26
T. Hattori et al. / Carbohydrate Research 353 (2012) 22–26
cellulose was 50,000. Proton decoupling fields of ca. 83 kHz were
employed in these experiments together with a spinning speed
of 12.0 kHz. Spectra were calibrated through the methyl carbon
resonance of hexamethylbenzene as an external reference at
17.35 ppm. High-pH anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) analysis was carried out
using a DIONEX DX-500 system (Dionex, Sunnyvale, CA) fitted with
a PAD (ED-40) and a CarboPac PA1 column (4.0 Â 250 mm). The
column was pre-equilibrated in 60% (v/v) deionized water (eluent
A) and 40% (v/v) 0.2 M NaOH (eluent B). Bound substance was then
eluted using the following gradient: 0–40 min, linear decrease in
ratio of eluent A from 60% (v/v) to 45% (v/v) with increase of eluent
C (0.1 M NaOH, 1.0 M sodium acetate) from 0% (v/v) to 15% (v/v)
with eluent B remaining constant at 40% (v/v); 40–50 min, 100%
(v/v) eluent C; 50–60 min, 60% (v/v) eluent A and 40% (v/v) eluent
4.5. In vitro preparation of cellulose-like substances
For the preparation of cellulose, a solution (200
ose (100 mg) in 50 mM acetate buffer (pH 4.0) was added to
200 L of enzyme solution (0.2 U) in the same buffer and the mix-
lL) of cellotri-
l
ture was maintained at 50 °C. After 1 h, the resulting insoluble sub-
stance was isolated by centrifugation and washed three times with
50% methanol to afford 30 mg of substance, which was directly ap-
plied to MALDI-TOF mass spectrometry, solid-state 13C NMR spec-
troscopy, and XRD analysis.
References
1. Liungdahl, L. G.; Eriksson, K. Ecology of Microbial Cellulose Degradation. VIII. In
Advances in Microbial Ecology; Marshall, K. C., Ed.; Plenum Press: New York,
1985; pp 237–299.
B. The flow rate was 1.0 mL/min. XRD were obtained with Cu K
from a powder XRD generator (JDX3530; Japan Electronic Organi-
zation Co. Ltd, Tokyo Japan) operating at 30 kV and 30 mA.
a
2. Atalla, R. H.; VanderHart, L. D. Science 1984, 223, 283–285.
3. Heux, L.; Dinand, E.; Vignon, M. R. Carbohydr. Polym. 1999, 40, 115–124.
4. Katharine, S. Nature 2011, 474, S12–S14.
9
5. Siro, I.; Plackett, D. Cellulose 2010, 17, 459–494.
6. Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.;
Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.;
Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.;
Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T. J. Mater. Sci.
2010, 45, 1–13.
7. John, M. J.; Thomas, S. Carbohydr. Polym. 2008, 71, 343–364.
8. Lin, F. C.; Brown, R. M., Jr.; Cooper, J. B.; Delmer, D. P. Science 1985, 230, 822–
825.
9. Bureau, T. E.; Brown, R. M., Jr. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 6985–6989.
10. Okuda, K.; Li, L.; Kudlicka, K.; Brown, R. M., Jr. Plant Physiol. 1993, 101, 1131–
1142.
11. Lai-Kee-Him, J.; Chanzy, H.; Müller, M.; Putaux, J.-L.; Imai, T.; Bulone, V. J. Biol.
Chem. 2002, 277, 36931–36939.
4.3. Enzyme assay
EG I activity was colorimetrically determined with Lac-b-pNP as
a substrate according to our previous method.37 A mixture contain-
ing 25
(pH 4.0, 50
(25 L) was incubated in a 96-well microplate for 20 min at
l
L of 10 mM substrates Lac-b-pNP in 50 mM acetate buffer
l
L volume) and an appropriate amount of enzyme
l
40 °C. One-tenth of the reaction mixture was removed at 5-min
intervals and immediately transferred to a microplate containing
190 lL of 1 M Na2CO3 to stop the reaction. The amount of pNP lib-
12. Colombani, A.; Djerbi, S.; Bessueille, L.; Blomqvist, K.; Ohlsson, A.; Berglund, T.;
Teeri, T. T.; Bulone, V. Cellulose 2004, 11, 313–327.
13. Cifuentes, C.; Bulone, V.; Emons, A. M. C. J. Integr. Plant Biol. 2010, 52, 221–233.
14. Sugiyama, J.; Persson, J.; Chanzi, H. Macromolecules 1991, 24, 2461–2466.
15. Saxena, I. M.; Brrown, R. M. J. Ann. Bot. 2005, 96, 9–21.
16. Rånby, B. G. Acta Chem. Scand. 1952, 6, 101–115.
erated was determined by measuring the absorbance at 405 nm
using a microplate reader (Ultrospec Visible Plate Reader II 96,
GE Healthcare Bio-Sciences, Little Chalfont, UK). One unit of activ-
ity was defined as the amount of enzyme releasing 1
per min.
l
mol of pNP
17. Sisson, W. Science 1938, 87, 350.
18. Kadokawa, J. Chem. Rev. 2011, 111, 4308–4345.
19. Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S. J. Am. Chem. Soc. 1991, 113,
3079–3084.
