Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source.
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.
Kim
Tae-Wan
TW
Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA.
Chokhawala
Harshal A
HA
Nadler
Dana C
DC
Nadler
Dana
D
Blanch
Harvey W
HW
Clark
Douglas S
DS
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
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D044602:Cellulases