title: Engineering ionic liquid-tolerant cellulases for biofuels production.
abstract: Dissolution of lignocellulosic biomass in certain ionic liquids (ILs) can provide an effective pretreatment prior to enzymatic saccharification of cellulose for biofuels production. Toward the goal of combining pretreatment and enzymatic hydrolysis, we evolved enzyme variants of Talaromyces emersonii Cel7A to be more active and stable than wild-type T. emersonii Cel7A or Trichoderma reesei Cel7A in aqueous-IL solutions (up to 43% (w/w) 1,3-dimethylimdazolium dimethylphosphate and 20% (w/w) 1-ethyl-3-methylimidazolium acetate). In general, greater enzyme stability in buffer at elevated temperature corresponded to greater stability in aqueous-ILs. Post-translational modification of the N-terminal glutamine residue to pyroglutamate via glutaminyl cyclase enhanced the stability of T. emersonii Cel7A and variants. Differential scanning calorimetry revealed an increase in melting temperature of 1.9-3.9°C for the variant 1M10 over the wild-type T. emersonii Cel7A in aqueous buffer and in an IL-aqueous mixture. We observed this increase both with and without glutaminyl cyclase treatment of the enzymes.
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA Graduate Group of Comparative Biochemistry at University of California, Berkeley, CA, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wolski
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Craig M
authors.initials: CM
authors.lastname: Dana
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA clark@berkeley.edu blanch@berkeley.edu.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA clark@berkeley.edu blanch@berkeley.edu.
authors.forename: Harvey W
authors.initials: HW
authors.lastname: Blanch
mesh_terms: D019881:Aminoacyltransferases; D056804:Biofuels; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry; D052578:Ionic Liquids / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D032901:Talaromyces / Q000201:enzymology / Q000235:genetics
chemical_list: D056804:Biofuels; D005656:Fungal Proteins; D007093:Imidazoles; D052578:Ionic Liquids; D002482:Cellulose; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D044602:Cellulases
keywords: biased clique; cellulase; directed evolution; ionic liquid; shuffling