Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans.
Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40 degrees C. The enzyme retained activity after incubation at pHs ranging from 8 to 11 for 12 h at 37 degrees C and 6 to 8 for 24 h at 37 degrees C. It retained 80% of its activity after incubation at 30 to 70 degrees C for 30 min and lost 50% of its activity after incubation for 15 min at 80 degrees C. Noticeable activation of the enzyme is observed when Fe(2+) ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu(2+), Fe(3+), Hg(2+), and Zn(2+) ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.
Peña-Montes
Carolina
C
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City, D.F. 04510, Mexico.
González
Augusto
A
Castro-Ochoa
Denise
D
Farrés
Amelia
A
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009930:Organic Chemicals / Q000032:analysis; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D002412:Cations; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009930:Organic Chemicals; D043182:Carboxylesterase; D010450:Endopeptidases; C087822:alkaline protease