Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties.

L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug

Anti-cancerous effect of enzyme on different human cancer cell lines <i>viz</i>. Jurkat clone E6-1, MCF-7 and K-562.

<p>Anti-cancerous effect of enzyme on different human cancer cell lines <i>viz</i>. Jurkat clone E6-1, MCF-7 and K-562.</p

Effect of chemical parameters on purified L-asparaginase a) Effect of metal ions (100 mM) on enzyme activity; b) Effect of inhibitors (10 mM) on enzyme activity (EDTA: Ethylenediaminetetraacetic acid, EGTA: Ethylene glycol tetraacetic acid, PCMB: p-chloromercuribenzoic acid, PMSF: Phenylmethanesulfonylfluoride, PBA c) Effect of serum and serum components (10 mM) on enzyme activity; d) Substrate (10 mM) specificity of enzyme.

<p>100% activity corresponds to 140 U of enzyme. Error bars represent SD of three experiments.</p

Stepwise purification of L-asparaginase from <i>Bacillus licheniformis.</i>

<p>Stepwise purification of L-asparaginase from <i>Bacillus licheniformis.</i></p

Molecular weight determination of purified enzyme (a) SDS PAGE for steps of purifying L-asparaginase; b) Plot of V_e/V_o against semi-log of molecular weight of proteins on Sephacryl TM S-200 high resolution column (16/60) for α-Lactalbumin (12.4 kDa), carbonic anhydrase (30 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), sweet potato β-amylase (200 kDa) and ferritin (450 kDa).

<p>Molecular weight determination of purified enzyme (a) SDS PAGE for steps of purifying L-asparaginase; b) Plot of V_e/V_o against semi-log of molecular weight of proteins on Sephacryl TM S-200 high resolution column (16/60) for α-Lactalbumin (12.4 kDa), carbonic anhydrase (30 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), sweet potato β-amylase (200 kDa) and ferritin (450 kDa).</p

Fluorescence spectroscopy showing the emission spectrum in the range of 300–400

<p> <b>nm (excitation wavelength: 292 nm) and unfolding transitions of L-asparaginase at 0 </b><b>M, 3 </b><b>M and 6 </b><b>M guanidine HCl.</b></p

Effect of physical parameters on purified L-asparaginase a) Effect of pH on enzyme activity; b) Effect of pH on the stability of enzyme; c) Effect of temperature on assay reaction; d) Heat stability of enzyme.

<p>100% activity corresponds to 140 U of enzyme. Error bars represent SD of three experiments.</p

CD spectra of purified L-asparaginase a) Far UV CD spectra of L-asparaginase at 0.1 mg/ml in 0.1 M tris HCL (pH-8.4); b) Melting temperature of enzyme (T₂₂₂_nm).

<p>c). Near UV CD spectra of purified L-asparaginase 1.0 mg/ml in 0.1 M Tris HCL (pH-8.4).</p