Purification and characterization of an extracellular non-aspartyl acid protease (pumAe) from Ustilago maydis

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Abstract

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 Kmvalue was 3.5 μM, and the Vmaxvalue was 11430 μmol h-1mg-1for Suc-R-P-F-H-L-L-V-Y-MCA.
Original languageAmerican English
Pages (from-to)408-411
Number of pages366
JournalCurrent Microbiology
DOIs
StatePublished - 1 Nov 2003

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Ustilago
Chromatography
chromatography
Purification
Gel Chromatography
purification
Peptide Hydrolases
Gels
Enzymes
gel
enzyme
Casein
Acids
collagen
Haploidy
acid
Hemoglobin
Ammonium Sulfate
hemoglobin
ammonium sulfate

Cite this

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title = "Purification and characterization of an extracellular non-aspartyl acid protease (pumAe) from Ustilago maydis",
abstract = "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 Kmvalue was 3.5 μM, and the Vmaxvalue was 11430 μmol h-1mg-1for Suc-R-P-F-H-L-L-V-Y-MCA.",
author = "Yuridia Mercado-Flores and Guadalupe Guerra-S{\'a}nchez and Lourdes Villa-Tanaca and C{\'e}sar Hern{\'a}ndez-Rodr{\'i}guez",
year = "2003",
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language = "American English",
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T1 - Purification and characterization of an extracellular non-aspartyl acid protease (pumAe) from Ustilago maydis

AU - Mercado-Flores, Yuridia

AU - Guerra-Sánchez, Guadalupe

AU - Villa-Tanaca, Lourdes

AU - Hernández-Rodríguez, César

PY - 2003/11/1

Y1 - 2003/11/1

N2 - 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 Kmvalue was 3.5 μM, and the Vmaxvalue was 11430 μmol h-1mg-1for Suc-R-P-F-H-L-L-V-Y-MCA.

AB - 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 Kmvalue was 3.5 μM, and the Vmaxvalue was 11430 μmol h-1mg-1for Suc-R-P-F-H-L-L-V-Y-MCA.

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