In Vitro Culture of Pseudananas sagenarius (Arruda) Camargo to obtain Endopeptidases

MARTIN M.I., TORRES, M.J., PÉREZ MARTÍNEZ A., HERNÁNDEZ M., MROGINSKI L. AND NATALUCCI C.L.

Mar del Plata

Congreso; XLIII Reunión Anual de la SAIB; 2007

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

Plants of Pseudananas sagenarius were culture in MS (Murashige & Skoog) to obtain the protease from the medium. A comparison between this protease and the endopeptidases isolated from fruits of the same plants were made. After 40 days in culture different biochemical assays of the medium were made. The cultures were maintained at 25 +/- 2 ºC; with 10.000 lux and 8 h photoperiod. Milk clotting activity was carried out at 37° C during 5 hours using unfatted powder milk in 10 mM CaCl2. The results confirm the presence of enzymes with milk clotting activity.The reaction mixture of caseinolytic activity contained casein 1% in 0.1 M Tris-HCl buffer, pH 8.0, containing 12 mM cysteine. The reaction was carried out at 37° C and stopped by the addition of trichloroacetic acid at different times. The activity was measure as change in absorbance at 280 nm. The activity increased gradually and linearly with time. The final activity was 0.003 Ucas/ml of the medium. In the SDS-PAGE a protein band was observed whit the molecular weight expected to cysteine endopeptidases and by isoelectric focusing a band with proteolytic activity was detected.The results confirm the presence of cysteine proteolytic enzymes and a protein profile similar to that obtain from fruits of P. sagenarius previously studied in our lab.