Synthetic multi-component enzyme mixtures for deconstruction of lignocellulosic biomass Goutami Banerjee, Suzana Car, John S. Scott-Craig, Melissa S. Borrusch, Mareike Bongers 1 , Jonathan D. Walton * Department of Energy Great Lakes Bioenergy Research Center and Department of Energy Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, USA article info Article history: Received 27 April 2010 Received in revised form 7 July 2010 Accepted 8 July 2010 Available online 13 July 2010 Keywords: Lignocellulose Trichoderma reesei Pichia pastoris Ammonia-fiber expansion Cellulase abstract A high throughput enzyme assay platform, called GENPLAT, was used to guide the development of an optimized mixture of individual purified enzymes from ten ‘‘accessory” and six ‘‘core” enzymes. Enzyme mixtures were optimized for release of Glu, Xyl, or a combination of the two from corn stover pretreated by ammonia-fiber expansion (AFEX). Assay conditions were a fixed enzyme loading of 15 mg/g glucan, 48 h digestion, and 50 °C. Five of the ten tested accessory proteins enhanced Glu or Xyl yield compared to the core set alone, and five did not. An 11-component mixture containing the core set and five acces- sory enzymes optimized for Glu released 52.1% of the available Glu, compared to 38.5% with the core set alone. A mixture optimized for Xyl released 39.9% of the Xyl, compared to 26.4% with the core set alone. We predict that there is still considerable opportunity for further improvement of synthetic mixtures. Furthermore, the strategy described here is applicable to the development of more efficient enzyme cocktails for any pretreatment/biomass combination and for detecting enzymes that make a heretofore unrecognized contribution to lignocellulose deconstruction. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction An essential step in the conversion of lignocellulosic biomass to ethanol or other liquid transportation fuels is the enzyme-cata- lyzed depolymerization of polysaccharides. However, the high cost of enzymes hinders the development of a viable lignocellulosic ethanol industry (Lynd et al., 2008). One approach to reduce the cost of enzymes is improving their specific activities, i.e., increasing the amount of Glu, Xyl, and other fermentable sugars released per unit of protein. Current commercial preparations, mainly derived from fermen- tation of the filamentous fungus Trichoderma reesei, contain more than 80 proteins (Nagendran et al., 2009; Banerjee et al., 2010a). Beyond the major cellulases and xylanases, little is known about the involvement of most of these proteins in biomass conversion. Reducing the levels of nonessential enzymes and enhancing the relative proportions of the critical enzymes are therefore potential strategies to increase the specific activities, and thereby lower the cost, of biomass-converting enzymes. One strategy to the rational improvement of enzyme cocktails is to construct enzyme mixtures de novo. This would allow complete control over the individual components and their relative propor- tions. In a previous paper, we described an experimental platform, named GENPLAT (for Great Lakes Bioenergy Research Center [GLBRC] Enzyme Platform), that can be used to construct and test synthetic enzyme mixtures (Banerjee et al., 2010b). Critical attri- butes of GENPLAT include the production of individual enzymes free of contaminating activities in a heterologous host such as Pichia pastoris, a liquid handling robot that can perform thousands of pipeting steps in a 96-well format, appropriate experimental de- sign, and automated Glu and Xyl assays. We demonstrated the capability of GENPLAT to develop an optimized mixture of six ‘‘core” enzymes, defined as cellobiohydrolase 1 (CBH1, GenBank CAA49596), cellobiohydrolase 2 (CBH2, P09787), endo-b1, 4-glucanase 1 (EG1, AAA34212), b-glucosidase (BG, AAA18473), endo-b1,4-xylanase 3 (EX3, BAA89465), and b-xylosidase (BX, CAA93248) (Banerjee et al., 2010b). In this paper we show that the hydrolytic efficiency of the core set can be significantly im- proved by the addition of other ‘‘accessory” enzymes. Of the ten tested accessory enzymes, five caused a significant increase in Glu and/or Xyl yield from AFEX-pretreated corn stover. The specific activity of an optimized 11-component mixture equalled commer- cial enzymes (Accellerase 1000 or Spezyme CP) for Glu yield and 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.028 Abbreviations: Glu, glucose; Xyl, xylose; GH, glycosyl hydrolase; CBH, cellobio- hydrolase; BG, b-glucosidase; EG, endo-b-1,4-glucanase; BX, b-xylosidase; EX, endo-b-1,4-xylanase; a-Glr, a-glucuronidase; Abf, arabinosidase; MW, molecular weight; AFEX, ammonia-fiber-expansion pretreatment; pNP, para-nitrophenyl; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; GC, gas chromatography. * Corresponding author. Tel.: +517 353 4885; fax: +517 353 9168. E-mail address: walton@msu.edu (J.D. Walton). 1 Present address: ETH Zürich, Rämistrasse 101, 8092 Zürich, Switzerland. Bioresource Technology 101 (2010) 9097–9105 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech