Poster Presentation 1B-49

An Automated Multidimensional Chromatographic System for Preparative-scale Purification of the Major Enzyme Components from a Trichoderma reesei Cellulase Complex

Alex Berlin,* Neil Gilkes, David Gregg, Renata Bura, Sarah Leung, Arwa Kurabi, Maobing Tu, Dan Xie, Douglas Kilburn and Jack Saddler

Forest Products Biotechnology Group

Department of Wood Science

University of British Columbia

2424 Main Mall

Vancouver, B.C. Canada  V6T 1Z4

Phone: (604)822-5936

Fax: (604)822-9159

E-mail: alberlin@interchange.ubc.ca

Cellulolytic microorganisms such as Trichoderma reesei secrete a complex mixture of hydrolytic enzymes. Investigation of cellulose hydrolysis reaction mechanisms often requires the purification of large amounts of individual enzyme components and typically involves elaborate and time-consuming chromatographic procedures. As part of our overall research effort into the hydrolysis of lignocellulosics for bioethanol production we have designed and implemented a chromatographic scheme for isolation of the four major components in the Trichoderma reesei cellulase complex. The separation system uses six sequential ion-exchange and gel-filtration columns and allows automated preparation of the target enzymes in large-scale quantities (≥100 mg) within a few hours. Enzyme identity can be confirmed by peptide sequencing and MALDI-TOF mass spectrometry. Unlike current genetic techniques for high-level fungal enzyme production, this approach provides enzymes in which native post-translational modifications are preserved.