title: Purification and characterization of a pineapple crown leaf thiol protease.
abstract: A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.
authors.affiliation: Department of Biochemistry, Manipur University, Canchipur, Imphal, India. rslaishram@yahoo.com
authors.forename: L Rupachandra
authors.initials: LR
authors.lastname: Singh
authors.forename: Th Premila
authors.initials: TP
authors.lastname: Devi
authors.forename: S Kunjeshwori
authors.initials: SK
authors.lastname: Devi
mesh_terms: D031230:Ananas / Q000201:enzymology*; D002364:Caseins / Q000494:pharmacology; D002846:Chromatography, Affinity; D003545:Cysteine / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D018515:Plant Leaves / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D002364:Caseins; C026018:azocasein; D003546:Cysteine Endopeptidases; D003545:Cysteine