title: New perspective on glycoside hydrolase binding to lignin from pretreated corn stover.
abstract: BACKGROUND
  Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.
  
  
  RESULTS
  In this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4-15 % depending on lignin concentration.
  
  
  CONCLUSIONS
  We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes' affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Yarbrough
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Mittal
authors.affiliation: Applied Chemicals and Materials Division, NIST, Boulder, CO 80305 USA.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mansfield
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Taylor
authors.affiliation: Department of Veterans Affairs, Boise, ID 83702 USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Hobdey
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Deanne W
authors.initials: DW
authors.lastname: Sammond
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Yannick J
authors.initials: YJ
authors.lastname: Bomble
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Crowley
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Decker
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Todd B
authors.initials: TB
authors.lastname: Vinzant
keywords: Biomass; Cellulase; Enzyme binding; Glycoside hydrolase; Lignin; Pretreatment