Purification, characterization, and antimicrobial activity of nontoxic trypsin inhibitor from Albizia amara Boiv.

Abstract

The present investigation describes the purification, and characterization of a novel, thermostable, nontoxic, proteinaceous trypsin inhibitor extracted from seeds of Albizia amara Boiv. The proteinaceous protease inhibitor (API) was purified via acetone fractionation, ion-exchange chromatography (diethylaminoethyl (DEAE) cellulose), and gel permeation chromatography (GPC; Sephadex G-100). The apparent molecular weight of the API is 49kDa and is identified as a serine protease inhibitor. The API remains active in a wide pH range (3.0–8.0), and showed thermal stability at 60°C. The API inhibits trypsin by a mixed inhibitory mechanism with an inhibition constant (Ki) of 1.24×10−8M using BApNA (Nα-benzoyl-dl-arginine-p-nitroanilide hydrochloride) as the substrate. The API also showed substantial activity in the presence of several metal ions, surfactant (Triton X-100), oxidizing agent (dimethyl sulfoxide; DMSO), and NaCl (5%). Moreover, API retained 85% trypsin inhibition activity upon storage at 4°C over a period of 6 months. The antimicrobial effectiveness of the API against Pseudomonas aeruginosa, Bacillus subtilis, Alternaria alternata, Alternaria tenuissima, and Candida albicans reported in this study suggests its utility as a potential antimicrobial component.