Isolation, Partial Purification and Characterization of Endoglucanase (EC.3.2.1.4) from Aspergillus niger S.L.1 Using Corn Cobs as Carbon Source

Abstract

Endoglucanase (EC3.2.1.4) from Aspergillus niger s.I.1 was isolated, partially purified and characterized. Aspergillus
niger was cultivated for endoglucanase production by the submerged culture technique using processed
corn cobs as carbon source. Xylose was added to the media for cellulose induction. Partial purification was conducted
using ammonium sulphate precipitation, and anion exchange chromatography on DEAE-Sephadex A-50
column. The enzyme was purified 5.26-fold from the crude extract. It migrated homogenously and had a relative
molecular weight of 46 KDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE). It had an optimum pH and temperature of 4.5 and 60oC, respectively. It also had the Km of 0.014
g/ml and Vmax of 0.377μmoles/hr. The Arrhenius plot of the enzyme activity gave activation energy (Ea) of
22.79 kJ/mole, Dixon plot gave extrapolated pKa values of 3.0 and 6.2 suggesting aspartate, glutamate and histidine
as possible amino acids in the catalysis of the enzyme. The activity of endoglucanase was enhanced in the
presence of MgCl2, CoCl2, FeCl3, CaCl2 and FeCl2. This indicates that the enzyme may be a metalloprotein and
that it requires metal ions for its optimum activity. Finally, it was observed that cellulose acetate inhibited the
activity of the enzyme on carboxymethylcelluloe(CMC) in a competitive manner, with an inhibition constant Ki
1.33x10-6 μmole/ml and 5.33x10-6 μmole/ml at the concentration of 0.01 g/ml and 0.04 g/ml respectively. The
significance of the findings has been discussed.