PROTEASE INHIBITOR FROM WHITE CRANBERRY BEANS (PHASEOLUS VULGARIS): ISOLATION, PURIFICATION AND CHARACTERIZATION
Objective: The main objective of the study was isolation, purification and characterization of protein protease inhibitor from the seeds of Phaseolus vulgaris and analysis of its antimicrobial potential.
Methods: The protease inhibitor was extracted by homogenizing seeds of Phaseolus vulgaris in 0.1 M phosphate buffer (pH-7.0). The crude extract of the inhibitor was purified by using ammonium sulphate precipitation followed by DEAE Cellulose ion exchange chromatography. The protease inhibitor was characterized to determine its optimum pH and pH stability, optimum temperature and temperature stability, stability in the presence of chemical modifiers, thermal stabilizers, metal ions, detergents, oxidising and reducing agents. The antimicrobial potential of the inhibitor against various bacterial species was confirmed using agar well diffusion method.
Results: The extracted protease inhibitor was purified to homogeneity with a 1.4 fold increase in the specific activity and 56 % purification yield. Inhibitor was optimally active at pH 7.0 and a temperature of 50 Â°C as well as showed considerable stability over pH ranging from 4.0-11.0 and up to a temperature of 70 Â°C for 4 h duration. The inhibitor was substantially active and stable in the presence of surfactants 1 % (v/v) Tween 20, 4 % (v/v) of oxidizing agents such as dimethyl sulphoxide (DMSO) and hydrogen peroxide (H2O2), 0.8 % (v/v) of reducing agents Î²-mercaptoethanol and Sodium thioglycolate. Metal ions Mg++, Ca++and Zn++enhanced the activity of inhibitor while CaCl2, glycine and glycerol promoted thermal stability of the inhibitor. Chemical modification of amino acids at the active site by diethyl pyrocarbonate (DEPC) and phenyl methyl sulphonyl fluoride (PMSF) led to decrease in the inhibitory activity. The stoichiometry of trysin-protease inhibitor interaction was 1:2 while 216 Î¼g of the inhibitor effected 50 % inhibition. The inhibitor displayed antimicrobial activity against Aeromonas hydrophilla, Citrobacter freundii and Acinetobacter baumanii.
Conclusion: The experimental results confirmed the anti proteolytic action of protease inhibitor extracted from P. vulgaris as well as its promising antimicrobial properties. Thus isolated protease inhibitor has significant industrial and therapeutic potential.
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