Purification and Characterization of an Extracellular Non-Aspartyl Acid Protease (pumAe) from Ustilago maydis

Mercado-Flores, Yuridia; Guerra-Sánchez, Guadalupe; Villa-Tanaca, Lourdes; Hernández-Rodríguez, César
October 2003
Current Microbiology;Oct2003, Vol. 47 Issue 5, p408
Academic Journal
The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45°C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The Km value was 3.5 μM, and the Vmax value was 11430 μmol h-1 mg-1 for Suc-R-P-F-H-L-L-V-Y-MCA.


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