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Membrane Bound Aminopeptidase B of a Potential Probiotic Pediococcus acidilactici NCDC 252: Purification, Physicochemical and Kinetic Characterization

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Abstract

An arginine aminopeptidase (EC 3.4.11.6) called aminopeptidase B was purified to apparent homogeneity from membrane extract of a potential probiotic Pediococcus acidilactici NCDC 252 using successive chromatographies on sephadex G-100 and phenylsepharose CL-4B. Purified enzyme was a heterotrimer with molecular mass of ~ 101.36 kDa. Predicted molecular weight of the enzyme from its gene (93.9 kDa) was close to the calculated molecular weight. The enzyme was optimally active at pH 7.5 and 40 °C. It was strongly inhibited by metal chelating agent and thiol protease inhibitors suggesting that enzyme is a metalloprotease with involvement of thiol. The Km and Vmax of enzyme for Arg-4mβNA were calculated to be 26 μM and 19.9 nmol/ml/min respectively. Its 3-D structure was modeled and validated using in-silico approach. In-silico analysis revealed Ser, His, Phe, Tyr and Thr to be present at active site of aminopeptidase B. Docking studies revealed that Arg-4mβNA binds with high affinity to the enzyme followed by Lys-4mβNA. The enzyme also hydrolyzed dipeptide-4mβNA derivatives containing hydrophobic amino acids and diaminocarboxylic acids (Arg, Lys and Asp) at the N-termini but not tripeptides, endopeptidase substrates and -βNA derivatives or peptides with proline and phenyl at their N-termini or C-termini.

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Acknowledgement

Pooja Attri is thankful to Kurukshetra University, Kurukshetra for financial help in the form of University Research Scholarship.

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Authors and Affiliations

  1. Kurukshetra University, Kurukshetra, India

    Pooja Attri, Drukshakshi Jodha & Poonam Bansal

  2. Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, 136119, India

    Jasbir Singh & Suman Dhanda

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  1. Pooja Attri
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  2. Drukshakshi Jodha
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  3. Poonam Bansal
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  4. Jasbir Singh
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  5. Suman Dhanda
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Correspondence to Suman Dhanda.

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Attri, P., Jodha, D., Bansal, P. et al. Membrane Bound Aminopeptidase B of a Potential Probiotic Pediococcus acidilactici NCDC 252: Purification, Physicochemical and Kinetic Characterization. Int J Pept Res Ther 27, 1641–1655 (2021). https://doi.org/10.1007/s10989-021-10197-w

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