PROTEASE INHIBITOR FROM WHITE CRANBERRY BEANS (PHASEOLUS VULGARIS): ISOLATION, PURIFICATION AND CHARACTERIZATION

Ashwini Puntambekar,Manjusha Dake

doi:10.22159/ijpps.2017v9i9.20472

Abstract

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 °C as well as showed considerable stability over pH ranging from 4.0-11.0 and up to a temperature of 70 °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 β-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 μg 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.

Highlights

Proteolytic enzymes belong to hydrolase category catalyze cleavage of specific peptide bonds in certain proteins
The protease inhibitor purified by salt precipitation using 70 % saturation with ammonium sulphate exhibited enhanced inhibitory activity against trypsin (59 %) compared to the crude preparation of extract
The data for yield and purification fold of protease inhibitor is presented in table 1

Summary

Introduction

Proteolytic enzymes belong to hydrolase category catalyze cleavage of specific peptide bonds in certain proteins. These are ubiquitously distributed among plants, animals and diverse microbial flora [1,2,3]. Some proteases are the key virulent factors in many pathogenic bacteria, parasites and viruses Inhibition of such proteases through selective action of protease inhibitors can provide an effective strategy to control pathogenesis. Protease inhibitors act as defensive proteins and have great demand in medicine and biotechnology They can be applied as therapeutics against emphysema, arthritis, and pancreatitis. Food preservation by increasing the shelf life can be feasible by the action of natural protease inhibitors originating from a plant source that could possibly inhibit extracellular and intracellular proteolytic enzymes during food storage [10]

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Results

Conclusion