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Partial purification and biochemical characterization of peroxidase from rosemary (Rosmarinus officinalis L.) leaves.

Aghelan Z, Shariat SZ - Adv Biomed Res (2015)

Bottom Line: PODs are a group of oxidoreductase enzymes that catalyze the oxidation of a wide variety of phenolic compounds in the presence of hydrogen peroxide as an electron acceptor.Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed for molecular weight (Mw) determination and Mw of the enzyme was found to be 33 kDa.The stability against high temperature and extreme pH demonstrated that the enzyme could be a potential POD source for various applications in the medicine, chemical and food industries.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

ABSTRACT

Background: In this study, it is aimed to purify POD from leaves of Rosmarinus officinalis L. and determine its some biochemical properties. PODs are a group of oxidoreductase enzymes that catalyze the oxidation of a wide variety of phenolic compounds in the presence of hydrogen peroxide as an electron acceptor.

Materials and methods: In this investigation, POD was purified 9.3-fold with a yield of 32.1% from the leaves of Rosemary by ammonium sulfate precipitation and ion-exchange chromatography. The enzyme biochemical properties, including the effect of pH, temperature and ionic strength were investigated with guaiacol as an electron donor. For substrate specificity investigation of the enzyme, Michaelis constant and the maximum velocity of an enzymatic reaction values for substrates guaiacol and 3,3', 5,5'-TetraMethyle-Benzidine were calculated from the Lineweaver-Burk graphs.

Results: The POD optimum pH and temperature were 6.0 and 40°C. The POD activity was maximal at 0.3 M of sodium phosphate buffer concentration (pH 6.0). Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed for molecular weight (Mw) determination and Mw of the enzyme was found to be 33 kDa. To investigate the homogeneity of the POD, native-PAGE was done and a single band was observed.

Conclusion: The stability against high temperature and extreme pH demonstrated that the enzyme could be a potential POD source for various applications in the medicine, chemical and food industries.

No MeSH data available.


Thermal stability of the purified peroxidase from rosemary leaves. The enzyme was incubated at the 40°C, 50°C and 60°C for fixed time intervals (10, 20, 30, 40, 50 and 60). Data are shown as a mean of the activity ± standard deviation of n = 3 experiments
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Figure 4: Thermal stability of the purified peroxidase from rosemary leaves. The enzyme was incubated at the 40°C, 50°C and 60°C for fixed time intervals (10, 20, 30, 40, 50 and 60). Data are shown as a mean of the activity ± standard deviation of n = 3 experiments

Mentions: Peroxidase from the leaves of rosemary was isolated and purified through two steps of ammonium sulphate fractionation and DEAE-cellulose chromatography. The fraction obtained in 80–90% saturation range showed the maximum activity. As the result of this step, the POD was purified to 3.0-fold with a yield of 66.2%. The enzyme then loaded on a DEAE-cellulose column and bound proteins were eluted with a linear gradient of 0.0–0.5 M Nacl in 0.02 M phosphate buffer (pH 8.0) at a flow rate of 0.7 ml/min [Figure 1]. The result of the purification of POD from the rosemary leaves is shown in Table 1. As can be seen in figure 2, this enzyme exhibited a single band on native-PAGE and SDS-PAGE that was suggestive of the homogeneity of the purified POD. The MW of the purified POD was estimated as 33 kDa according to the MW markers on SDS-PAGE [Figure 2]. The effect of temperature on the purified POD was studied at different temperatures from 10°C to 80°C. The enzyme was heat stable over a wide range of temperatures (0–70°C). The optimum temperature of the POD was found to be 40°C for guaiacol [Figure 3]. In addition, this enzyme can be stable at 60°C for 10 min [Figure 4]. The effect of pH on the enzyme was determined by measuring the activity at different pH range of 2.0–9.0 [Figure 5]. The enzyme showed the highest activity in pH 6.0. The effect of ionic strength on the POD activity was determined at a fixed concentration of guaiacol and H2O2 by different concentrations of sodium phosphate buffer, pH 6.0 (0.05–2 M). The maximum activity was observed in 0.3 M of phosphate buffer concentration at pH 6.0 [Figure 6]. Km and Vmax values were calculated for guaiacol, TMB and H2O2 substrates from Lineweaver-Burk graphs. The Km values of the POD for guaiacol, TMB and H2O2 substrates were 28.8, 1.06 and 8.5 mM, respectively. Indeed, Vmax values were 0.312, 0.194 and 1.15 mM/min for above mentioned substrates, respectively [Figure 7].


