Binding characteristics of <i>Trichoderma reesei</i> cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

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Binding characteristics of Trichoderma reesei  cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

Publication Type:

Journal Article

Source:

Biotechnology and Bioengineering, Wiley Subscription Services, Inc., A Wiley Company, Volume 108, Number 8, p.1788-1800 (2011)

ISBN:

1097-0290

Keywords:

Deconstruction

Abstract:

Abstract Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4°C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme–substrate combinations over the protein loading range tested (i.e., 25–450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites Ebm (mg/g glucan) and association constant Ka (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed. Biotechnol. Bioeng. 2011; 108:1788–1800. © 2011 Wiley Periodicals, Inc.