Abstract
Acid phosphatases play a crucial role in food processing industries to reduce phosphate content of food. Here in acid phosphatase from the seeds of Macrotyloma uniflorum has been purified to homogeneity using UNOsphere-S cation exchange chromatography followed by gel filtration with 81.85 fold purification. Molecular weight of purified enzyme was 55,000 (± 1040) Daltons under physiological conditions. It was a heterodimer of subunits having molecular weights 27,093 and 28,241 Daltons as determined by MALDI-TOF analysis. The optimum pH and temperature for the purified enzyme was 5.0 and 50 °C respectively. The enzyme was stable in the pH range 3.5-5.5 and showed temperature stability up to 60 °C. Substrate specificity of enzyme was checked with different substrates namely, p-nitrophenyl phosphate (p-NPP), ATP, ADP, glucose 6-phosphate, glucose-1-phosphate, fructose 6-phosphate, phenyl phosphate, α-naphthyl-phosphate, pyridoxyl phosphate and β-glycerophosphate. Enzyme showed high substrate specificity towards p-NPP, phenyl phosphate, ATP and α-naphthyl phosphate. Km and Vmax of enzyme were found to be 0.934 mM and 1.333 mM/min respectively with respect to p-NPP as a substrate. Chemical modification studies showed that tryptophan was present at the active site of the enzyme.