Oryzasin as an Aspartic Proteinase Occurring in Rice Seeds: Purification, Characterization, and Application to Milk Clotting

An aspartic proteinase in rice seeds (oryzasin) was purified by (NH 4 ) 2 SO 4 fractionation, DEAE-celluose anion exchange chromatography, Sephadex G-100 gel filtration, Mono Q anion exchange chromatography, and pepstatin-affinity chromatography. SDS−PAGE showed the affinity-purified enzyme to have two molecular forms, 57 and 53 kDa, together with their probable autolysates appearing as two small bands at 35 and 25 kDa. Compared with the other three bands, the 57 kDa band reacted strongly on western blot analysis. The affinity-purified oryzasin pH optimum for hydrolysis is 3.0 and is completely inhibited by pepstatin but not affected by other proteinase inhibitors such as EDTA, leupeptin, PMSF, and E-64. The milk-clotting activity of oryzasin was investigated using the crude enzyme obtained by precipitation at 30% and 60% (NH 4 ) 2 SO 4 saturation. The enzyme clotted a skim milk solution at pH 6.3, yielding the same κ-casein digest pattern as those of chymosin and pepsin producing a 12 kDa band.