JOURNAL OF PURE AND APPLIED MICROBIOLOGY, August 2014. Vol. 8(4), p. 2523-2533 * To whom all correspondence should be addressed. Tel: +966-6401000 ext 25479; Fax: +966-6952521; E-mail: mahmoodrasool@yahoo.com Process Development to Augment the Production of Microbial Extra Cellular Protease using Response Surface Methodology Muhammad Asif 1 , Taskeen Niaz 2 , Arif Malik 2 , Ahmad Zaheer 3 , Abrar Hussain 1 , Humera Aslam Awan 4 and Mahmood Rasool 5 * 1 Department of Biotechnology and Informatics, BUITEMS, Quetta, Pakistan. 2 Institute of Molecular Biology & Biotechnology (IMBB), the University of Lahore, Pakistan. 3 National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan. 4 Department of Biological Sciences, Forman Christian College University, Lahore, Pakistan. 5 Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia. (Received: 03 February 2014; accepted: 21 March 2014) Alkaline proteases are the most important group of extremoenzymes which have a wide array of applications in various industries such as detergent, food, pharmaceutical, leather and silk. For the purpose of maximum alkaline protease production, response surface methodology was performed using Bacillus licheniformis. The sources used were glucose as carbon source, peptone as nitrogen source, sodium phosphate as phosphorus source and zinc sulfate and magnesium sulfate as micronutrients salt solution; pH and temperature selected for optimization. Response surface methodology central composite deigns, involving 2 6 full factorial half fractions, designed 53 experiments with different combination using six sources and analysis of data was done by SPSS 16. The multiple linear regression model equation was articulated by the coefficient of the determination R 2 and statistical significance of the variables were checked by p (<0.05) value, if lesser the p value then higher the significant of the subsequent coefficient. Importance of the regression coefficient was tested by t test. Applying statistical analysis multiple linear regression using quadric equation shows that the effect of carbon (C), micronutrient salt solutions (M), phosphorous (P 2 ), carbon and phosphorous (CP), carbon and micronutrient salt solutions (CM), carbon and temperature (CT), nitrogen and micronutrient salt solutions (NM), nitrogen and temperature (NT), micronutrient salt solutions and pH (MpH), were given significant results and enhance the production of alkaline proteases by Bacillus licheniformis. Keywords: Alkaline Protease, Response Surface Methodology, Bacillus licheniformis. Proteases are the most important group of industrial enzymes that are used in different processes, i.e. in the detergent and food industries 1 , pharmaceutical, leather processing 2-3 , cosmetic, brewing, silk, recovery of silver from used x-ray films and for treatment of house hold wastes 4-6 . The molecular weight of proteases ranges from 18 – 90 kDA 7 . Proteases dominate the worldwide enzyme market, accounting for a two-third share of the detergent industry 8 . Microorganisms are the major source for enzyme production 9-10 and the genus Bacillus are one of the major and most omnipresent genera of bacteria, which contains 65 species, and the new species continually being described. The genus covers all aerobic or facultative anaerobic, spore- forming, rod-shaped bacteria, and is regularly encountered and cultivated from soil samples as their primary habitat 11 . The importance of the Bacillus strains is due to production of important enzymes in a very short time of period into the fermentation broth 2 .