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Alkaline protease has tremendous applications in different industries, and to fulfil industrial and commercial enzyme requirements, overproduction and optimization of production medium are prerequisites. Protease production by Bacillus amyloliquefaciens SP1, isolated from apple tree rhizosphere was studied. Plackett Burmann design and response surface methodology was used to optimize medium for alkaline protease production. Higher levels of protease activity were observed in presence of disaccharides owing to diauxic growth and inorganic nitrogen sources proved to be less favourable. After screening through one-factor-at-a time experiments and Plackett Burmann design, casein, yeast extract, maltose and KH2PO4 were identified as the most significant variables. Statistical design based three-dimensional (3-D) and contour plots were generated to understand the relationship between enzyme activity and medium variables. The protease production was found to increase from 1730 to 3630 µg/ml/min in optimized medium i.e. approx. 2.1 fold increased compared with original medium. The study assumes significance in the ability of bacterium to survive in wide variety of salts and agricultural wastes and yield optimum levels of extracellular protease. [ABSTRACT FROM AUTHOR]