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

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.