImageReady CS2 (Version 9.0, Adobe Systems

23Inc.). The linear movement of individual molecule was tracked and analyzed by the use of a24

newly developed routine in Igor Pro (Version 6.4, WaveMetrics, Inc.), which was basically25

designed to follow the center of the moving particle. AFM data analysis software Gwyddion2

1(Version 2.2, http://gwyddion.net/) was used for making the 3-dimensional movies in Fig. 42and movies S8 and S9 as described previously (32).

34Adsorption of TrCel7A on crystalline celluloses- Cladophora cellulose (either cellulose Iα or

5cellulose IIII, 0.1% w/v) was incubated with various concentrations of enzymes (final6concentration, Abs280=0.04-0.8) in 1 ml of 50 mM sodium acetate buffer, pH 5.0, at 30 ˚C

7with inversion for 120 min. The free protein concentration [F] (µM) at equilibrium was

8measured after centrifugation (15,000g x 30 sec) by measuring the absorbance at 280 nm of9the supernatant (900 µl). An absorption coefficient at 280 nm of 88,250 M-1cm-1 was used for

10TrCel7A as

11nmol/mg-cellulose) was calculated by subtraction of the amount of free enzyme from the12amount of added enzyme. The amount of adsorbed enzyme was plotted versus free enzyme

13

concentration, based on a two-binding-site model for TrCel7A analysis, using the following

14equation:

15A=A1•[F]/(1/Kad1+[F])+A2•[F]/(1/Kad2+[F])16

where A1 and A2 are the adsorption maxima of high- and low-affinity binding

17(nmol/mg-cellulose); Kad1 and Kad2 are the adsorption constants of the high- and low-affinity

18

binding sites (µM-1).

described

previously

(26).

The

amountofadsorbed

enzyme(A,1920

Synergistic hydrolysis of crystalline cellulose-TrCel7A and TrCel6A (the TrCel6A/TrCel7A

21

mixing ratio was 0/2.0, 0.5/1.5, 1.0/1.0, 1.5/0.5, or 2.0/0 μM) were incubated in 50 mM

22sodium acetate buffer, pH 5.0, with 0.1 % cellulose IIII (14) at 30˚C for 30 min, then23

centrifuged (x 15,000 g). The soluble products thus obtained were analyzed by HPLC

24

(LC-2000 series; Jasco, Tokyo, Japan), using a CoronaTM Charged Aerosol DetectorTM (ESA25

Biosciences, Chelmsford, MA) as described previously (33). The supernatant was separated3

1on Shodex® Asahipak NH2P-50 (Showa Denko K.K., Kanagawa, Japan) with a linear gradient2of acetonitrile/H2O (60/40 to 50/50, v/v). The amount of products was quantified using

3cellooligosaccharides with degree of polymerization (DP) = 2-7 (Seikagaku Corporation,4Tokyo, Japan) as standards.

54

1

Captions for Supporting Online Movies

2Movie S1. HS-AFM images of TrCel7A (final conc. 2.0 μM) sliding on crystalline cellulose

3Iα. x/y=150/90 nm, 10x playback, total time 153 s. Scale bar and height lookup table (LUT) of4

the images are shown in Fig. 1A.

5
6Movie S2. HS-AFM images of TrCel7A (final conc. 2.0 μM) on cellulose IIII. x/y=180/100

7nm, 10x playback, total time 466 s. Scale bar and height LUT of the images are shown in Fig.

82C.

910

Movie S3. HS-AFM images of TrCel7A (final conc. 2.0 μM) on cellulose IIII. x/y=72/40 nm,

115x playback, total time 154 s. Scale bar and height LUT of the images are included in the12images.

13
14

Movie S4. HS-AFM images of TrCel7A (final conc. 2.0 μM) on cellulose IIII. x/y=150/75 nm,

1510x playback, total time 90 seconds. Scale bar and height LUT of the images are shown in Fig.

163B.

1718

Movie S5. HS-AFM images of TrCel7A (final conc. 2.0 μM) on cellulose IIII peeling the19surface of the same cellulose fiber of movie S4. x/y=150/75 nm, 10x playback, total time 372

20

seconds. Scale bar and height LUT of the images are shown in Fig. 3B.

2122

Movie S6. HS-AFM images of cellulose IIII before the enzyme addition. x/y=250/125 nm,

23

10x playback, total time 18 seconds

2425

Movie S7. HS-AFM images of crystalline cellulose IIII with the addition of TrCel6A (10 s)
51and TrCel7A (505 s) with final concentrations of 2.0 μM each. x/y=250/125 nm, 10x

2playback, total time 832 seconds.

