Analytical and Bioanalytical Chemistry

, Volume 398, Issue 3, pp 1257–1262 | Cite as

A novel FRET approach for in situ investigation of cellulase–cellulose interaction

Technical Note


A novel real-time in situ detection method for the investigation of cellulase–cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)–cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase–cellulose study using cellulosic fiber.


FRET Cellulase Cellulose Carboxymethyl cellulose (CMC) 



The authors would like to gratefully acknowledge the financial support from Department of Energy Office of Biological and Environmental Research through the BioEnergy Science Center (DE-AC05-00OR22725).


  1. 1.
    Lynd LR, Laser MS, Bransby D, Dale BE, Davison B, Hamiton R, Himmel M, Keller M, McMillan JD, Sheehan J, Wyman CE (2008) Nat Biotechnol 26:169–172CrossRefGoogle Scholar
  2. 2.
    Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Gairney J, Eckert CA, Frederick WJ Jr, Hallet JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinske T (2006) Science 311:484–489CrossRefGoogle Scholar
  3. 3.
    Wilson DB (2008) In: Himmel ME (ed) Biomass recalcitrance. Blackwell, Oxford, pp 374–392Google Scholar
  4. 4.
    Velleste R, Teugjas H, Väljamäe P (2010) Cellulose 17(1):125–138CrossRefGoogle Scholar
  5. 5.
    Aronson NN, Halloran BA, Alexyev MF, Amable L, Madura JD, Pasupulati L, Worth C, van Roey P (2003) Biochem J 376:87–95CrossRefGoogle Scholar
  6. 6.
    Rudsander UJ, Sandstrom C, Piens K, Master ER, Wilson DB, Brumer H III, Kenne L, Teeri TT (2008) Biochemistry 47(18):5235–5241CrossRefGoogle Scholar
  7. 7.
    Moran-Mirabal JM, Santhanam N, Corgie SC, Craighead HG, Walker LP (2008) Biotechnol Bioeng 101(6):1129–1141CrossRefGoogle Scholar
  8. 8.
    Hildén L, Väljamäe P, Johansson G (2005) J Biotechnol 118(4):386–397CrossRefGoogle Scholar
  9. 9.
    Boisset C, Fraschini C, Schülein M, Henrissat B, Chanzy H (2000) Appl Environ Microbiol 66:1444–1452CrossRefGoogle Scholar
  10. 10.
    Turon X, Rojas OJ, Deinhammer RS (2008) Langmuir 24(8):3880–3887CrossRefGoogle Scholar
  11. 11.
    Enebro J, Momcilovic D, Siika-aho M, Sigbritt K (2009) Cellulose 16:271–280CrossRefGoogle Scholar
  12. 12.
    Gadella TWJ (2009) FRET and flim techniques, 1st edn. Elsevier, Oxford, UKGoogle Scholar
  13. 13.
    Karlsson J, Saloheimo M, Siika-Aho M, Tenkanen M, Penttila M, Tjerneld F (2001) Eur J Biochem 268(24):6498–6507CrossRefGoogle Scholar
  14. 14.
    Miller GL (1959) Anal Chem 31:426–428CrossRefGoogle Scholar
  15. 15.
    Wirick MG (1968) J Polym Sci A Polym Chem 6(7):1965–1974CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.BioEnergy Science Center, Institute of Paper Science and Technology, School of Chemistry and BiochemistryGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Department of Biomedical EngineeringEmory UniversityAtlantaUSA

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