Binding Modules Alter the Activity of Chimeric Cellulases: Effects of Biomass Pretreatment and Enzyme Source

作者：Kim Tae Wan; Chokhawala Harshal A; Nadler Dana; Blanch Harvey W; Clark Douglas S^*

来源：Biotechnology and Bioengineering, 2010, 107(4): 601-611.

DOI：10.1002/bit.22856

• carbohydrate binding module
• cell free protein synthesis
• biofuels
• cellulase
• pretreatment

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 mariner. 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 mu g/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.

• 出版日期2010-11-1