Prescott's Microbiology 8th Edition by Joanne Willey, Linda Sherwood, Christopher J. Woolverton, Lansing Prescott, John Harley, Donald Klein

Exercise 4

The gram-positive bacterium Bacillus thuringiensissubsp. is-raelensisis one of the many bacteria that accumulates PHB. To harvest the energy stored in this inclusion body, the cell must dismantle the PHB. This is accomplished by depo-lymerizing PHB into smaller 3-hydroxybutyrate molecules. The enzyme that catalyzes PHB depolymerization in B. thuringiensissubsp. israelensiswas originally thought to degrade a different lipid that is not associated with PHB granules. How do you think it was shown that the enzyme in question was specific for PHB? Do you think the cell produces this enzyme all the time? Why or why not?

Step 1 of 2

An enzyme that breaks down poly-hydroxybutyrate (PHB) into smaller 3-hydroxybutyrate molecules is encoded by the gene phaZ. PHB is a type of carbon storage molecule that lumps into inclusion bodies in the cell. When external carbon sources are limited, the cell can utilize the PHB inclusion bodies for energy.

To show that phaZ was specific for PHB, the enzyme could be over-expressed and purified. The purified enzyme can then be mixed with PHB to see whether it can break it down to 3-hydroxybutyrate. Other similar carbon storage molecules, like glycogen, can be used to demonstrate that PhaZ is specific for PHB. In the paper, Tseng et al used a turbidometric assay and observed a decrease in absorption at 650nm to determine whether PhaZ was breaking down PHB. The authors also determined that PhaZ was able to cleave ester bonds that joined the PHB molecules.