A low molecular weight alkaline serine protease from the seeds of Nelumbo nucifera:: Purification and characterization

A low molecular weight alkaline serine protease from the seeds of Nelumbo nucifera was purified to electrophoretic homogeneity. Three steps of purification consisting of ammonium sulfate precipitation, gel filtration and ion exchange chromatography techniques were employed to achieve purified alkaline serine protease. Its molecular mass was estimated by SDS-PAGE to be 24 kDa. The optimum pH of the enzyme was 8.0 using 1% casein as a substrate. The enzyme exhibited maximum activity at 40 degrees C. The K-m and V-max of the enzyme were found to be 2.8 x 10(-4)M and 200 mu M/min. respectively. The purified enzyme was strongly inhibited by PMSF, indicating the presence of serine residue at the active site and was weakly inhibited by EDTA. The enzyme activity was enhanced in the presence of Ca++ and weakly inhibited by Cu++. Na+ showed no effect on the enzyme activity while slight changes in the enzyme activity were observed in the presence of K+ and Mg++.