34Movie S8. Three-dimensional HS-AFM images of cellulose IIII incubated with TrCel6A. This

5movie was created from the first 480 s of movie S7. TrCel6A was added (10 s) at a final

6

concentration of 2.0 μM. Height LUT of these images is shown in Fig. 4.

7
8Movie S9. Three-dimensional HS-AFM images of cellulose IIII incubated with

9TrCel6A+TrCel7A. This movie was created from the latter part of movie S7. After 495 s from10

the addition of TrCel6A (5 s from the initial frame in this movie), TrCel7A was added at a11

final concentration of 2.0 μM.

6References and Notes
1. M. E. Himmel et al., Biomass recalcitrance: Engineering plants and enzymes for biofuels
production. Science 315, 804 (2007). doi:10.1126/science.1137016 Medline
2. J. Jalak, P. Väljamäe, Mechanism of initial rapid rate retardation in cellobiohydrolase
catalyzed cellulose hydrolysis. Biotechnol. Bioeng. 106, 871 (2010).
doi:10.1002/bit.22779 Medline3. B. Yang, D. M. Willies, C. E. Wyman, Changes in the enzymatic hydrolysis rate of Avicel
cellulose with conversion. Biotechnol. Bioeng. 94, 1122 (2006). doi:10.1002/bit.20942
Medline4. D. N. S. Hon, Cellulose: A random walk along its historical path. Cellulose 1, 1 (1994).
doi:10.1007/BF00818796
5. R. Wolfenden, Y. Yuan, Rates of spontaneous cleavage of glucose, fructose, sucrose, andtrehalose in water, and the catalytic proficiencies of invertase and trehalas. J. Am. Chem.
Soc. 130, 7548 (2008). doi:10.1021/ja802206s Medline
6. T. T. Teeri, Crystalline cellulose degradation: New insight into the function ofcellobiohydrolases. Trends Biotechnol. 15, 160 (1997). doi:10.1016/S01677799(97)01032-9
7. T. T. Teeri et al., Trichoderma reesei cellobiohydrolases: Why so efficient on crystallinecellulose? Biochem. Soc. Trans. 26, 173 (1998). Medline
8. B. Henrissat, A. Bairoch, New families in the classification of glycosyl hydrolases based onamino acid sequence similarities. Biochem. J. 293, 781 (1993). Medline
9. C. Divne et al., The three-dimensional crystal structure of the catalytic core ofcellobiohydrolase I from Trichoderma reesei. Science 265, 524 (1994).
doi:10.1126/science.8036495 Medline
10. J. Rouvinen, T. Bergfors, T. Teeri, J. K. Knowles, T. A. Jones, Three-dimensional structureof cellobiohydrolase II from Trichoderma reesei. Science 249, 380 (1990).
doi:10.1126/science.2377893 Medline
11. K. Igarashi et al., High speed atomic force microscopy visualizes processive movement of
Trichoderma reesei cellobiohydrolase I on crystalline cellulose. J. Biol. Chem. 284,36186 (2009). doi:10.1074/jbc.M109.034611 Medline
12. T. Ando et al., A high-speed atomic force microscope for studying biologicalmacromolecules. Proc. Natl. Acad. Sci. U.S.A. 98, 12468 (2001).
doi:10.1073/pnas.211400898 Medline
13. J. Ståhlberg, G. Johansson, G. Pettersson, A new model for enzymatic hydrolysis of cellulose
based on the two-domain structure of cellobiohydrolase I. Biotechnology (N. Y.) 9, 286(1991). doi:10.1038/nbt0391-286
14. K. Igarashi, M. Wada, M. Samejima, Activation of crystalline cellulose to cellulose III(I)
results in efficient hydrolysis by cellobiohydrolase. FEBS J. 274, 1785 (2007).
doi:10.1111/j.1742-4658.2007.05727.x Medline

