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Purification, kinetic and functional characterization of membrane bound dipeptidyl peptidase-III from NCDC 252: a probiotic lactic acid bacteria

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Abstract

Pediococcus acidilactici is a probiotic lactic acid bacteria possessing studied in-vitro probiotic properties. Study of membrane proteins is crucial in developing technological and health applications of probiotic bacteria. Genome analysis of Pediococcus acidilactici revealed about more than 60 proteases/peptidases which need characterization. Dipeptidyl peptidase-III (DPP-III) is studied for first time in prokaryotes and it is a membrane protein in P. acidilactici that has been purified to apparent homogeneity. The enzyme was purified 81.66 fold with 36.75% yield. The specific activity of purified DPP-III was 202.67 U/mg. The protein moved as single band on native PAGE. The purity was also confirmed by in-situ gel assay. However SDS–PAGE analysis revealed it as high molecular weight heterotetramer with molecular weight of 108 kDa. The enzyme was maximally active at pH 8.5 and at 37 C. Purified DPP-III specifically hydrolyzed Arg-Arg-4-βNA with micromolar affinity (Km = 9.0 µM) and none of studied endopeptidase and monopeptidase substrate was hydrolyzed. Inhibition study revealed purified DPP-III to be a serine protease with involvement of metal ion at active site. The significance of this enzyme as membrane protein is yet to be studied.

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Acknowledgements

Pooja Attri is thankful to Kurukshetra University, Kurukshetra for providing University Research Scholarship (URS).

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Authors and Affiliations

  1. Department of Biochemistry, Kurukshetra University, Kurukshetra, India

    Suman Dhanda

  2. https://www.researchgate.net/profile/Pooja_Attri

    Pooja Attri

  3. https://www.researchgate.net/profile/Drukshakshi_Jodha

    Drukshakshi Jodha

  4. https://www.researchgate.net/profile/Jasbir_Singh12

    Jasbir Singh

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  1. Pooja Attri
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Correspondence to Suman Dhanda.

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Attri, P., Jodha, D., Singh, J. et al. Purification, kinetic and functional characterization of membrane bound dipeptidyl peptidase-III from NCDC 252: a probiotic lactic acid bacteria. Mol Biol Rep 45, 973–986 (2018). https://doi.org/10.1007/s11033-018-4245-1

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  • DOI: https://doi.org/10.1007/s11033-018-4245-1

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