Purification and partial characterization of phytase from rice bean (Vigna umbellata Thunb.) germinated seeds

Rice bean (Vigna umbellata Thunb.) phytase activity increased during germination and reached maximum at 72 h. The phytate content in seeds decreased with increase in germination time. Phytase was purified 32 fold from 72-h germinated cotyledons with final specific activity 2.22 U/mg. Native PAGE revealed a single band. On SDS PAGE, it revealed two bands with molecular mass 66 and 44 kDa. The native molecular mass was 110 kDa on size exclusion chromatography. The A(280)/(260) ratio was 1.88. When the enzyme was excited at 295 nm, the emission maximum was observed at 330 nm. The FTIR results suggest that Lys, Tyr, Phe, Trp, Ser, Gln and Asn residues on the enzyme's surface. The enzyme was stored at 4 A degrees C, showed 12 % residual activity on 35th day which was improved to 53.6 and 65.7 %, respectively in the presence of additives ascorbic acid and acetaminophen. The optimum pH and temperature of enzyme were 4.0 and 40 A degrees C, respectively. The energy of activation was 32.2 kJ/mol. The values of K (m) and V (max) were 0.197 mM and 2.35 mu mol/min/mg protein, respectively with sodium phytate as substrate. Phytase showed broad substrate specificity. The k (cat)/K (m) ratio was the highest for sodium phytate.