World Applied Sciences Journal 12 (6): 751-757, 2011 ISSN 1818-4952 © IDOSI Publications, 2011 Corresponding Author: Hafiz Muhammad Nasir Iqbal, Enzyme Biotechnology Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan, Tel: +92-307-6715243, E-mail: nasir_pk99@hotmail.com. 751 Purification and Kinetic Parameters Characterization of an Alkaline Protease Produced from Bacillus subtilis through Submerged Fermentation Technique Ishtiaq Ahmed, Muhammad Anjum Zia and Hafiz Muhammad Nasir Iqbal Enzyme Biotechnology Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan Abstract: This paper reported the purification and kinetic parameters characterization of an alkaline protease produced from Bacillus subtilis through submerged fermentation process using rice husk as growth supporting substrate (by-product of rice industry) collected from Pearl Rice Mill Hafiz Abad, Faisalabad. Bacillus subtilis was cultured in fermentation medium under some pre-optimized growth conditions. Maximum alkaline protease activity of 216±4.32U/mL was obtained when fermentation medium of rice husk was inoculated with 4% (4mL/100mL) inoculum size, 7% substrate concentration at pH 11 for 48 h fermentation time period with 2% molasses as additional supplement material for fermentation medium. An alkaline protease was purified 1.49-fold with specific activity of 74.66 U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 27kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed 10 and 45°C as an optimum pH and temperature respectively. Using casein as substrate, the enzyme showed maximum activity (V ) of 148U/mL with its corresponding K value of 58µM. Among max M activators/inhibitors EDTA and Ca gave enhancing effect on purified alkaline protease where as SDS, Tween- 2+ 81, Na and Hg caused enzyme inhibition and inactivation to variable extents. The specific activity and + 2+ substrate affinity of this alkaline protease from Bacillus subtilis is greater than those of other reported Bacillus sp; therefore, it was concluded that it may be potentially useful for industrial purposes. Key words: Alkaline protease Bacillus subtilis Purification SDS-PAGE Characterization INTRODUCTION attention in attempts to exploit their physiological and Proteases (EC 3.4.21-24) are enzymes that hydrolyze of the most important groups of industrial enzymes used proteins and form a bulky cluster of enzymes which are in detergent, protein, brewing, meat, photographic, ubiquitous in nature and most central category of leather, dairy industries, pharmaceutical and food industry enzymes from an industrial point of view. An extensive [7, 8]. They used in pharmaceutical and food industry for range of microorganisms has great potential to produce peptide synthesis in leather industry for dehairing and in alkaline proteases under suitable growth conditions [1]. detergent industry as an additive of detergent formulation Bacteria are the most dominant group of alkaline protease [2, 9] to substitute currently used toxic chemicals, is a producers with the genus Bacillus being the most relatively new development and has conferred added prominent and serve as an ideal source of these enzymes biotechnological importance [6]. [2, 3] because of their rapid growth and limited space Alkaline proteases have traditionally detained the required for their cultivation [1]. An ease with which they predominant share of the industrial enzyme market can be genetically manipulated to generate new enzymes accounting for about 60-65% of total global sale of with altered properties that is desirable for their diverse enzymes [6, 10, 11]. About 35% of total microbial enzymes applications [4]. used in detergent industry are derived from bacterial The vast diversity of proteases, in contrast to their sources and most of them produced by Bacillus sp. mode of action and specificity, has attracted worldwide [12, 13]. B. subtilis is a highly favorable bacterium for biotechnological application [5, 6]. Proteases are the one