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.