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 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

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

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

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

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

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

<i>irtA</i>, <i>irtB</i> and <i>Rv2895c</i> are upregulated under low iron conditions as evident from semi-quantitative RT-PCR analysis.

<p>Agarose gel showing PCR amplifications of the reverse transcribed cDNA from <i>M.tb</i> cultures grown under low iron (upper panel) and iron replete (lower panel) conditions. The bands were densitometrically scanned and analyzed by ImageJ software. The histograms represent fold increase (n = 3; ±SD) in the expression of i<i>rtA</i> (A), i<i>rtB</i> (B) and <i>Rv2895c</i> (C) under low iron conditions compared with the corresponding RT-PCR levels from the RNA isolated from <i>M.tb</i> H37Rv grown under iron replete condition. A mean of three independent experiments is shown.</p

Strategy for targeted knockout of <i>msmeg_6554</i>.

<p>(A) <i>msmeg_6554</i>, a homologue of <i>M.tb irtA</i> in <i>M.smeg</i> was disrupted by introducing hygromycin cassette as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002087#s4" target="_blank">materials and methods</a>. A 3.3 kb insert at the <i>msmeg_6554 locus</i> was incorporated to create the knockout. (B) PCR amplifications indicating the disruption <i>of msmeg_6554</i>. Lane 2, negative control; lane 3, amplification from <i>M.smeg</i> mc²155 genomic DNA; lane 4, amplification from PCK48hyg vector; lane 5, KO48-SCO, single crossover; lane 6, KO48-DCO, double crossover. (C) RT-PCR of <i>msmeg_6553</i> on RNA isolated from mc²155▵6554 grown under iron replete (lane 1) and iron depleted (lane 2) conditions.</p

<i>M.smeg msmeg_6554</i> knockout is incapable of siderophore export which can however be complemented by <i>M.tb irtA</i> as evident from siderophore production by CAS assay.

<p>(A) Block 1, mc²155; 2, mc²155▵6554 on CAS agar plate or 5, in iron depleted; or 8, normal iron replete media. 3, mc²155▵6554 was complemented with pMtb1348; and 6, grown in iron depleted media; or 9, in iron replete media. <i>M.smeg</i> mc²155 strain grown under iron depleted (4) and normal conditions (7) were used as control. The cultures were incubated for 3 days in each case. (B) The graphs indicate the growth pattern of the three strains in iron replete media (1) and iron depleted media (2) in terms of O. D. (A₅₄₀) of the culture monitored from 6 hrs of incubation till 48 hrs. (C) Siderophore concentration in the culture supernatants was measured as a function of CAS reactivity after 1 and 3 days of growth of the wild type mc²155 (M1); mc²155▵6554 (M2); or mc²155▵6554 complemented with <i>M.tb irtA</i> gene (M3) in iron depleted media.</p