20. Fort, S.; Boyer, V.; Gerffe, L.; Davies, G. J.; Moroz, O.; Christiansen, L.; Schülein,
M.; Cottaz, S.; Driguez, H. J. Am. Chem. Soc. 2000, 122, 5429–5437.
21. Lee, J. H.; Brown, R. M., Jr.; Kuga, S.; Shoda, S.; Kobayashi, S. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 7425–7429.
22. Egusa, S.; Kitaoka, T.; Goto, M.; Wariishi, H. Angew. Chem., Int. Ed. 2007, 46,
2063–2065.
4.4. Analysis of EG I-mediated transglycosylation
Analysis of the time course of transglycosylation and character-
ization of the resulting products was performed by four different
methods as outlined below.
23. Egusa, S.; Kitaoka, T.; Igarashi, K.; Samejima, M.; Goto, M.; Wariishi, H. J. Mol.
Catal. B: Enzym. 2010, 67, 225–230.
24. Hiraishi, M.; Igarashi, K.; Kimura, S.; Wada, M.; Kitaoka, M.; Samejima, M.
Carbohydr. Res. 2009, 344, 2468–2473.
25. Kono, H.; Numata, Y.; Erata, T.; Takai, M. Polymer 2004, 45, 2843–2852.
26. Kono, H.; Numata, Y. Polymer 2004, 45, 4541–4547.
27. Malm, E.; Bulone, V.; Wickholm, K.; Larsson, P. T.; Iversen, T. Carbohydr. Res.
2010, 345, 97–100.
28. Yasutake, N.; Totani, K.; Harada, Y.; Haraguchi, S.; Murata, T.; Usui, T. Biosci.,
Biotechnol., Biochem. 2003, 67, 1530–1536.
29. Yasutake, N.; Totani, K.; Harada, Y.; Haraguchi, S.; Murata, T.; Usui, T. Biochem.
Biophys. Acta 2003, 1620, 252–258.
30. Biely, P.; Vršanska˘, M.; Claeyssens, M. Eur. J. Biochem. 1991, 200, 157–163.
31. Kleywegt, G. J.; Zou, J. Y.; Divne, C.; Davies, G. J.; Sinning, I.; Stâhlberg, J.;
Reinikainen, T.; Srisodsuk, M.; Teeri, T. T.; Jones, T. A. J. Mol. Biol. 1997, 272,
383–397.
32. Nishiyama, Y.; Langan, P.; Chanzy, H. J. Am. Chem. Soc. 2002, 124, 9074–9082.
33. Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P. J. Am. Chem. Soc. 2003, 125,
14300–14306.
34. Wada, M.; Chanzy, H.; Nishiyama, Y.; Langan, P. Macromolecules 2004, 37,
8548–8555.
35. Langan, P.; Sukumar, N.; Nishiyama, Y.; Chanzy, H. Cellulose 2005, 12, 551–562.
36. Dinand, E.; Vignon, M.; Chanzy, H.; Heux, L. Cellulose 2002, 9, 7–18.
37. Ogata, M.; Kameshima, Y.; Hattori, T.; Michishita, K.; Suzuki, T.; Kawagishi, H.;
Totani, K.; Hiratake, J.; Usui, T. Carbohydr. Res. 2010, 345, 2623–2629.
4.4.1. HPAEC-PAD
To a solution (200
(100 mg) in 50 mM acetate buffer (pH 4.0) was added 200
zyme solution (0.2 U) in the same buffer. The mixture was then
incubated at 50 °C. The amount of transglycosylation product as
a function of time was examined on the 0.1 mL scale. Aliquots of
5 lL were taken and mixed with 145 lL of H2O. The reaction was
then immediately quenched by heating in a boiling water bath
l
L) of cellobiose (110 mg) or cellotriose
L of en-
l
for 15 min and the mixture was clarified by filtration through a
0.45 lm filter unit. The reaction mixture (water soluble part) was
subsequently analyzed by HPAEC-PAD.
4.4.2. MALDI-TOF mass spectrometry, solid-state 13C NMR
spectroscopy, and XRD analysis
Synthetic artificial cellulose was directly analyzed by MALDI-
TOF mass spectrometry, solid-state 13C NMR spectroscopy, and
XRD analysis without derivatization.