Purification and Kinetic Characterization of Statistically Optimized Cellulase Produced from Aspergillus niger

Bio-conversion of cellulosic based biomass materials for cellulase production is one among the major increasing demands for various biotechnological applications. Aspergillus niger was cultured in corn cobs based fermentation medium under some pre-optimized growth conditions. After four days of still culture incubation a large magnitude of cellulase (28.3±0.24U/mL) was achieved when the SSF medium containing 10 g corn cobs inoculated with 5 mL of inoculum at 30°C. The effects of different minerals were optimized through Response Surface Methodology (RSM) by adopting a Central Composite Design (CCD). The crude cellulase was purified 5.71 fold with specific activity of 232.5U/mg using ammonium sulfate precipitation, and Sephadex-G-100 gel filtration column chromatography. Enzyme was found to be a monomeric protein as evident by single band corresponding to 43 kDa on SDS-PAGE. Characterization revealed that the purified cellulase was optimally active and thermally more stable at pH 7 and 70oC, respectively. Using carboxymethyl cellulose as substrate, the enzyme showed maximum activity (Vmax) of 45.5U/mL with its corresponding Km value of 25μM. Among activators/inhibitors, different metal ions (EDTA, Hg2+ and Zn+2) showed inhibitory effect up to different extents as different concentrations, whereas, the enzyme was activated by Co2+ and Mg2+ at the concentration of 5mM.