Purification, characterization and anticancer evaluation of l -methioninase from Trichoderma harzianum
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ORIGINAL ARTICLE
Purification, characterization and anticancer evaluation of l‑methioninase from Trichoderma harzianum Nisha Salim1 · A. Santhiagu1 · K. Joji1 Received: 11 July 2020 / Accepted: 19 October 2020 © King Abdulaziz City for Science and Technology 2020
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 L i+ and inhibited by C u2+, 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 I C50 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. Keywords Antitumor · Cytotoxicity · Electrophoresis · Kinetic analysis · Sulforhodamine B assay · Thermodynamics
Introduction l-methioninase is a potential therapeutic enzyme that catalyzes the degradation of l-methionine to methanethiol, α-ketobutyrate and ammonia. The enzyme requires pyridoxal-l-phosphate for γ elimination of l-methionine (Tanaka
Accession number The rDNA sequence of Trichoderma harzianum was deposited to gene bank under accession number MH828332.1. * A. Santhiagu [email protected] Nisha Salim [email protected] K. Joji [email protected] 1
Bioprocess Laboratory, School of Biotechnology, National Institute of Technology, Calicut, India
et al. 1977), α, β elimination of l-cysteine and its analogs (Tanaka et al. 1985). Methionine is an essential amino acid in humans which performs a crucial role in mammalian metabolisms such as biosynthesis of protein, glutamine and polyamine (Wise and Thompson 2010; Halpern et al. 1974). The increased requirement of plasma methionine for protein synthesis and regulation of
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