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Purification and Characterization of Carboxylesterase from the Seeds of Jatropha curcas

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Abstract

Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The Km of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).

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Abbreviations

JCSE:

Jatropha curcas seed esterase

PAGE:

Polyacrylamide gel electrophoresis

SDS-PAGE:

Sodiumdodecylsulphate–polyacrylamide gel electrophoresis

CM-cellulose:

Carboxymethyl-cellulose

rpm:

Revolution per minute

DBLS:

Diazo blue B lauryl sulphate

kDa:

Kilodalton

Ops:

Organophosphates

IEF:

Iso-electrofocusing

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Acknowledgments

The first author, T. Subramani wishes to gratefully acknowledge, UGC sponsored Rajiv Gandhi National fellowship to carry out present work.

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Authors and Affiliations

  1. Department of Biochemistry, Bangalore University, Central College Campus, 560085, Bangalore, Karnataka, India

    T. Subramani, N. Ramachandra Swamy & K. R. Siddalinga Murthy

  2. Department of Biotechnology, PES Institute of Technology, BSK III Stage, Hosakerahalli, Bangalore, 560085, Karnataka, India

    K. S. Chandrashekharaiah

Authors
  1. T. Subramani
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  2. K. S. Chandrashekharaiah
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  3. N. Ramachandra Swamy
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  4. K. R. Siddalinga Murthy
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Correspondence to K. S. Chandrashekharaiah or K. R. Siddalinga Murthy.

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Subramani, T., Chandrashekharaiah, K.S., Swamy, N.R. et al. Purification and Characterization of Carboxylesterase from the Seeds of Jatropha curcas . Protein J 31, 120–128 (2012). https://doi.org/10.1007/s10930-011-9380-7

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