548 Journal of Basic Microbiology 2010, 50, 548 – 556 © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com Research Paper Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology Rathinasamy Periasamy and Thayumanavan Palvannan Department of Biochemistry, Periyar University, Salem, Tamil Nadu, India Production of laccase using a submerged culture of Pleurotus orstreatus IMI 395545 was opti- mized by the Taguchi orthogonal array (OA) design of experiments (DOE) methodology. This approach facilitates the study of the interactions of a large number of variables spanned by factors and their settings, with a small number of experiments, leading to considerable savings in time and cost for process optimization. This methodology optimizes the number of impact factors and enables to calculate their interaction in the production of industrial enzymes. Eight factors, viz. glucose, yeast extract, malt extract, inoculum, mineral solution, inducer (1 mM CuSO 4 ) and amino acid (L-asparagine) at three levels and pH at two levels, with an OA layout of L18 (2 1 × 3 7 ) were selected for the proposed experimental design. The laccase yield obtained from the 18 sets of fermentation experiments performed with the selected factors and levels was further processed with Qualitek-4 software. The optimized conditions shared an enhanced laccase expression of 86.8% (from 485.0 to 906.3 U). The combination of factors was further validated for laccase production and reactive blue 221 decolorization. The results revealed an enhanced laccase yield of 32.6% and dye decolorization up to 84.6%. This methodology allows the complete evaluation of main and interaction factors. Keywords: Laccase / Taguchi DOE methodology / Pleurotus ostreatus IMI 395545 / Optimization / Dye decolorization Received: March 14, 2010; accepted: April 29, 2010 DOI 10.1002/jobm.201000095 Introduction * Laccase (benzenediol:oxygen oxidoreductase [EC.1.10.3.2]) belongs to a group of enzymes called blue copper oxi- dases capable of oxidizing phenols and aromatic amines by reducing molecular oxygen to water. Laccase is widespread in nature and has been found in plants, fungi, bacteria and insects [1]. Laccase plays an impor- tant role in the global carbon cycle and could help in degrading a wide range of xenoaromatics such as tex- tile dyes [2], polychlorinated biphenyls, polycyclic aro- matic hydrocarbons, pesticides and synthetic polymers [3, 4]. Extensive studies made on fungal laccase have proved its potential in the various fields of biotechnol- ogy and created a great market demand for commercial Correspondence: Dr. T. Palvannan, Department of Biochemistry, Peri- yar University, Salem – 636 011, Tamil Nadu, India E-mail: pal2912@yahoo.com Phone: +91-427-2345766, -2345520 Fax: +91-427-2345124 applications like wastewater detoxification [5, 6], deter- gent manufacturing and transformation of antibiotics and steroids [7]. The wide range of applications of lac- case in the biotechnological and textile industries cre- ates the need for large amounts of enzymes at low cost to meet the market demand. The main limitation for the extensive industrial ap- plication of laccase is its high cost. To attain the pro- duction of a large amount of enzyme at low cost, media optimization plays a crucial role. The optimization of fermentation media to generate a balanced proportion of various nutrients is very important to get optimum microbial growth and enzyme yield [8]. Laccase expres- sion by fungi is found to be influenced by the applied culture conditions, such as the nature of the carbon source, the concentration of a carbon source, the pH of the fermentation broth, the presence of inducers, the presence of lignocellulosic materials and a nitrogen source [5, 9 – 11]. It is essential to optimize all the cul- ture conditions and the composition of the production