The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase.
Intracellular proteases of Yarrowia lipolytica have been scarcely studied. These enzymes may play an important role in nitrogen metabolism, posttranslational processing, nutritional stress, dimorphism, etc.; biochemical and genetic control of these enzymes can help in obtaining high-level expression of recombinant proteins in heterologous systems. In this study, we report the presence of three proteases: aminopeptidase yylAPE, carboxypeptidase yylCP and dipeptidyl aminopeptidase yylDAP, measured under several nutritional conditions. Yarrowia lipolytica produced the highest level of intracellular proteolytic enzymes, i.e. yylAPE, yylCP and yylDAP, in media with peptone during stationary growth phase. When soluble extracts were subjected to PAGE, and the three activities were revealed in gels with the corresponding substrates, only one band of activity was detected for each one. The three enzymes were affected by serine protease inhibitors. Chelating agents affected mainly APE activity. The aminopeptidase was purified by selective fractionation with ammonium sulfate and three chromatographic steps (anion exchange, hydrophobic interaction and gel filtration chromatography). The enzyme had a molecular mass of 97 kDa; optimal pH and temperature were 7.0 and 37 degrees C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The K(m) value was 0.86 microM and V(max) value was 990.8 micromoL min(-1) mg(-1) for Lys-pNA.
Hernández-Montañez
Zahuiti
Z
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, IPN, Mexico, DF, Mexico.
Araujo-Osorio
Janet
J
Noriega-Reyes
Yamilet
Y
Chávez-Camarillo
Griselda
G
Villa-Tanaca
Lourdes
L
D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013696:Temperature; D025062:Yarrowia / Q000201:enzymology*
D005656:Fungal Proteins; D015842:Serine Proteinase Inhibitors; D002268:Carboxypeptidases; D010450:Endopeptidases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases