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 PARENT SESSION
3D Biodegradation and biotransformation: routes and pathways
9:00 AM to 7:00 PM, Monday, 07 May 2001
(M/EH078) Cellulose Degradation: Mechanistic Studies of Thermobifida fusca Endocellulase Cel6A.
Salminen, Outi1,2, Wilson, David1, 1 2
ABSTRACT- Cellulases are enzymes that degrade cellulose, the most abundant organic raw material found in nature. The physically complex structure of cellulose poses a difficult task to cellulose degrading microorganisms in nature. Efficient microbiological degradation requires synergistic action of a set of mechanistically related enzymes. Cel6A (previously named E2) is one of six cellulose degrading hydrolases that have been isolated from the thermophilic soil bacterium Thermobifida fusca (previously Thermomonospora fusca). Cel6A is a 43 kD protein with two domains, a 30 kD N-terminal catalytic domain (cd) and a C-terminal cellulose binding domain connected by a flexible linker. The structure of the Cel6A cd has been solved by X-ray crystallography to 1 resolution. An active site framing loop, in which a conservative aspartic acid residue D79 resides, appears open in the crystal structure holding D79 11 away from the site of substrate cleavage. PCR site directed mutagenesis studies have, however, shown that D79 mutants have a significant loss of activity suggesting that the loop in which D79 is located may move during catalysis bringing this residue into close proximity to the bond being cleaved. Evidence for such loop mobility has been sought by multidimensional NMR, which offers the ability to look at proteins in their natural environment thereby allowing conformational changes to be visualized. To meet the requirements set by isotopic labeling a recombinant Cel6A expression system in Escherichia coli was sought. Study of several E. coli expression systems, and their optimization for recombinant protein overproduction, resulted in a system that produces active, folded, and labeled recombinant Cel6A protein efficiently and cheaply. Due to its thermostability and broad pH optimum, Cel6A cellulase has promise for economical use in environmental biotechnology including cellulosic waste conversion into useful chemicals and energy.
Key words: cellulase, hydrolase, nmr
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