Experiments are being conducted in our lab regarding production of synthetic cellulose. Cellulose is natures most abundant macromolecule. It is a polymer composed of molecules of glucose strung together by b1-4 linkages. This summer, we continued work in imaging the enzyme that produces cellulose in the bacterial strain Acetobacter xylinum. Broth cultures of Acetobacter xylinum XY201 were grown shaking at 28°C for three days then crushed in a French press. A drop of the membrane preparation obtained from the press was incubated with a drop of a UDP glucose/activator solution on a formvar coated TEM grid. After incubation, the grids were negatively stained with UA. To prove cellulose was being produced a cellobiohydrolase/gold complex was added to the grid for three minutes after incubation.
Images were obtained of the enzyme cellulose synthase. As seen in figure 1, it has a horeshoe structure. Enzymes were found associated with cellulose microfibrils on the grid indicating they produced cellulose on the grid or were attracted to cellulose contaminants already present on the grid.
The enzyme degraded rapidly when exposed to the electron beam and was difficult to observe. Measurements of several molecules indicate that the enzyme is between 5nm and 10nm in diameter. Figure 2 shows cellulose synthase particles near a microfibril of cellulose labeled with a cellobiohydrolase/gold complex.
Cellulose synthase is an enzyme with a diameter between 5 and 10nm. It is found most often associated with cellulose microfibrils even when cellulose is not being produced (i.e. no monomer has been added) indicating an affinity for cellulose as well as glucose. More structural studies of this molecule need to be undertaken. Three dimensional imaging was attempted, but the molecules degrade before a series of tilted images can be obtained. These 3D experiments should be continued to gain a more complete understanding of the structure of the enzyme. More accurate measurements can also be obtained when more molecules are observed and measured.
We would like to thank Dr. Krystyna Kudlika, Dr. Inder Saxena, and Lilian Liao for their aid with the project. This work is supported in part by Welch Foundation Grant F1217 to R.M.B. and the Office of Naval Research grant N00014-95-1-0933.
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