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Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander

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Abstract

Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699 bp flanked by 5′UTR and 3′UTR of 134 bp and 198 bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4 kDa, which established a specific activity of 2617 units mg−1 of protein and under native condition yield bands of 52 kDa and 110 kDa, confirming the dimeric and tetrameric state of the protein. The Km and Vmax of 0.078 ± 0.008 mM and 1052.3 ± 33.59 units mg−1 of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4–9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and β-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.

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Acknowledgements

This research was financially supported in the form of fellowship by the Department of Science and Technology (DST) under INSPIRE scheme, Grant No IF140298.

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  1. Molecular Biology and Proteomics Laboratory, Department of Biotechnology, Indian Institute of Technology, Roorkee, 247667, India

    Rashmi Gangwar, Poonam Kumari, Apurva Chatrath & Ramasare Prasad

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RG and RP conceived the idea and designed the experiments. RG along with PK and AC performed the experiment and analysed the data. RG and RP wrote the manuscript. RP gave all the critical revisions. All authors read and approved the final manuscript.

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Correspondence to Ramasare Prasad.

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Gangwar, R., Kumari, P., Chatrath, A. et al. Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander. Mol Biol Rep 47, 3251–3270 (2020). https://doi.org/10.1007/s11033-020-05374-x

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