title: Purification and biochemical characterisation of acid phosphatase-I from seeds of Nelumbo nucifera.
abstract: 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°C and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 μM, 10 μmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn(2+), W(2+); weakly inhibited by Cu(2+), Mo(2+) and Cr(6+) and moderately activated by Mg(2+). The enzyme was shown to be thermolabile as it lost 50% of its activity at 50°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.
authors.affiliation: a Department of Bioscience , COMSATS Institute of Information Technology Islamabad , Islamabad , Pakistan.
authors.forename: Sanaullah
authors.initials: S
authors.lastname: Khan
authors.affiliation: c Department of Chemistry , University of Science and Technology , Bannu , Pakistan.
authors.forename: Shahnaz
authors.initials: S
authors.lastname: Khan
authors.affiliation: a Department of Bioscience , COMSATS Institute of Information Technology Islamabad , Islamabad , Pakistan.
authors.forename: Sajida
authors.initials: S
authors.lastname: Batool
authors.affiliation: b Department of Biotechnology , University of Science and Technology , Bannu , Pakistan.
authors.forename: Mushtaq
authors.initials: M
authors.lastname: Ahmed
mesh_terms: D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D031653:Nelumbo / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*; D013696:Temperature
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D008670:Metals; D000135:Acid Phosphatase
keywords: Nelumbo nucifera seeds; acid phosphatase-I; biochemical characterisation; purification