Tae-Wan Kim and Harshal A. Chokhawala contributed equally to this work.
Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source†
Article first published online: 9 JUL 2010
Copyright © 2010 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 107, Issue 4, pages 601–611, 1 November 2010
How to Cite
Kim, T.-W., Chokhawala, H. A., Nadler, D., Blanch, H. W. and Clark, D. S. (2010), Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source. Biotechnol. Bioeng., 107: 601–611. doi: 10.1002/bit.22856
- Issue published online: 13 SEP 2010
- Article first published online: 9 JUL 2010
- Manuscript Accepted: 21 JUN 2010
- Manuscript Revised: 15 JUN 2010
- Manuscript Received: 9 APR 2010
- Energy Biosciences Institute
- carbohydrate binding module;
- cell-free protein synthesis;
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. Biotechnol. Bioeng. 2010;107:601–611. © 2010 Wiley Periodicals, Inc.