title: Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.
abstract: BACKGROUND
  Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.
  
  
  RESULTS
  In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.
  
  
  CONCLUSIONS
  Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 USA ; Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, 164 Food Safety and Toxicology Building, East Lansing, MI 48824 USA ; Biomass Conversion Research Lab (BCRL), MBI Building, 3900 Collins Road, East Lansing, MI 48910 USA.
authors.forename: Dahai
authors.initials: D
authors.lastname: Gao
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 USA.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Haarmeyer
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 USA ; Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, 164 Food Safety and Toxicology Building, East Lansing, MI 48824 USA ; Biomass Conversion Research Lab (BCRL), MBI Building, 3900 Collins Road, East Lansing, MI 48910 USA.
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Balan
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 USA ; Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824 USA.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Whitehead
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 USA ; Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, 164 Food Safety and Toxicology Building, East Lansing, MI 48824 USA ; Biomass Conversion Research Lab (BCRL), MBI Building, 3900 Collins Road, East Lansing, MI 48910 USA.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Dale
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 USA ; Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, 164 Food Safety and Toxicology Building, East Lansing, MI 48824 USA ; Biomass Conversion Research Lab (BCRL), MBI Building, 3900 Collins Road, East Lansing, MI 48910 USA ; Department of Chemical & Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Room C-150A, Piscataway, NJ 08854 USA.
authors.forename: Shishir Ps
authors.initials: SP
authors.lastname: Chundawat
keywords: Cellulase adsorption; Cellulosic biofuels; Enzymatic saccharification; Lignin; Non-specific enzyme binding