title: Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627.
abstract: Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein(-1) extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 degrees C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 degrees C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.
authors.affiliation: Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Sun
authors.forename: Jay J
authors.initials: JJ
authors.lastname: Cheng
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Skory
authors.forename: William S
authors.initials: WS
authors.lastname: Adney
authors.forename: Steven R
authors.initials: SR
authors.lastname: Thomas
authors.forename: Brent
authors.initials: B
authors.lastname: Tisserat
authors.forename: Yufuko
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Yuri T
authors.initials: YT
authors.lastname: Yamamoto
mesh_terms: D002021:Buffers; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D030821:Plants, Genetically Modified; D013696:Temperature
chemical_list: D002021:Buffers; D002480:Cellulase