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

Abstract

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.

Immobilization of Phytase from Rice Bean (Vigna umbellata Thunb.) on Glutaraldehyde Activated Chitosan Microspheres

“…The other chemicals were of AR Grade from India. Phytase purified from germinated rice bean seeds was used (Belho et al, 2016). De-ionized water was collected from the Milli-Q system (Millipore USA).…”

“…The free phytase activity was assayed by discontinuous spectrophotometric method, using 100 mM sodium acetate buffer, pH 4.0, as assay buffer as described earlier (Belho et al, 2016). For the assay of immobilized phytase activity, the reaction mixture contained 1.5 mM sodium phytate (0.35 mL), and the assay buffer (0.05 mL) was maintained at 40 °C.…”

“…The reaction was stopped by removal of the microspheres. The rest of the process was as described earlier (Belho et al, 2016).…”

“…The study of the phytase is therefore considered to be very important. It has been purified from some plant sources and characterized (Gibson & Ullah, 1988;Konietzny et al, 1995;Mahajan & Dua, 1997;Phillippy, 1998;Greiner et al, 1998Greiner et al, , 2001Greiner & Alminger 1999;Greiner, 2002;Andriotis & Ross, 2003;Belho et al, 2016;Belho & Ambasht, 2017). Immobilization of phytase has been pursued mostly from microbial sources like Aspergillus ficuum (Liu et al, 1999;Moraes et al, 2008), A. niger (Menezes-Blackburn et al, 2011;Trouillefou et al, 2015;Coutinho et al, 2020), A. heteromorphus (Lata et al, 2014), Escherichia coli (Menezes-Blackburn et al, 2011;Cho et al, 2011;El-Shora et al, 2019), Candida krusei (Quan et al 2003), and mushrooms (Onem & Nadaroglu, 2014;Onem et al, 2016).…”

A novel purple acid phytase from an earthworm cast bacterium

“…Depending on optimum pH, acid and alkaline phytases can be distinguished. In general, acid phytases exhibit rather broad substrate specificity, whereas alkaline phytases show narrow substrate selectivity . So far, phytases have found application solely as feed supplements.…”