Sunday, August 12, 2012
Columbia Hall, Terrace Level (Washington Hilton)
Fengkun Du, Oscar Galagarza, Yaoyao Liu, Trudy A. Tucker and George E. Pierce, Department of Biology, Georgia State University, Atlanta, GA
Nitrile hydratase (NHase) is a cobalt or iron (Co/Fe) containing enzyme that transforms nitriles to the corresponding amide. Both
Rhodococcus rhodochrous DAP 96253 and DAP 96622 can be induced with multiple inducers such as urea, Co, Fe and nickel (Ni). The effect of various metals and urea on NHase activity in
R. rhodochrous DAP 96253 and DAP 96622 was investigated. The data showed that when rhodococcal
cells were multiply induced with Co and urea, both
R. rhodochrous DAP 96253 and DAP 96622 strains expressed the highest level of nitrile hydratase activity (~200 units/mg-cdw for DAP 96253 and ~50units/mg-cdw for DAP 96622). The second highest nitrile hydratase activity was achieved when cells were induced with Co only (~7.4 units/min·mg-cdw for DAP 96253 and ~20 units/mg-cdw for DAP 96622).
Protein profiles of R. rhodochrous DAP 96253 and DAP 96622 grown on Co and urea were examined using gel electrophoresis and analyzed with ImageQuant TL. The NHase bands (both α and β subunits) accounted for 59.8% of the total cytosolic protein in R. rhodochrous DAP 96253. Protein profiles of the two strains induced with Co and urea were significantly different on SDS PAGE. Differences were also noted with ammonium sulfate precipitation. Most NHase from DAP 96253 precipitated between 45-70% ammonium sulfate saturation, while most NHase from DAP 96622 precipitated between 35-45% ammonium sulfate saturation.
Preliminary NHase purification from DAP 96253 showed that the NHase(s) may form protein complex(es) with other proteins. NHase activity after DEAE anion exchange was approximately 1500 units/mg-protein.
