Production, partial purification and characterization of cellulase by aspergillus niger from submerged fermentation of grape bagasse

Thesis Abstract

The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study the ability of selected cellulosic substrate to induce cellulase production by Aspergillus niger and the ability of the induced enzyme to hydrolyze the cellulosic substrate were assessed. The enzyme was produced by submerged fermentation technique in which grape bagasse was the cellulosic substrate which served as a carbon source. Crude enzyme was harvested after 5 days of growth with activity of 8.2μmole/min for enzyme produced by Aspergillus niger. Cellulase produced from Aspergillus niger was subjected to a three step purification process 50% ammonium sulphate precipitation, dialysis and gel column chromatography for characterization of the cellulase. The gel column chromatography yielded two peaks. Gel elution fractions were assayed for total cellulase activity and protein concentration. The 2 peaks indicate isoforms of the enzyme produced by Aspergillus niger. The total cellulase activity as well as β-glucosidases activity was characterized using filter paper and cellobiose as substrate. The partially purified enzyme showed that total cellulase activity had an optima pH and temperature of 5.5 and 50oC for isoform A and 5.0 and 55oC for isoform B using filter paper as substrate. Similarly, β-glucosidases activity had an optima pH 5.5 and 6.0 with optima temperature of 50oC for both isoforms using cellobiose as substrate. Kinetic parameter showed a Vmax and Km of 90.9μmole/min and 0.09mM cellobiose and 83.3μmole/min and 0.08mM cellobiose forr both isoforms respectively. This kinetic study showed that grape bagasse is a good substrate for cellulase from Aspergillus niger and can be utilized as substrate for cellulase production. These results obtained in this study have established suitable conditions for maximizing the production of cellulase which is used for conversion of high cellulosic waste into wealth as found in bioethanol production.