Journal of Rafsanjan University of Medical Sciences

  Alkaline Protease Production and its Purification from a Soil Separated Alkalophilic Bacillus

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  HR. Falahatpisheh MSc [1] , M. Jalali PhD [2] , Naser Badami PhD [3] , N. Mardani BSc [4]

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  Background and Objective: 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 addtion 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.

  Materials and Methods: 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 λ=275 nm after alkaline hydrolysis of casein as a substrate.

  Results: 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.

  Conclusion : 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.

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  Key words: Alkaline Proteas, Alkalophilic bacillus, Purification, Alkaline protease activity

  [1] - Academic Member Dept. of Biochemistry, Tehran University of Medical Sciences, Tehran

  (Corresponding author), Tel: (021)22376426 , Fax: (021)22360660, E-mail:hfalahatpishe@yahoo.com

  [2] - Assotiated Professor Dept. of Biochemistry, Tehran University of Medical Sciences, Tehran

  [3] - Assotiated Professor Dept. of Microbiology, Tehran University of Medical Sciences, Tehran

  [4] - BSc in Public Health Sciences, Dept. of Microbiology, Tehran University of Medical Sciences, Tehran