|
Poster Presentation 6-42
Expression of Endoglucanase E1 in Transgenic Duckweed Lemna minor
Ye Sun1, William S. Adney2, Ben A. Bergmann3, Jiayang Cheng1, Stephen R. Decker2, Shelby Freer4, Michael E. Himmel2, Yufuko Nishimura3, Christopher D. Skory4, Anne-Marie Stomp3, Steven Thomas2, Brent Tisserat4 and Yuri T. Yamamoto3
1Department of Biological and Agricultural Engineering North Carolina State University Weaver Labs 277, Raleigh, NC 27695-7625
2National Bioenergy Center National Renewable Energy Laboratory
3Department of Forestry, North Carolina State University
4National Center for Agricultural Utilization Research USDA - Agricultural Research Service Fermentation Biotechnology Research
Telephone: (919) 515-6744; Fax: (919) 515-7760; E-mail: ysun@eos.ncsu.edu
Transgenic duckweed (Lemna minor) that expresses Acidothermus cellulolyticus E1 endoglucanase was generated using Agrobacterium-mediated transformation. Out of 15 independent transgenic lines, one line with the highest CMC-degrading activity was selected for further studies. The 2-week-old transgenic duckweed fronds cultured in SH medium were ground in sodium citrate buffer (pH 4.8), and the expression of endoglucanase E1 in the supernatant was examined using immunoblotting and enzyme assays. The E1 produced in transgenic duckweed was active and the expression level was 0.24% of total soluble protein. The recombinant E1 protein was thermostable and had higher activity at 70°C compared to room temperature. The average E1 activity at 70°C in duckweed extracts was 0.21 mmol min-1 g-1 fresh duckweed or 0.17 mmol min-1 mg-1 soluble protein. A 5-min heat treatment of the extract at 65°C effectively removed most other proteins without reducing the enzyme activity. This may be useful for downstream processing.
|