Abstract
l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na+, K+) and phosphate ion activated the enzyme activity, while divalent cations (Mg2+, Mn2+, Zn2+, Pb2+, Ca2+, Co2+, Hg2+, Cd2+, Cu2+) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K m, V max, K cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22 × 102 M−1s−1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC50 value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).
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Acknowledgments
We would like to thank Dr. N. Bawa (NBE Institute, Delhi, India, and Dr. M. V. Jagannadham (Molecular Biology Unit, IMS, BHU, Varanasi) for providing laboratory facilities for this research work. The author Mrs. Priyanka Singh is also thankful to INSPIRE-DST, Government of India, for financial support, and the School of Biochemical Engineering, Institute of Technology, BHU, Varanasi, India, for providing laboratory and technical support.
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Singh, P., Banik, R.M. Biochemical Characterization and Antitumor Study of l-Glutaminase from Bacillus cereus MTCC 1305. Appl Biochem Biotechnol 171, 522–531 (2013). https://doi.org/10.1007/s12010-013-0371-3
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DOI: https://doi.org/10.1007/s12010-013-0371-3