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Purification and Characterization of Anti-cancer l-Glutaminase of Bacillus cereus Strain LC13

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

The present investigation was carried out in order to isolate a bacterial strain from marine soil which could produce l-glutaminase. The isolated organism identified as Bacillus cereus strain LC13 by 16s rRNA analysis was able to produce the enzyme under optimal conditions of pH 7.0, 37 °C and 0.3 % l-glutamine. Maltose (0.4 %, w/v) was best carbon source supplement. A 5.4-fold increase (from 6.1 to 69.1 U/mL) was achieved after optimization. Ammonium sulphate precipitation and ion exchange chromatography were used to purify the protein, and 12.7-purification fold (from 4.6 to 58.6 U/mg protein) was achieved from cell free extract with a recovery of 49.4 %. The molecular weight of the enzyme was found to be 35 ± 1 kDa as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and confirmed by liquid chromatography–mass spectrometry. The purified l-glutaminase was stable at physiological pH and temperature, activated by Na+, K+, slightly inhibited by Cu2+, Co2+, Fe2+, Fe3+, Zn2+, inhibited by Hg2+, and no effect was observed with Ca2+, Mg2+, Pb2+, and Mn2+. Phenylmethylsulphonyl fluoride, sodium azide, ethylenediaminetetraacetic acid and N-acetyl imidazole had no effect on enzyme whereas iodoacetamide, tosyl-l-lysylchloromethylketone, N-ethylmaleimide, p-Chloromercuribenzoic acid and N-bromosuccinimide strongly inhibited the enzyme, indicating the involvement of cysteine and histidine in the catalytic activity. The enzyme possesses low Km value of 0.4 mM (with Vmax of 61.34 U/ml) which defines higher affinity for its substrate. The l-glutaminase has also shown good radical scavenging activity which could have application in the medical field, like anti-tumour agent.

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Authors and Affiliations

  1. School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, 560078, India

    Sunil S. More

  2. Department of Biochemistry, Center for Post Graduate Studies, Jain University, Bangalore, 560011, India

    Radhika Swamy, Niveditha Mohan, Markuli Navyashree & Bhavya Janardhan

  3. Department of Biotechnologies, Faculty of Applied Fundamental Sciences, INES-Ruhengeri, 155, Ruhengeri, Rwanda

    Francois N. Niyonzima

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  1. Sunil S. More
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  2. Radhika Swamy
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More, S.S., Swamy, R., Mohan, N. et al. Purification and Characterization of Anti-cancer l-Glutaminase of Bacillus cereus Strain LC13. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88, 695–705 (2018). https://doi.org/10.1007/s40011-016-0808-x

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  • DOI: https://doi.org/10.1007/s40011-016-0808-x

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