Partial purification and biochemical characterization of peroxidase from rosemary (Rosmarinus officinalis L.) leaves.

Aghelan Z, Shariat SZ - Adv Biomed Res (2015)

Thermal stability of the purified peroxidase from rosemary leaves. The enzyme was incubated at the 40°C, 50°C and 60°C for fixed time intervals (10, 20, 30, 40, 50 and 60). Data are shown as a mean of the activity ± standard deviation of n = 3 experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4550948&req=5

Figure 4: Thermal stability of the purified peroxidase from rosemary leaves. The enzyme was incubated at the 40°C, 50°C and 60°C for fixed time intervals (10, 20, 30, 40, 50 and 60). Data are shown as a mean of the activity ± standard deviation of n = 3 experiments
Mentions: Peroxidase from the leaves of rosemary was isolated and purified through two steps of ammonium sulphate fractionation and DEAE-cellulose chromatography. The fraction obtained in 80–90% saturation range showed the maximum activity. As the result of this step, the POD was purified to 3.0-fold with a yield of 66.2%. The enzyme then loaded on a DEAE-cellulose column and bound proteins were eluted with a linear gradient of 0.0–0.5 M Nacl in 0.02 M phosphate buffer (pH 8.0) at a flow rate of 0.7 ml/min [Figure 1]. The result of the purification of POD from the rosemary leaves is shown in Table 1. As can be seen in figure 2, this enzyme exhibited a single band on native-PAGE and SDS-PAGE that was suggestive of the homogeneity of the purified POD. The MW of the purified POD was estimated as 33 kDa according to the MW markers on SDS-PAGE [Figure 2]. The effect of temperature on the purified POD was studied at different temperatures from 10°C to 80°C. The enzyme was heat stable over a wide range of temperatures (0–70°C). The optimum temperature of the POD was found to be 40°C for guaiacol [Figure 3]. In addition, this enzyme can be stable at 60°C for 10 min [Figure 4]. The effect of pH on the enzyme was determined by measuring the activity at different pH range of 2.0–9.0 [Figure 5]. The enzyme showed the highest activity in pH 6.0. The effect of ionic strength on the POD activity was determined at a fixed concentration of guaiacol and H2O2 by different concentrations of sodium phosphate buffer, pH 6.0 (0.05–2 M). The maximum activity was observed in 0.3 M of phosphate buffer concentration at pH 6.0 [Figure 6]. Km and Vmax values were calculated for guaiacol, TMB and H2O2 substrates from Lineweaver-Burk graphs. The Km values of the POD for guaiacol, TMB and H2O2 substrates were 28.8, 1.06 and 8.5 mM, respectively. Indeed, Vmax values were 0.312, 0.194 and 1.15 mM/min for above mentioned substrates, respectively [Figure 7].

Bottom Line: PODs are a group of oxidoreductase enzymes that catalyze the oxidation of a wide variety of phenolic compounds in the presence of hydrogen peroxide as an electron acceptor.Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed for molecular weight (Mw) determination and Mw of the enzyme was found to be 33 kDa.The stability against high temperature and extreme pH demonstrated that the enzyme could be a potential POD source for various applications in the medicine, chemical and food industries.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

ABSTRACT

Background: In this study, it is aimed to purify POD from leaves of Rosmarinus officinalis L. and determine its some biochemical properties. PODs are a group of oxidoreductase enzymes that catalyze the oxidation of a wide variety of phenolic compounds in the presence of hydrogen peroxide as an electron acceptor.

Materials and methods: In this investigation, POD was purified 9.3-fold with a yield of 32.1% from the leaves of Rosemary by ammonium sulfate precipitation and ion-exchange chromatography. The enzyme biochemical properties, including the effect of pH, temperature and ionic strength were investigated with guaiacol as an electron donor. For substrate specificity investigation of the enzyme, Michaelis constant and the maximum velocity of an enzymatic reaction values for substrates guaiacol and 3,3', 5,5'-TetraMethyle-Benzidine were calculated from the Lineweaver-Burk graphs.

Results: The POD optimum pH and temperature were 6.0 and 40°C. The POD activity was maximal at 0.3 M of sodium phosphate buffer concentration (pH 6.0). Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed for molecular weight (Mw) determination and Mw of the enzyme was found to be 33 kDa. To investigate the homogeneity of the POD, native-PAGE was done and a single band was observed.

Conclusion: The stability against high temperature and extreme pH demonstrated that the enzyme could be a potential POD source for various applications in the medicine, chemical and food industries.

No MeSH data available.