TY - JOUR
T1 - The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase
AU - Hernández-Montañez, Zahuiti
AU - Araujo-Osorio, Janet
AU - Noriega-Reyes, Yamilet
AU - Chávez-Camarillo, Griselda
AU - Villa-Tanaca, Lourdes
PY - 2007/3
Y1 - 2007/3
N2 - 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°C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The Km value was 0.86 μM and Vmax value was 990.8 μmoL min-1 mg -1 for Lys-pNA.
AB - 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°C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The Km value was 0.86 μM and Vmax value was 990.8 μmoL min-1 mg -1 for Lys-pNA.
KW - APE
KW - Aminopetidase
KW - E.C.3.4.11.15
KW - Proteases
KW - Yarrowia lipolytica
UR - http://www.scopus.com/inward/record.url?scp=33846991293&partnerID=8YFLogxK
U2 - 10.1111/j.1574-6968.2006.00578.x
DO - 10.1111/j.1574-6968.2006.00578.x
M3 - Artículo
C2 - 17227470
AN - SCOPUS:33846991293
SN - 0378-1097
VL - 268
SP - 178
EP - 186
JO - FEMS Microbiology Letters
JF - FEMS Microbiology Letters
IS - 2
ER -