title: Detecting cellulase penetration into corn stover cell walls by immuno-electron microscopy.
abstract: In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild-, moderate- and high-severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme- and polymer-specific antibodies. Low severity dilute-acid pretreatment (20 min at 100 degrees C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120 degrees C) allowed the enzymes to penetrate approximately 20% of the cell wall, and the high severity (20 min pretreatment at 150 degrees C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute-acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high-resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes.
authors.affiliation: Chemical and Biosciences Center, Golden, Colorado 80401, USA. bryon_donohoe@nrel.gov
authors.forename: Bryon S
authors.initials: BS
authors.lastname: Donohoe
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Selig
authors.forename: Sridhar
authors.initials: S
authors.lastname: Viamajala
authors.forename: Todd B
authors.initials: TB
authors.lastname: Vinzant
authors.forename: William S
authors.initials: WS
authors.lastname: Adney
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
mesh_terms: D002424:Caustics / Q000494:pharmacology; D002473:Cell Wall / Q000737:chemistry* / Q000187:drug effects; D002480:Cellulase / Q000032:analysis*; D008031:Lignin / Q000032:analysis; D016253:Microscopy, Immunoelectron / Q000379:methods; D013464:Sulfuric Acids / Q000494:pharmacology; D014990:Xylans / Q000032:analysis; D003313:Zea mays / Q000737:chemistry* / Q000187:drug effects
chemical_list: D002424:Caustics; D013464:Sulfuric Acids; D014990:Xylans; D008031:Lignin; D002480:Cellulase; C033158:sulfuric acid