Purification and Characterization of Peroxidase from Sweet Gourd (Cucurbita moschata Lam. Poiret)

Figures & data

Table 1 Levels of purification of sweet gourd (Cucurbita moschata L.) peroxidase obtained after the application of different purification steps leading to the improvement in enzyme activity

Purification steps	Total volume (mL)	Enzyme activity (EU/mL.min)	Total enzyme activity (EU/mL.min)	Protein (mg/mL)	Total protein (mg)	Specific activity (EU/mg)	Yield (%)	Purification fold
Homogenate	23	6660	153,180	0.804	18.5	8280	100	1
(NH4)2SO4 precipitation	4	7000	28,000	0.749	3	9333.3	18	1.127
CM-Sephadex ion exchange chromatography	15	1240	18,600	0.22	3.3	84,545.4	12	10.2

Figure 1 Cation exchange chromatography of POD from sweet gourd (Cucurbita moschata L.): elution profile of unbound fraction from CM-Sephadex A-50 obtained in 0.1 M sodium phosphate buffer (pH 8.0) as 5-mL fractions.

Figure 2 Photograph of SDS-PAGE. Line 1: molecular weight markers. Line 2: the peroxidase from CM-Sephadex cation exchange chromatography. (Color figure available online.)

Figure 3 The effect of incubation period on the peroxidase activity from sweet gourd (Cucurbita moschata L.) at different pH values: (a) acetate buffers; (b) phosphate buffers; (c) tris/HCl buffers.

Table 2 Optimum pH, optimum temperature, optimum ionic strength, stable pH, thermal stabilization, and substrate specificity of peroxidase from sweet gourd (Cucurbita moschata L.)

Kinetic and optimum properties
Optimum pH	7.2
Optimum temperature (°C)	50
Optimum ionic strength (M)	0.4
Stable pH	8.0
Stable temperature (°C)	40
For guaiacol Km (mM)	17.1
For guaiacol V max (EU/mL.min)	15,500
For H2O2 Km (mM)	3.8
For H2O2 V max (EU/mL.min)	15,889

Figure 5 Thermal inactivation change of the peroxidase activity from sweet gourd (Cucurbita moschata L.).

Figure 5 Kinetic behavior of the two substrate reactions for sweet gourd (Cucurbita moschata L.) POD: (a) plot of guaiacol substrate; (b) plot of H₂O₂ substrate.