title: Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass.
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 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.
authors: Biomass Conversion Research Lab, Department of Chemical Engineering and Materials Science, Michigan State University, University Corporate Research Park, 3900 Collins Road, Lansing, Michigan 48910, USA. gaodahai@msu.edu
authors: Dahai
authors: D
authors: Gao
authors: Shishir P S
authors: SP
authors: Chundawat
authors: Nirmal
authors: N
authors: Uppugundla
authors: Venkatesh
authors: V
authors: Balan
authors: Bruce E
authors: BE
authors: Dale
mesh_terms: D000143:Acids / Q000737:chemistry; D000641:Ammonia / Q000737:chemistry; D018533:Biomass; D044602:Cellulases / Q000378:metabolism*; D008031:Lignin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D014242:Trichoderma / Q000201:enzymology*; D003313:Zea mays / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000143:Acids; C036909:lignocellulose; D000641:Ammonia; D008031:Lignin; D044602:Cellulases