Purification and characterisation of an acid protease from Aspergillus carbonarius

Abstract

The aim of this study is to purify and characterize an acid protease from Aspergillus carbonarius. The protease was purified to apparent homogeneity by 4 M sucrose concentration, ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl Sepharose CL-4B and gel filtration chromatography through Sephadex G-100. A 10-fold purification with specific activity of 485.47 Umg-1 protein was achieved. SDS-PAGE and zymogram analysis of the protease indicated an estimated molecular mass of 72 kDa. The optimum temperature and pH for the proteolytic activity were 40°C and pH 3.0, respectively. The enzyme was active over a wide range of temperature from 40 to 80°C. The enzyme retained about 70% of its original activity (40°C) at 90 and 100°C. The enzyme was most stable at pH of 3.0 and temperature of 50°C. The enzyme has a relatively broad pH range of 4.0 to 10.0 after 30 min. The enzyme was slightly significantly (P < 0.001) stimulated by K+, Ba2+ while Ca2+ and Zn2+ moderately enhanced it. Mn2+, Fe2+ and Cu2+ strongly significantly (p<0.001) enhanced the enzyme activity while it was significantly (p<0.001) inhibited by Hg2+. The enzyme was not affected by Sr2+ and Mg2+ but Co2+ and Na+ elicited slight repression of the enzyme activity. The protease was strongly significantly (p < 0.001) inhibited by E-64, IAA and DIMSO while EDTA and 2-ME appreciably enhanced the activity of the protease. The enzyme is a cysteine protease as indicated by its inhibition studies. The protease may find potential applications in food and brewing industries as well as in meat tenderization. Key words: Aspergillus carbonarius, protease, purification, characterization, kinetic properties.