Abstract
L-glutaminase is a therapeutic enzyme used in the treatment of acute lymphoblastic leukemia (ALL). In this study, the extracellular L-glutaminase produced by Bacillus subtilis strain JK-79 was purified to homogeneity. The purified L-glutaminase has specific activity of 937 U mg−1 with KM and VMax value of 8 × 10-2 mM and 200 µM/ml min, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed it to have a molecular mass of 64 kDa. Further biochemical characterization revealed that the purified enzyme attained maximal activity at pH 7 and temperature 37°C. The enzyme was stable at 37°C for 60 min at physiologic-ph. The enzyme showed high specificity towards L-glutamine. The effect of metal ions on enzyme activity showed that NaCl, KCl and CaCl2 enhanced the activity while AgCl3 and CuCl2 strongly inhibited the activity of the enzyme. The in-vitro antioxidant activity of the enzyme was investigated and the IC50 value for DPPH and ABTS assay were 400 and 600 µg/ml, respectively. The purified enzyme showed cytotoxic activity against various Leukemic cell lines tested with IC50 value of 231, 480, and 500 µg/ml for K562, U932 and Jurkat cell lines, respectively. Interestingly, L-glutaminase also revealed cytotoxic effect on other cancer cell lines such as MCF-7, OV1063 and HCA 7 with IC50 value of 500, 526 and 750 µg/ml, respectively. Hence this enzyme from marine bacteria would possibly be an attractive candidate for further pharmaceutical use as a broad spectrum anti-tumor drug. Key words: L-glutaminase, purification, characterization, anti-oxidant activity, anti-cancer activity.
Highlights
L-glutaminase (L-glutamine amide hydrolase EC.3.5.1.2) has identified applications in many fields
This study focused on purification and characterisation of L-glutaminase from potent marine microbe Bacillus subtilis strain JK-79
L-glutaminase produced extracellularly by marine B. subtilis strain JK 79 was purified to apparent homogeneity using a combination of ammonium sulphate precipitation and chromatographic procedures
Summary
L-glutaminase (L-glutamine amide hydrolase EC.3.5.1.2) has identified applications in many fields. L-glutaminase plays an important role in cellular metabolism of plants, animals and microorganisms such as bacteria, fungi and yeast (Brosnan et al, 1995; Riberg et al, 1995; Gurung et al 2013; El-Ghonemy, 2014) L-glutaminase has replaced the use of monosodium glutamateas flavor enhancer in Chinese foods (Tachiki et al, 1998), and used in the manufacture of threonine by gamma glutamyl transfer reactions (Sabu et al, 2000b)
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