Purification and biochemical characterisation of acid phosphatase-I from seeds of Nelumbo nucifera

Acid phosphatase-I (Apase-I) from seeds of Nelumbo nucifera was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50 degrees C and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 mu M, 10 mu mol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn2+, W2+; weakly inhibited by Cu2+, Mo2+ and Cr6+ and moderately activated by Mg2+. The enzyme was shown to be thermolabile as it lost 50% of its activity at 50 degrees C after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase.