Purification and Properties of Glucose 6-Phosphate Dehydrogenase From Coriander (Coriandrum Sativum) Leaves

Abstract

Glucose‐6‐phosphate dehydrogenase (D‐glucose‐6‐phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from coriander (Coriandrum sativum) leaves; the kinetic behavior and some properties of the enzyme were also investigated. The purification was done at 4C and involved two steps: ammonium sulfate fractionation, and DEAE‐Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 26.4% and had a specific activity of 1.826 U/mg protein. Optimum pH, stable pH, optimum temperature, molecular weight, KM and Vmax values for NADP+ and glucose 6‐phosphate (G6‐P) were also determined. The overall purification was about 74‐fold. SDS‐PAGE of the purified enzyme showed a single band. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. The molecular mass was estimated to be 74.4 kDa by SDS‐PAGE and 73.2 kDa by Sephadex G‐200 gel filtration column chromatography. The enzyme had an optimum pH at 8.5 and was stable at pH 8.0 in 0.1 M Tris‐HCl buffer. The optimum temperature was at 30C. The KM values for NADP+ and G6‐P were 0.026 mM and 0.116 mM, respectively. The Vmax values for these substrates were 0.035 EU/mL and 0.038 EU/mL, respectively.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

“…The subunit molecular weight of pigeon pea glucose-6-phosphate dehydrogenase was found to be quite close to the molecular weight of enzyme obtained from Cryptococcus neoformans (50 KDa) [32], A. vinelandii (52 KDa) [33], dog liver (52.5 KDa) [34], A. aculeatus (52 ± 1.1 KDa) [15], goat erythrocyte (52 KDa) [35]. However, the value is lower than as reported from Pseudomonas W6 [29], pig liver [20], turkey erythrocyte [36], coriander [24] and rainbow trout [37]. The result revealed that pigeon pea glucose-6-phosphate dehydrogenase is a homodimer with molecular weight of 113 KDa and subunit molecular weight approximately equals to 55 KDa.…”

“…than reported from microbial and animal sources [15] [35] [38]. The V max value was found to be greater than the value reported from coriander (0.038 U/ml) [24]. The V max value from other plant sources has not been reported.…”

“…The K m and V max value for glucose-6-phosphate was found to be 2.68 mM and 0.11 U/ml respectively. The K m value was found to be higher than other reported K m values in plant sources such as pea leaves (2000 µM) [23], potato tuber (260 µM) [11] and coriander (116 µM) [24]. The K m value was also greater log Molecular Weight logM.Wt.…”

“…The results are shown in Figure 8. The K m and V max for NADP + using Lineweaver-Burk plot was found to be 0.75 mM and 0.13 U/ml which was greater than that of values reported from pea leaves (500 µM) [23], potato (6 µM) [11] coriander (26 µM, 0.035 U/ml) [24], rat liver (100 µM) [40], Acetobacter hansenii (340 µM) [41], goose erythrocyte (7.4 µM, 0.286 U/ml) [40], turkey erythrocyte (17.1 µM, 0.37 U/ml) [36] and rainbow trout erythrocyte (166 µM, 0.275 U/ml) [37]. However, the K m value of NADP + was found to be less than that of glucose-6-phosphate which indicates that glucose-6-phosphate dehydrogenase shows more affinity towards NADP [42].…”

“…The reports on purification of glucose-6-phosphate dehydrogenase from plant sources are very less. However, some attempts have been made for the purification of enzyme from plant sources, such as from spinach leaf [21], soybean nodules [22], potato tuber [11], pea leaves [23] and coriander [24]. The enzyme is remarkable for its genetic diversity and many variants produced due to missense mutation have been described with wide ranging levels of enzyme activity and associated clinical symptoms.…”