115. J. Lehtiö et al., The binding specificity and affinity determinants of family 1 and family 3cellulose binding modules. Proc. Natl. Acad. Sci. U.S.A. 100, 484 (2003).
doi:10.1073/pnas.212651999 Medline
16. Y. S. Liu et al., Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces. J.
Biol. Chem. 286, 11195 (2011). doi:10.1074/jbc.M110.216556 Medline
17. M. Wada, H. Chanzy, Y. Nishiyama, P. Langan, Cellulose IIII crystal structure and hydrogenbonding by synchrotron x-ray and neutron fiber diffraction. Macromolecules 37, 8548
(2004). doi:10.1021/ma0485585
18. Y. Nishiyama, J. Sugiyama, H. Chanzy, P. Langan, Crystal structure and hydrogen bonding
system in cellulose Iα from synchrotron x-ray and neutron fiber diffraction. J. Am. Chem.
Soc. 125, 14300 (2003). doi:10.1021/ja037055w Medline
19. L. E. R. Berghem, L. G. Pettersson, U. B. Axiofredriksson, The mechanism of enzymatic
cellulose degradation. Characterization and enzymatic properties of a β-1,4-glucan
cellobiohydrolase from Trichoderma viride. Eur. J. Biochem. 53, 55 (1975).
doi:10.1111/j.1432-1033.1975.tb04041.x
20. B. Nidetzky, W. Steiner, M. Hayn, M. Claeyssens, Cellulose hydrolysis by the cellulases
from Trichoderma reesei: A new model for synergistic interaction. Biochem. J. 298, 705(1994). Medline
21. C. Divne, J. Ståhlberg, T. T. Teeri, T. A. Jones, High-resolution crystal structures reveal how
a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I fromTrichoderma reesei. J. Mol. Biol. 275, 309 (1998). doi:10.1006/jmbi.1997.1437 Medline
22. A. Koivula et al., The active site of cellobiohydrolase Cel6A from Trichoderma reesei: Theroles of aspartic acids D221 and D175. J. Am. Chem. Soc. 124, 10015 (2002).
doi:10.1021/ja012659q Medline
23. H. Chanzy, B. Henrissat, Undirectional degradation of valonia cellulose microcrystals
subjected to cellulase action. FEBS Lett. 184, 285 (1985). doi:10.1016/00145793(85)80623-2
24. T. Imai, C. Boisset, M. Samejima, K. Igarashi, J. Sugiyama, Unidirectional processive actionof cellobiohydrolase Cel7A on Valonia cellulose microcrystals. FEBS Lett. 432, 113(1998). doi:10.1016/S0014-5793(98)00845-X Medline
25. T. M. Wood, S. I. McCrae, Biochem. J. 171, 61 (1972).
26. K. Igarashi, M. Wada, R. Hori, M. Samejima, Surface density of cellobiohydrolase oncrystalline celluloses. A critical parameter to evaluate enzymatic kinetics at a solid-liquidinterface. FEBS J. 273, 2869 (2006). doi:10.1111/j.1742-4658.2006.05299.x Medline
27. A. Koivula et al., Immunoaffinity chromatographic purification of cellobiohydrolase IImutants from recombinant trichoderma reesei strains devoid of major endoglucanase
genes. Protein Expr. Purif. 8, 399 (1996). doi:10.1006/prep.1996.0116 Medline
28. H. Palonen, M. Tenkanen, M. Linder, Dynamic interaction of Trichoderma reeseicellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis.
Appl. Environ. Microbiol. 65, 5229 (1999). Medline229. M. Shibata, H. Yamashita, T. Uchihashi, H. Kandori, T. Ando, High-speed atomic forcemicroscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin.
Nat. Nanotechnol. 5, 208 (2010). doi:10.1038/nnano.2010.7 Medline
30. D. Yamamoto et al., High-speed atomic force microscopy techniques for observing dynamic
biomolecular processes. Methods Enzymol. 475, 541 (2010). doi:10.1016/S00766879(10)75020-5 Medline
31. N. Kodera, D. Yamamoto, R. Ishikawa, T. Ando, Video imaging of walking myosin V by
high-speed atomic force microscopy. Nature 468, 72 (2010). doi:10.1038/nature09450
Medline32. K. Igarashi, M. Samejima, Biosci. Industry 68, 312 (2010).
33. K. Igarashi, T. Ishida, C. Hori, M. Samejima, Characterization of an endoglucanase
belonging to a new subfamily of glycoside hydrolase family 45 of the basidiomycete
Phanerochaete chrysosporium. Appl. Environ. Microbiol. 74, 5628 (2008).
doi:10.1128/AEM.00812-08 Medline
Acknowledgements: The authors are grateful to K. Tokuyasu of the National Food ResearchInstitute, J. Ståhlberg of the Swedish University of Agriculture Sciences, and A. Isogai ofthe University of Tokyo for their critical suggestions 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 and21688023), T.U. (21023010 and 21681017), and T.A. 20221006) from the Japanese
Ministry of Education, Culture, Sports, and Technology; by a Grant for Development ofTechnology for High Efficiency Bioenergy Conversion Project to M.S. (07003004-0)
from the New Energy and Industrial Technology Development Organization; and by aGrant for Development of Biomass Utilization Technologies for Revitalizing Rural Areasto M.S. from the Japanese Ministry of Agriculture, Forestry and Fisheries.

3