Observing Thermobifida fusca cellulase binding to pretreated wood particles using time-lapse confocal laser scanning microscopy

Paul Zhu, Jose M. Moran-Mirabal, Jeremy S. Luterbacher, Larry P. Walker, Harold G. Craighead

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

We report on studies of Thermobifida fusca cellulases Cel5A, Cel6B and Cel9A binding to pretreated wood particles using Confocal Laser Scanning Microscopy (CLSM). Hydro-thermal pretreated wood particles were immobilized on borosilicate substrates before fluorescently-labeled cellulase solutions at various concentrations were added. Time-lapse CLSM revealed that cellulases Cel5A, Cel6B and Cel9A quickly bound to certain areas of wood particles, slowly diffused into and adsorbed to less accessible areas, but showed little affinity for other areas of the wood. Cellulase-to-substrate association constants were estimated using a transient enzyme binding kinetics model, and were found to be in agreement with published values. In order to accurately account for the fluorescence signal of labeled enzyme mixed with wood autofluorescence, we also developed a spectral deconvolution method to separate signals from multiple fluorochromes.

Original languageEnglish (US)
Pages (from-to)749-758
Number of pages10
JournalCellulose
Volume18
Issue number3
DOIs
StatePublished - Jun 2011

Keywords

  • Cellulose
  • CLSM
  • Enzyme binding kinetics
  • Transient model

ASJC Scopus subject areas

  • Polymers and Plastics

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