Purification, characterization and optimization conditions of protease produced by Aspergillus brasiliensis strain BCW2

Abstract

Proteases are a group of enzymes in high demand due to their wide and different biotechnological applications. However, the cost of production of proteases on industrial scale is high. Hence, this study was aimed at screening fungal strains having ability to produce protease using cheaper agricultural wastes, purifying and characterizing the protease produced. Fungi isolated were screened for protease production and identified using traditional and molecular means. Optimum conditions for protease production, purification and characterization were determined using standard methods. Proteolytic activity of the selected isolate BCW2 measured in zone of hydrolysis was 38 mm. The selected isolate shared 99% sequence homology with Aspergillus brasiliensis JC-A3. Hence, the isolate was tagged as Aspergillus brasiliensis BCW2. Production of protease by solid state fermentation was optimum at pH 9.0, temperature of 30 °C, 10% orange peel as carbon source, 2% yeast as nitrogen source, 60% moisture content and an incubation period of 72 h. The purified protease has a molecular weight of 68 KDa, a yield of 28% and 13.3 fold. The partially purified protease was stable between pH 4-6 and temperature of 30-40 °C, exhibited enhanced activity with Ca2+, and Tween-20 and had specificity to gelatin as substrate. The suitability of orange peel as a substrate for protease production by the strain Aspergillus brasiliensis BCW2 will reduce production cost. The use of orange peel as organic substrate is first being reported through this work. The purified enzyme has de-clotting and blood stains removal properties, making the enzyme open up to wider biotechnological applications. GenBank accession number for the strain A. brasiliensis BCW2 ITS rRNA gene sequence is MF599164.1.

Keywords

• Solid state fermentation
• Protease
• Optimum growth conditions
• Organic substrate
• Purification
• Characterization