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Artocarpus integer leaf protease: Purification and characterisation

Siti Balqis, Z., Rosma, A.
Food chemistry 2011 v.129 no.4 pp. 1523-1529
Artocarpus integer, ammonium sulfate, chromatography, cysteine, enzyme activity, enzyme inhibition, enzymes, filtration, gels, leaves, meat, pH, temperature
The presence of a protease in Artocarpus integer leaves, which are traditionally used as a meat tenderiser, was verified by the presence of a band at 69kDa, using caseinolytic zymography. Purification by temperature phase partitioning with Triton X-114, ammonium sulphate precipitation and gel filtration chromatography yielded a preparation with a 12-fold increase in enzyme purity and a final specific activity of 76.67U/mg. The cysteinic nature of this enzyme was confirmed through inhibition of enzyme activity by E-64 and iodoacetamide and enhancement of activity by cysteine and 2-mercaptoethanol. The protease retained 70% of its activity over a broad pH range (pH 6–12), with optimal activity recorded at pH 10 and 40°C. The enzyme was stable at temperatures up to 70°C, with 80% of its activity intact. Addition of 5mM Ca²⁺ stimulated enzyme activity and a kinetic study of the enzyme yielded Kₘ and Vₘₐₓ values of 0.304mg/mL and 0.735mg/mL/min, respectively.