The isolation and characterisation of proteases from Euphorbia tirucalli, E. triangularis, and Carica papaya latex.

Abstract

Plant proteases play an important role in the food and industrial sectors from meat tenderisers to milk clotting agents and even anti-parasitic agents. Proteases have been identified in plant latex, but many proteases have not been isolated and characterised. This research aimed to isolate and characterise proteases from the latex of Euphorbia tirucalli, E. triangularis, and Carica papaya. Three-phase partitioning (TPP) of E. tirucalli plant latex revealed the presence of two active proteases on gelatin-containing zymograms, that were subsequently separated by size exclusion chromatography. These proteases were classified as a 75 kDa serine protease (E. tiru SP), inhibited by PSMF, and SBTI and a 37 kDa cysteine protease (E. tiruCP), inhibited by E-64. Analysis of E. triangularis latex by TPP and p-aminobenzamidine affinity chromatography showed the presence of three serine proteases inhibited by PMSF and SBTI, E. laris SP1 (>97.4 kDa), E. laris SP2 (68 kDa) and E. laris SP3 (38 kDa). These proteases showed stability in constant ionic strength buffers from pH 4 to 9, both with and without the presence of the reducing agent cysteine. Papain was isolated from the latex of Carica papaya for use as a control protease in zymograms on account of the anomalous behaviour of commercial papain preparations on these gels. Papain was isolated by two methods: ammonium sulfate precipitation followed by TPP and CM-cellulose cation exchange chromatography. The isolated papain was detected by rabbit anti-papain antibodies in a dot blot and western blot and showed inhibition by E-64. The latex from all three plant species showed milk clotting activity with higher activity in the presence of calcium chloride. These findings suggest that isolated plant latex proteases from the Euphorbia species can be used in the food industry as milk clotting agents. Further characterisation of these isolated proteases should identify further uses in biotechnology.