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Extraction and Purification of Ipomoea Peroxidase Employing Three-phase Partitioning

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Abstract

Three-phase partitioning (TPP) is a novel separation process used for the extraction and purification of biomolecules. The biomolecules are recovered in a purified form at the interface (precipitate), while the contaminants partition in t-butanol and aqueous phases. Peroxidase from the leaves of Ipomoea palmata was purified by using TPP. The ratio of the crude extract to t-butanol of 1:1 and 30% ammonium sulfate at 37 °C resulted in about 160% activity recovery and twofold purification in the aqueous phase of the first cycle of TPP. On subjecting the aqueous phase to the second cycle of TPP, a purification of 18-fold was achieved with about 81% activity recovery. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed substantial purification, and the molecular weight of peroxidase was found to be 20.1 KDa. The present study shows a higher degree of purification and activity yield as a primary purification process in comparison with existing literature values, thus demonstrating TPP as an attractive downstream process for the purification of peroxidase.

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Acknowledgments

The authors are thankful to Dr. V.Prakash, Director, CFTRI, Mysore. One of the authors, Mr. A. V. Narayan, is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, for Senior Research Fellowship and also to Dr. S. C. Sharma, Principal, R. V. College of Engineering, Bangalore, for their encouragement.

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Authors and Affiliations

  1. Department of Biotechnology, R.V. College of Engineering, Bangalore, 560 059, India

    A. V. Narayan

  2. Department of Food Engineering, Central Food Technological Research Institute, Mysore, 570 020, Karnataka, India

    M. C. Madhusudhan & K. S. M. S. Raghavarao

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  1. A. V. Narayan
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  2. M. C. Madhusudhan
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  3. K. S. M. S. Raghavarao
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Correspondence to K. S. M. S. Raghavarao.

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Narayan, A.V., Madhusudhan, M.C. & Raghavarao, K.S.M.S. Extraction and Purification of Ipomoea Peroxidase Employing Three-phase Partitioning. Appl Biochem Biotechnol 151, 263–272 (2008). https://doi.org/10.1007/s12010-008-8185-4

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