title: Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source.
abstract: Improving the catalytic activity of cellulases requires screening variants against solid substrates. Expressing cellulases in microbial hosts is time-consuming, can be cellulase specific, and often leads to inactive forms and/or low yields. These limitations have been obstacles for improving cellulases in a high-throughput manner. We have developed a cell-free expression system and used it to express 54 chimeric bacterial and archaeal endoglucanases (EGs), with and without cellulose binding modules (CBMs) at either the N- or C-terminus, in active enzyme yields of 100-350 µg/mL. The platform was employed to systematically study the role of CBMs in cellulose hydrolysis toward a variety of natural and pretreated solid substrates, including ionic-liquid pretreated Miscanthus and AFEX-pretreated corn stover. Adding a CBM generally increased activity against crystalline Avicel, whereas for pretreated substrates the effect of CBM addition depended on the source of cellulase. The cell-free expression platform can thus provide insights into cellulase structure-function relationships for any substrate, and constitutes a powerful discovery tool for evaluating or engineering cellulolytic enzymes for biofuels production.
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA.
authors.forename: Tae-Wan
authors.initials: TW
authors.lastname: Kim
authors.forename: Harshal A
authors.initials: HA
authors.lastname: Chokhawala
authors.forename: Dana C
authors.initials: DC
authors.lastname: Nadler
authors.forename: Dana
authors.initials: D
authors.lastname: Nadler
authors.forename: Harvey W
authors.initials: HW
authors.lastname: Blanch
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018533:Biomass; D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D015870:Gene Expression; D006109:Poaceae / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D044602:Cellulases