Isolation, Partial Purification and Characterization of Molybdenum-Reducing Enzymes from an Antartica Bacterium (Gamma-Proteobacterium Strain Dr.Y1)
Ahmad, Siti Aqlima (2006) Isolation, Partial Purification and Characterization of Molybdenum-Reducing Enzymes from an Antartica Bacterium (Gamma-Proteobacterium Strain Dr.Y1). Masters thesis, Universiti Putra Malaysia.
Bacterial Isolate no. J7A was isolated from Jubany Station, Antarctica and it has the capability to reduce the heavy metal molybdenum (molybdate) to molybdenum blue in a solid medium agar, pH 7 at 10°C, after for 4 days of incubation. Isolate J7A was identified as Gram-negative and gamma-Proteobacterium Strain Dr.Y1 through moleculare phylogenetics analysis of the sequenced 16s rRNA gene. The optimization studies were carried out to optimize the production of molybdenum blue. The combination of 1% (w/v) glucose, 0.3% (w/v) ammonium sulphate, 0.1% (w/v) of yeast extract, 30mM molybdate, and low phosphate medium at pH 7 give the optimum production of Molybdenum blue. Partial purification and characterization were conducted on molybdenum reducing enzyme with anion exchange chromatography using Macro-Prep High-QTM column and gel filtration chromatography using Agilent ZorbaxTM (GF-250) column. Three bands were visualized on the gel filtration fraction at 39, 36 and 33 kDa using the SDS polyacrylamide-gel electrophoresis (SDS-PAGE) suggesting that purification was not achieved. In enzyme kinetic studies, NADH serves as the substrate for electron donor and 12-MP act as the substrate for electron acceptor. The Km and Vmax for NADH were 0.4838 mM and 21.51 units/mg enzyme respectively. While the values for 12-MP were 5.347 mM and 64.04 units/mg enzyme respectively. The characterization of Mo-reducing enzyme studies were carried out at the optimum pH of 7.5 using 50mM Tris-HCl at 15°C. The enzyme is stable at -20°C for six days in Tris-HCL buffer at pH 7.5.
Repository Staff Only: Edit item detail