Production and purification of a protease from an alkalophilic Bacillus sp. 2-5 strain isolated from soil

This research has focused on isolation and characterization of a strain of Bacillus sp. from alkaline soil, which was able to produce extracellular alkaline protease at pHs ranging from 8 to 11 and temperatures of 20 to 50§C. Also the impact of different carbon and nitrogen sources were investigated. The yield and fold of enzyme purification was 24% and 50 times, respectively. Molecular weight of purified enzyme was measured by SDS-PAGE as 24.7 kDa. The alkaline protease produced by Bacillus sp. 2 - 5 showed the most caseinolytic activity [without any gelatinolytic activity] at pH>10

[Alkaline protease production and its purification from a soil separated alkalophilic bacillus]

Proteases are among the most important industrial enzymes. Alkaline proteases are used primarily as cleansing additives. As alkaline pH and temperature stability are two main important factors of enzyms for their addition to detergents' formula, it is desirable to search for new proteases with novel properties from different sources. The purpose of this study was to isolate an alkalophilic bacillus sp. with possibility of alkaline protease production and then purification of the produced alkaline protease. Isolation and purification of proper colonies from soil samples were performed. After characterization of taxonomic and biochemical specifications of colonies, they were cultivated in a specific liquid culture medium [alkaline pH] and then selected bacillus 2-5 was cultivated in a proper culture medium. The enzyme was isolated and purified as fallows: 1- Ammonium sulfate precipitation [saturation percentage, 55%] 2- Ultra filtration 3- Cation exchange chromatography [CM- cellulose]. Alkaline protease activity was checked by determination of equal concentration of tyrosine as a product at lamda=275 nm after alkaline hydrolysis of casein as a substrate. Bacillus 2-5 was selected because only it had growth and alkaline protease activity. It had both amylolitic and proteolytic activities at alkaline pHs but no gelatinolytic activity was found. Purification progression was demonstrated by gel electrophoresis [PAGE]. Molecular weight of alkaline protease by SDS-PAGE and was measured by using protein standard solutions this was 24700 Dal. The yield of purification was 24% and parification, was 50 times gain factor, as found by other researchers. The purified enzyme was monomer because electrophoretic mobility at PAGE was the same as SDS-PAGE