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Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

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Abstract

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266 × 103 units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25 kDa. They showed temperature optima at 50°C and pH optima at 5.0. km for SPases was reported to be 1.05 mM and 0.186 mM for guaiacol and H2O2 respectively. The Vmax/Km value for o-dianisidine was 449 while for H2O2 it was 5 × 105. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5 μM) but ferulic acid inhibits its activity at a much lower concentration (0.02 μM). Enzymes were stimulated by metal cations like Cu2+, Ca2+ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn2+ and Mg2+. Alanine stimulated the enzyme activity (up to 33%; at 0–100 μM) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13–57% at 0–100 μM).

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Abbreviations

SPase:

Sapindus peroxidase

HRP:

Horse radish peroxidase

CM-cellulose:

Carboxymethyl cellulose

Vo:

Void volume

Ve:

Elution volume

kDa:

kiloDalton

Native-PAGE:

Native-Polyacrylamide Gel Electrophoresis

SDS-PAGE:

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

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Acknowledgments

One of us (KP) is highly grateful to D.D.U. Gorakhpur University for providing annual grants.

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Authors and Affiliations

  1. Department of Biotechnology, Panjab University, Chandigarh, 160 014, India

    Jagtar Singh

  2. Department of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh, India

    Pradeep Kumar

  3. Department of Biotechnology, SGGS College, Sector-26, Chandigarh, India

    Amit Joshi

  4. Department of Biotechnology, GGDSD College, Sector-32, Chandigarh, India

    Navneet Batra

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  1. Jagtar Singh
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  2. Pradeep Kumar
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  3. Navneet Batra
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  4. Amit Joshi
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Correspondence to Jagtar Singh.

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Singh, J., Kumar, P., Batra, N. et al. Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi . J. Plant Biochem. Biotechnol. 21, 11–16 (2012). https://doi.org/10.1007/s13562-011-0065-2

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  • DOI: https://doi.org/10.1007/s13562-011-0065-2

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