PARTIAL PURIFICATION AND CHARACTERIZATION OF SWEET CORN GERM LIPOXYGENASE

Lipoxygenase (LPO) extracted from the germ fraction of sweet corn (Zea mays L. var. Jubilee) was purified by acetone powder preparation; extraction with 0.1 M Tris-HCl, pH 8; 40–60% fractionation with ammonium sulfate; and conventional column chromatography on Sephacryl S-300 HR and Fast Protein Liquid Chromatography (FPLC) on a Mono Q column. A 124-fold purification was achieved with 26.3% recovery. Further purification was achieved by FPLC chromatofocusing on a Mono P column and size exclusion chromatography on Superose-12, with a resultant 436-fold purification and 16.8% recovery. The apparent molecular weight and isoelectric point (pI) determined by FPLC on Superose-12 and Mono P columns were 90,500 and 5.06, respectively. Lipoxygenase-catalyzed formation of conjugated dienes was inhibited by both synthetic (BHA, BHT) and natural phenolic antioxidants (quercetin, chlorogenic acid) at a concentration of 0.2 mM with 57.2, 16.3, 61.4 and 32.3% inhibition, respectively. The activation energy for thermal inactivation of sweet corn germ lipoxygenase from the ammonium sulfate preparation at 55–70C was 56.3 kcal/mol.     

Lipoxygenase in Sweet Corn Germ: Isolation and Physicochemical Properties

Off-flavor and off-aroma development, which may be catalyzed by lipoxygenase (LPO), are common in frozen stored sweet corn. Lipoxygenase activity in the germ fraction of sweet com (Zea nruys L. cv. Jubilee) was determined and compared with that in the degermed fraction. Lipoxygenase activity/g germ was about three times greater than that of the degermed fraction, Optimized procedures for isolation of lipoxygenase from the germ fraction were developed. Lipoxygenase was isolated by preparation as an acetone powder, extraction with 0.2M TrisHCI, pH 8.0 (4°C), fractionation with 40–60% saturated ammonium sulfate and dialysis. Optimum pH was 6–7 and temperature 50°C for activity of partially purified lipoxygenase. The enzyme appeared stable at pH 5–8 and ～90% of original activity was inactivated after heating in pH 7 buffer at 70°C for 3 min.