Biochemical and functional characterization of partially purified proteolytic preparation obtained from Hohenbergia penduliflora Mez Stems

CARVAJAL, C.; PÉREZ, A.; MORA, M.; TREJO, S.; MARTIN, M.I.; TORRES, M.J.; NATALUCCI AND HERNÁNDEZ, M

Ciego de Avila

Congreso; VII International Congress on Biotechology and Agriculture; 2007

Centro de Bioplantas

Plant genome encodes hundreds of proteases, but only a few plant proteinases have been totally purified and characterized. Bromeliaceae family plants usually contain high concentration of thiol proteases. Plant proteolytic enzymes have been found to be useful in different industries. Nowadays peptidases of plant origin are of special interest in medicine and industry because they are active at a very wide range of temperature and pH (1). Bromelain, in particular, exhibits therapeutic effects: anti-inflammatory, digestive, antimetastasis and anti-tumoral activities (2,3). In recent years, it is including in the drug group modifiers of the biological answer (4). Therefore, is important to find new sources to obtain similar proteasas. In the present study, partially purified preparation obtained from crude extract of Hohenbergia penduliflora Mez stems was characterized biochemical and functionally. The stable and active enzymatic product was recovered by ethanol precipitation. Maximum proteolytic activity was achieved at pH 8. Sodium chloride increased up to 3 M reduced proteolytic activity of partially purified preparation. Enzymatic preparation was stable at ionic strength values evaluated (no appreciable decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution at 37 °C for casein hydrolysis during 10 min), and exhibited high thermal stability (inactivation required heating for 20 min at 75 °C). Carboxipeptidasa activity was not detected in this preparation. Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. It was completely inhibited by E64 and activated by adition of cysteine. SDS-PAGE showed one protein band between 20 and 31 KDa. Isoelectric focusing and zimogram evidenced the acid characteristic of proteases that are present in this preparation.Mez stems was characterized biochemical and functionally. The stable and active enzymatic product was recovered by ethanol precipitation. Maximum proteolytic activity was achieved at pH 8. Sodium chloride increased up to 3 M reduced proteolytic activity of partially purified preparation. Enzymatic preparation was stable at ionic strength values evaluated (no appreciable decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution at 37 °C for casein hydrolysis during 10 min), and exhibited high thermal stability (inactivation required heating for 20 min at 75 °C). Carboxipeptidasa activity was not detected in this preparation. Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. It was completely inhibited by E64 and activated by adition of cysteine. SDS-PAGE showed one protein band between 20 and 31 KDa. Isoelectric focusing and zimogram evidenced the acid characteristic of proteases that are present in this preparation. Palabras clave: plant cysteine peptidases, Bromeliaceae.plant cysteine peptidases, Bromeliaceae.