Fractionation and purification of the enzymes stored in the latex of Carica papaya.
The latex of the tropical species Carica papaya is well known for being a rich source of the four cysteine endopeptidases papain, chymopapain, glycyl endopeptidase and caricain. Altogether, these enzymes are present in the laticifers at a concentration higher than 1 mM. The proteinases are synthesized as inactive precursors that convert into mature enzymes within 2 min after wounding the plant when the latex is abruptly expelled. Papaya latex also contains other enzymes as minor constituents. Several of these enzymes namely a class-II and a class-III chitinase, an inhibitor of serine proteinases and a glutaminyl cyclotransferase have already been purified up to apparent homogeneity and characterized. The presence of a beta-1,3-glucanase and of a cystatin is also suspected but they have not yet been isolated. Purification of these papaya enzymes calls on the use of ion-exchange supports (such as SP-Sepharose Fast Flow) and hydrophobic supports [such as Fractogel TSK Butyl 650(M), Fractogel EMD Propyl 650(S) or Thiophilic gels]. The use of covalent or affinity gels is recommended to provide preparations of cysteine endopeptidases with a high free thiol content (ideally 1 mol of essential free thiol function per mol of enzyme). The selective grafting of activated methoxypoly(ethylene glycol) chains (with M(r) of 5000) on the free thiol functions of the proteinases provides an interesting alternative to the use of covalent and affinity chromatographies especially in the case of enzymes such as chymopapain that contains, in its native state, two thiol functions.
Azarkan
Mohamed
M
Laboratoire de Chimie Générale, Unité de Chimie des Protéines (CP 609), Faculté de Médecine, Université Libre de Bruxelles, Campus Erasme, 808 Route de Lennik, B-1070, Brussels, Belgium.
El Moussaoui
Anouar
A
van Wuytswinkel
Delphine
D
Dehon
Géraldine
G
Looze
Yvan
Y
D029441:Carica / Q000201:enzymology*; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D007840:Latex
D002411:Cation Exchange Resins; D007840:Latex; D010450:Endopeptidases