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°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°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 μmol/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.

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The immobilization of phytase from rice bean was optimized on the glutaraldehyde-activated chitosan microsphere, and characterized. The optimum percent immobilization was 77.48%, when microspheres were prepared with 1.5% chitosan, activated with glutaraldehyde (0.5%, for 4 h) and with the enzyme protein (0.2 mg/mL, and coupling time 4 h). The immobilized phytase exhibited elongated fibres and pores. FTIR results suggest the formation of a Schiff base with a band at 1638 cm-1. The pH and temperature optimum of immobilized phytase was 4.0 and 40 °C, respectively. The energy of activation was 34.5 kJ/mol. The Km and Vmax values of immobilized phytase were 0.62 mM and 3.42 μmol/min. The immobilized enzyme when incubated at 50 °C, retained 59% activity after 75 min. The immobilized phytase showed 80% activity retention, after 14 days, when stored at 4 °C. The immobilized phytase could be reused for 4 cycles with 58% activity retention. The immobilized phytase, when incubated for 60 min in the presence trypsin and pepsin, showed 92.9 and 95% activity retention, respectively. The properties of immobilized phytase did not alter with respect to pH, and temperature optima. Immobilized phytase exhibited proteolytic resistance when incubated with pepsin for 1 h, and this can find application in animal feed.

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