Purification And Characterization Of Protease From Artocarpus Integer Leaf

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dc.contributor.authorZulfigar, Siti Balqis
dc.date.accessioned2019-06-27T01:20:32Z
dc.date.available2019-06-27T01:20:32Z
dc.date.issued2012-07
dc.description.abstractThe presence of a protease in Artocarpus integer leaves, which can be used as a meat tenderiser, was verified by the presence of a band at 69 kDa, using caseinolytic zymography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Purification by temperature-phase partitioning with 6 % (v/v) Triton X-114, 44 % (w/v) ammonium sulphate precipitation and gel filtration chromatography yielded a preparation with a 12-fold increase in enzyme purity and a final specific activity of 76.67 U/mg. The purified protease was maximally active at 40ºC and at pH 10.0. The enzyme hydrolysed natural substrates in the order of gelatin < BSA < casein when tested at the optimal pH and temperature.The cysteinic nature of this enzyme was verified through the inhibition of the activity by E-64 and iodoacetamide at the concentration of 1 mM with the residual activity of 30 + 1.6 % and 9 + 0.8 % respectively. The enhancement of activity up to 212.1 % and 186.7 % were obtained after 15 min incubation with 5 mM cysteine and 30 mM 2-mercaptoethanol respectively. The activity of the enzyme was affected by the metal ions’ concentrations. The enzyme was generally inhibited with the presence of Cu2+, Mg2+ and Mn2+. However, lower degree of inhibition of Mn2+ was detected when the concentration of the cation was increased to 10 mM. Ca2+ suppressed the activity at 1 mM and activated the enzyme at 5 and 10 mM. The presence of Zn2+ activated the activity of A. integer leaf protease at the low concentration of 1 mM (108. 4 + 3.3 %) and suppressed the activity at the higher concentrations of 5 and 10 mM with 74.8 + 4.1% and 64.3 + 2.1 % relative activity respectively. The enzyme was stable at temperatures up to 70 ºC, with 80 % of its activity intact and the energy of inactivation (Eia) was calculated as 89 + 11 kJ/mol.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/8414
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectPurification and characterization of proteaseen_US
dc.subjectfrom artocarpus integer leafen_US
dc.titlePurification And Characterization Of Protease From Artocarpus Integer Leafen_US
dc.typeThesisen_US
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