DAHIYA et al : OPTIMIZATION OF GROWTH PARAMETERS OF PHYTASE PRODUCING FUNGUS USING RSM 955 Journal of Scientific & Industrial Research Vol. 68, November 2009, pp. 955-959 *Author for correspondence Tel: 01662263312; Fax: 091-1662-276240 E-mail: namitasingh71@gmail.com Optimization of growth parameters of phytase producing fungus using RSM Sonia Dahiya, Namita Singh* and J S Rana Department of Bio & Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar 125 001, India Received 01 September 2008; revised 24 July 2009; accepted 27 July 2009 This study presents optimum parameters for phytase enzyme secretion by fungus (NSF-7 & NSF-9) using RSM based on Box-Behnken model. Fisher’s statistical testing was performed for analysis of variance (ANOVA) for quadratic regression equations of both fungus. Optimum conditions for phytase secretion by NSF-7 and NSF-9, respectively, were: pH, 4.4-4.7, 4.0-4.5; temperature, 28-32°, 32-35°C; and substrate concentration, 0.29-0.32, 0.27-0.30 g/100 ml. Keywords: Box-Behnken design, Inorganic phosphate, Optimization, Phytase Introduction Phytases (myo-inositol hexakisphosphate 3-phosphorylase, E C 3.1.3.8 and myo-inositol hexakisphosphate 6-phosphorylase, E C 3.1.3.26) are classified as family of histidine acid phosphatases 1 , which hydrolyse phytic acid (myo-inositol hexakisphosphate) to inorganic phosphate and myo-inositol phosphate derivatives. Phytic acid is major phosphorus form in seed-based animal feed formulations 2 . However, mono-gastric animals (pigs and poultry), which have very low or no phytase in digestive tracts, cannot utilize phytic acid present in cereal grains/legumes. Phytase also acts as an antinutrient by complexing with proteins and inhibiting enzymes such as ±-amylase, trypsin, acid-phosphatase and tyrosinase 3 . Supplementations with inorganic phosphorus, along with phytate phosphorus excretion, impose a global ecological problem (eutrophication). Phytase can liberate phytate bound phosphorus in pig’s digestive tract. Supplementing feed grade phytase to pig feeds may reduce the need for inorganic phosphorus supplementation and reduce phosphorus excretion. Conventional methods for optimization of phytase parameters are extremely time-consuming and expensive 4,5 . Response surface methodology (RSM) is efficient in handling large number of design parameters 6 . RSM in biotechnological processes is gaining immense importance for optimization of enzyme production 4,7,8 . This study presents optimum parameters (pH, temperature and substrate concentration) for phytase enzyme secretion by fungus (NSF-7 & NSF-9) using RSM based on Box-Behnken model. Materials and Methods Microorganism and Culture Conditions Several fungi were isolated from composting soil, rotten fruits, plant debris and bottom of phytate rich plants. Fungi were grown on potato dextrose agar (PDA) medium at 37°C and preserved at -20°C in spore glycerol stock. Fungal strains (11) were screened for phytase enzyme production quantitatively on PSM (Phytase screening medium) with calcium phytate as sole source of phosphorus 9 and qualitatively by determining amount of inorganic phosphate liberated at 37°C and pH, 5.0±0.5 by Fiske and Subbarow 10 . Fungal biomass was estimated gravimetrically by filtering culture through a pre-weighed dry Whatmann no. 1 filter paper. Mycelium was thoroughly washed with distilled water and dried at 80°C. Optimization of Growth Parameters by Response Surface Methodology (RSM) Independent variables (pH of medium, temperature of medium and substrate concentration) were optimized by RSM using a Box-Behnken design of experiments (Table 1). Each variable was studied at three different levels (-1, 0, +1) for pH (3.0, 5.0, and 7.0), temperature (20°, 45°, and 70°C) and substrate concentration (0.1, 0.3, 0.5 g/100ml). Experimental design included 17 flasks separately for each strain, with five replicates having all three variables at their central coded values. Upon