Purification of 5′-phosphodiesterase from Adzuki (Vigna angularis L.) bean

Abstract

Adzuki bean is high in 5′-phosphodiesterase (5′-PDE), an enzyme that can form flavor enhancing 5′-nucleotides. In this work, 5′-PDE from adzuki bean is purified. Acetone precipitation (1 crude extract:1.25 cold acetone) is used as the first purification step as it is commonly used in enzyme purification to reduce unwanted proteins, before advancing into an application of the Fast Protein Liquid Chromatography system (FPLC). Three major techniques of purification were carried out in sequence as follows: desalting (using a Hi Prep 26/10 desalting column), anion exchange chromatography (using a Hi-Trap DEAE Fast Flow column), and gel filtration chromatography (using a Superose 12 10/300 GL column). The purification protocol adopted yielded 13.37% 5′-PDE with a purification fold of 6.7 and specific activity of 35.2 µmol p-nitrophenol/min/mg protein. The molecular weight of adzuki bean 5′-PDE was estimated using gel filtration chromatography by comparing the fraction that contained the highest 5′-PDE activity with the molecular weights of standard proteins (1.35–669 kDa). Native PAGE was conducted to determine the homogeneity of proteins in the gel filtration fraction showed one band. SDS-PAGE analysis indicated that the enzyme was composed of two identical polypeptide chains (subunits), each with a molecular weight of 62 kDa. Therefore, the estimated molecular weight of the native protein using SDS-PAGE was 124 kDa.