Plant protease Inhibitors (PIs) play key roles in regulation of many biological activities and being less toxic, more potent and specific compared to chemical inhibitors. A new proteinaceous trypsin inhibitor (MUTI) was isolated and purified from Macrotyloma uniflorum seeds. MUTI with a molecular mass of 25 KDa was purified to homogeneity via three sequential purification steps i.e., ammonium sulphate precipitation to CNBr activativated Sepharose 4B coupled trypsin affinity chromatography. Purified MUPI showed optical specific activities of 665µmols of tyrosine released/ml/min. Overall, there was a remarkable increase in the fold of purification. MUTI is stable to denaturation by heat (upto 80֩C), pH (4 – 10). The inhibitory activity increased to two fold in the presence of mercuric chloride and got reduced by half in the presence of ferric chloride. SDS, Dithiothreitol, β-mercaptoethanol, Hydrogen peroxide, Triton X 100 enhanced its inhibitory activity whereas activity was reduced in the presence of DMSO. Chemical modification of the inhibitor by N-ethylmaleimide and DEPC decreased its activity but the activity was increased considerably when modified with PMSF. Presence of protease inhibitor activity was confirmed by reverse zymography and Dot- blot. Also MUPI has antifungal and antimicrobial properties. MUPI inhibited Phytophthoracapsici by 16.6% and Rhizoctoniasolani by 27.7%. Antibacterial activity was shown against Staphylococcus aureus and Pseudomonas aeruginosa. MUPI retained 90% inhibition upon storage at 4°C for over a period of six months. Thus MUPI can be effectively exploited to increase the shelf life of seafood.
Keywords: Antifungal, Antimicrobial, Protease Inhibitor, Macrotyloma uniflorum seeds.
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