International Journal of Bio-Science and Bio-Technology Vol. 4, No. 4, December, 2012 27 Statistical Optimization of Media Components for Enhanced Production of the Recombinant Staphylokinase Variant from Salt Inducible E.Coli GJ1158 Seetha Ram Kotra 1 *, Anmol Kumar 1 , KRS Sambasiva Rao 1 and KK Pulicherla 2 1 Department of Biotechnology, Acharya Nagarjuna University Nagarjuna Nagar, Guntur, Andhra Pradesh, India 2 Department of Biotechnology, RVR & JC College of Engineering, Chowdavaram, Guntur, Andhra Pradesh, India *Corresponding author: kotraseetharam@ymail.com Abstract Different types of nutritional factors are influencing the production of recombinant staphylokinase variant from Msak - Hirulog p RSET-A GJ1158, which was constructed in our lab. The optimal conditions for the production of recombinant staphylokinase variant by using submerged fermentation at 37 0 C for 6 hours after induction with 300 mM NaCl at flask level with a working volume of 100 ml. Initially, all nutritional factors were selected by one factor- at-a-time method. The significance of each factor with respect to recombinant staphylokinase production was identified by Taguchi (7 factors viz.,glucose, K 2 HPO 4 , TMM, KH 2 PO 4 , NH 4 Cl, yeast extract and MgSO 4 )and the enzymatic activity was increased remarkably by 1.32 folds compared to the test tube level (7580 to 10,081 U/mL). Based on taguchi results glucose, K 2 HPO 4 , TMM and KH 2 PO 4 are the most influencing parameters. The outcome of taguchi design showed that the further optimization using response surface methodology (Central Composite Design) with 30 experiments increased the yield of 1.98 folds (10,081to 19,928 U/ml). For the first time we optimized the components concentration for enhanced production of sak variants and having maximum activity. Keywords: recombinant staphylokinase, one factor –at–a –time method, Taguchi, central composite design, enzyme activity 1. Introduction Staphylokinase (Sak), an extracellular protein produced by Staphylococcus aureus strains, is a promising blood clot dissolving agent [1]. Sak is a profibrinolytic agent that forms a 1:1 stoichiometric complex with plasminogen that, after conversion to plasmin, activates other plasminogen molecules to plasmin [2]. Now-a-days sak has gain lot of importance in thrombotic complications due to its mechanism of action and less side effects. Still now no investigations have been performed on use of statistical methods for optimization of media components for the enhanced production of recombinant staphylokinase.Optimization by one-factor-at-a-time method (classical method) involves changing individual variable while fixing the others at a certain arbitrary levels [3]. From large number of factors, a statistical design enables easy selection of important parameters. Initially, one-factor-at-a-time method was used to investigate the effect of media constituents, such as carbon and nitrogen source. Later, the concentration of the medium components were optimized by using orthogonal matrix method and by Design Expert Version 6.0.10 version.