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Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute‐acid pretreated lignocellulosic biomass
Author(s) -
Gao Dahai,
Chundawat Shishir P.S.,
Uppugundla Nirmal,
Balan Venkatesh,
Dale Bruce E.
Publication year - 2011
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.23140
Subject(s) - cellulase , chemistry , trichoderma reesei , corn stover , lignocellulosic biomass , lignin , glucan , hydrolysis , chromatography , biochemistry , organic chemistry
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 E bm (mg/g glucan) and association constant K a (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.