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Purification, characterization and anticancer evaluation of l-methioninase from Trichoderma harzianum

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Abstract

The Trichoderma harzianum l-methioninase was purified 7.15-fold with a recovery of 47.9% and the specific activity of 74.4 U/mg of protein. The purified enzyme has an apparent molecular mass of 48 kDa on SDS-PAGE and exhibited maximum activity at pH 8 and 35 °C. The enzyme was catalytically stable below 50 °C and at a pH range of 6.0–8.5. The thermal inactivation of l-methioninase exhibited first-order kinetics with the k value between 5.71 × 10–4 min−1 and 1.83 × 10–2 min−1. The studies on thermodynamic parameters of l-methioninase indicated the compaction and aggregation of the enzyme molecule during denaturation. This is the first report of thermodynamic analysis of thermal inactivation in l-methioninase. The enzyme activity was enhanced by Li+ and inhibited by Cu2+, Co2+, Fe2+, Hydroxylamine and PMSF. The purified enzyme showed Km, Vmax and kcat value of 1.19 mM, 21.27 U/mg/min and 16.11 s−1, respectively. The l-methioninase inhibited the growth of human cell lines hepatocellular carcinoma (Hep-G2) and breast carcinoma (MCF-7) with IC50 values of 14.12 μg/ml and 20.07 μg/ml, respectively. The in vivo antitumor activity of l-methioninase was evaluated against DAL cell lines bearing in Swiss albino mice. The enzyme effectively reduced tumor volume, packed cell volume, viable cell count and restored hematological parameters, serum enzyme and lipid profile to normal levels compared to DAL control mice. The present study has demonstrated the high efficacy of Trichoderma harzianum l-methioninase against cancer cell lines in vitro and in vivo conditions. The purified l-methioninase has significant thermal stability and better catalytic properties than the enzyme purified from other sources.

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Acknowledgements

One of the authors Ms. Nisha Salim is thankful to UGC for providing research fellowship. The authors wish to acknowledge Dr K. Rathinasamy, and Ms Shabeeba of National institute of Technology Calicut for providing facilities for cell culture studies.

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Authors and Affiliations

  1. Bioprocess Laboratory, School of Biotechnology, National Institute of Technology, Calicut, India

    Nisha Salim, A. Santhiagu & K. Joji

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  1. Nisha Salim
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  2. A. Santhiagu
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  3. K. Joji
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Contributions

AS conceived the original idea and designed the study. NS performed the experiment and wrote the paper. KJ provided help in performing the experiment.

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Correspondence to A. Santhiagu.

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The author declares that they have no conflict of interest. All the authors consent to the submission of this manuscript to the journal.

Ethical approval

All the procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted and ethical approval was obtained from Committee for the purpose of control and supervision of experiments on animals (CPCSEA). (Reg No: 688/PO/Re/S/02/CPCSEA.), Approval No: NCP/IAE/2018-19/29.

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Accession number

The rDNA sequence of Trichoderma harzianum was deposited to gene bank under accession number MH828332.1.

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Salim, N., Santhiagu, A. & Joji, K. Purification, characterization and anticancer evaluation of l-methioninase from Trichoderma harzianum. 3 Biotech 10, 501 (2020). https://doi.org/10.1007/s13205-020-02494-w

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