International Journal of ChemTech Research CODEN (USA): IJCRGG ISSN: 0974-4290 Vol.7, No.01, pp 355-368, 2014-2015 Laccase production using mixed substrates containing lignocellulosic materials by Pleurotus ostreatus in submerged liquid culture Bakkiyaraj Selvaraj, Arrivukkarasan Sanjeevirayar* and Aravindan Rajendran Biochemical Engineering Laboratory, Department of Chemical Engineering, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India. Abstract: Enhanced production of the industrially important enzyme laccase from the white rot fungus Pleurotus ostreatus MTCC1804 was performed in the submerged liquid culture. The preliminary media component indispensable to increase the laccase production were screened by the classical or one factor at a time technique. The consequence of different lignocellulosic substrates on laccase production in single and combined or mixed substrate mode were investigated under controlled fermentation conditions. From the experimental study it was revealed that, the mixed substrates including rice bran, sugarcane bagasse and glucose rendered maximum laccase activity of 6471U/l than the productivities of individual substrates. Screening of the appropriate nitrogen source permitted to substantial improvement in the laccase production of 8433 U/l by the combined nitrogen sources viz, yeast extract and L-aspargine monohydrate of organic nitrogen sources. The stimulatory effect of various aromatic inducers on the production of extracellular laccase yield was observed. Out of eleven aromatic inducers explored, 2,5 xylidine enhanced the laccase production to 17542 U/l. The statistical analysis for screening of essential media components by Plackett-Burman design was implemented and maximum laccase production of 37452 U/l was obtained in the 9 th day of fermentation process. KeywordsLaccase, Pleurotus ostreatus, Lignocellulosic substrates, 2,5 xylidine, Plackett-burmann design. 1.0 Introduction Laccases (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) are one of the blue multi copper enzymes (BMCE), which belong to the ligninolytic enzymatic system of fungi, particularly from the white rot fungi. They catalyze the oxidation of number of organic and inorganic compounds including monophenols, diphenols, polyphenols, methoxyphenols and aromatic amines, with the concomitant four electron reduction of oxygen to water 1,2 . Laccases are widely distributed among fungi 3,4,5 , higher plants 6,7,8 , in some bacteria 9,10 and in several insects 11,12 . Laccases from various sources plays several physiological functions in different manners. In fungi, they are involved in various biological functions viz, morphogenesis, fungal-plant pathogen/host interaction, stress defense, and lignin degradation 2,13 . Plant laccases are participated in the lignification process 14 , Bacterial laccases involved in pigment synthesis 15 and in insects they are involved in cuticle sclerotization process 11,12 . Among the microbial populations, fungi are recognized as the prominent source by the researchers, due to their extreme facility in producing a large variety of extracellular enzymes and easy handling. They are the specific organisms responsible for lignocellulose degradation; among them wood-rotting Basidiomycetous fungi are identified as the best degrader of lignocellulosic materials 16 . The outstanding ability of secreting the varieties of hydrolytic and oxidative enzymes by the white rot fugal group makes them to grow efficiently on the lignocellulosic substrates and they are very indispensable for lignin, cellulose and hemicellulose degradation. The hydrolytic enzymes often identified from the cultures of white rot fungal broth are endo-1, 4- ß- D-glucanase (EC 3.2.1.4), exo-1,4-ß-D-glucanase (EC 3.2.1.91), and xylanase (EC 3.2.1.8). The lignin modifying enzymes such as lignin peroxidase (EC 1.11.1.14), manganese-dependent peroxidase (EC 1.11.1.13),