ISSN 2169-3048
International Journal of Biochemistry and Biotechnology ISSN 2169-3048 Vol. 4 (5), pp. 564-573, June, 2015. © International Scholars Journals
Full Length Research Paper
Solid State Fermentation for the production of Laccase by Neurospora sitophila using agro-wastes and its partial purification
Saqib Hussain Hadri1*, Muhammad Javaid Asad1, Muhammad Gulfraz1, Muhammad Asghar3, Nasir Mahmood Minhas2, Raja Tahir Mahmood1, Shehnaz Zakia4 and Nasir Mahmood5
1Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan.
2Department of Plant Breeding and Genetics, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan.
3Department of Chemistry and Biochemistry, University of Agriculture Faisalabad, Pakistan.
4Pakistan Museum of Natural History, Islamabad, Pakistan.
5Pakistan Science Foundation, Islamabad, Pakistan.
Corresponding Author. E-mail: [email protected]
Abstract
Laccase is the enzyme that has many industrial and biotechnological. Keeping in mind the importance of this enzyme on commercial and industrial levels, the present study was designed to optimize laccase production by Neurospora sitophila using agro-wastes including rice straw, sugarcane bagasse and corn cobs. Among the different conditions optimized, for the enhanced production of the laccase, include fermentation period, moisture level and inoculum size and were found to be 96 hours, 70% (for corn cobs and rice straw), 60% (for sugarcane bagasse) and 5mL respectively. Peptone, yeast extract and tween-20 were used as nitrogen source, inducer and surfactant respectively, to check their effect on the production of laccase. Peptone had negative effect in all concentrations while 0.4 % yeast extract and 0.2 % tween-20 enhanced the laccase production. Ammonium sulfate was used for the partial purification of the laccase and 60% ammonium sulfate was found to be the best concentration for this purpose with a 7.5 fold increase in the laccase activity as compared to the crude extract while using corn cobs as agriculture waste. Protein concentration of crude extract and after each purification step was determined by Biuret-protein assay. Optimum temperature and pH for the laccase activity were found to be 30oC and 5 respectively. Km and Vmax using guaiacol were found to be 0.666mM and 20.8 µM/min respectively.
Key words: Laccase, Neurospora sitophila, Guaiacol, solid state fermentation, ammonium sulfate.