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Biochemical characterization and antitumor study of L-glutaminase from Bacillus cereus MTCC 1305
1School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi, India.
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 (Mg(2+), Mn(2+), Zn(2+), Pb(2+), Ca(2+), Co(2+), Hg(2+), Cd(2+), Cu(2+)) 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 × 10(2) M(-1)s(-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).
Insights
Bacillus cereus MTCC 1305 produced extracellular L-glutaminase, a homotetrameric enzyme with specificity for L-glutamine. This enzyme demonstrated anticancer activity against hepatocellular carcinoma (Hep-G2) cell lines.
Area of Science:
- Biochemistry
- Enzymology
- Biotechnology
Background:
- L-glutaminase is an enzyme with potential therapeutic applications.
- Bacillus cereus is a known source of extracellular enzymes.
Purpose of the Study:
- To purify and characterize L-glutaminase from Bacillus cereus MTCC 1305.
- To evaluate the enzyme's anticancer activity against hepatocellular carcinoma (Hep-G2) cell lines.
Main Methods:
- Extracellular L-glutaminase was produced by Bacillus cereus MTCC 1305 and purified.
- Enzyme properties including molecular weight, substrate specificity, optimal pH, temperature, and stability were determined.
- Kinetic parameters (Km, Vmax, Kcat) were calculated.
- Anticancer activity was assessed using Hep-G2 cell lines.
Main Results:
- Purified L-glutaminase is a homotetrameric enzyme (140 kDa) specific for L-glutamine.
- Optimal activity was observed at pH 7.5 and 35°C, with notable stability at elevated temperatures.
- The enzyme was activated by monovalent cations and reducing agents, but inhibited by divalent cations and thiol-binding agents.
- Kinetic parameters were determined as Km = 6.25 mM, Vmax = 100 μmol/min/mg protein, and Kcat = 2.22 × 10^2 M⁻¹s⁻¹.
- L-glutaminase exhibited significant inhibition of Hep-G2 cell growth with an IC50 value of 82.27 μg/ml.
Conclusions:
- The purified L-glutaminase from Bacillus cereus MTCC 1305 is a well-characterized enzyme with potential as an anticancer agent.
- Further research into its therapeutic efficacy and mechanism of action is warranted.
