49F-22 |
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M. I. CORTÉS-VÁZQUEZ, V1, J. L. Muñoz-Sánchez, and R. Briones-Martínez. (1) Biotecnología/Laboratorio de Biofísica Molecular y Enzimología, Centro de Desarrollo de Productos Bióticos - IPN, Km 8 Carretera Yautepec-Jojutla, 62731 Yautepec, Morelos, 62731, Mexico Plant proteases occur in numerous, mainly tropical, plants, and they are of special interest in pharmaceutical and food industries. Preparations of papain and bromelain are currently employed in the manufacture of protein hydrolysates, and for processing meat and fish residues. Several Bromeliaceae species have been studied in our laboratory in a systematic way looking for Mexican plants as sources for industrial proteinases. In this search a new cysteine endopeptidase from Bromelia hemisphaerica fruit juice, named hemisphaericin, has been purified and characterized. These enzyme is constituted by cationic, neutral and anionic components, all of them active. In other studies it has showed good properties for food applications. In spite of the technological studies fulfilled up to this point, allowing the uncover of practical potential for hemisphaericin in macromolecular substrate hydrolysis, the complete interpretation of the obtained results requires a better understanding of substrate specificity properties of the enzyme. Our objective was the purification and substrate specificity studies of hemisphaericin cationic form: the main active fraction. Gel filtration on Sephadex G-75 and ionic-exchange chromatography was used for cationic hemisphaericin purification. Molecular weight was determined by SDS-polyacrylamide gel electrophoresis. Kinetic analysis of chromogenic substrates (p-nitroanilides) hydrolysis and inhibition was performed using spectrophotometric and pH-stat titulation methods. Our results showed that the cationic form of hemisphaericin has a molecular mass of 24 kDa. Enzyme activity was inhibited by E-64, but not by EDTA. Substrate specificity resulted in like-trypsin and like-chymotrypsin. The observed evidences showed the possibility that hemisphaericin cationic fraction may belong to a class of enzymes with singular dual specificity behavior, similar to that reported for cruzain and cathepsin-D peptidases. Like another Bromeliaceae proteases, hemisphaericin C have a potential as a biocatalyst in food uses. Its peculiar substrate specificity poses these enzyme with practical and scientific interest in food industry.
Session 49F, Fruit & Vegetable Products: Fresh vegetables
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