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

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

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

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

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

Hydroxyethylamine Based Phthalimides as New Class of Plasmepsin Hits: Design, Synthesis and Antimalarial Evaluation

<div><p>A novel class of phthalimides functionalized with privileged scaffolds was designed, synthesized and evaluated as potential inhibitors of plasmepsin 2 (K_i: 0.99 ± 0.1 μM for <b>6u</b>) and plasmepsin 4 (K_i: 3.3 ± 0.3 μM for <b>6t</b>), enzymes found in the digestive vacuole of the plasmodium parasite and considered as crucial drug targets. Three compounds were identified as potential candidates for further development. The listed compounds were also assayed for their antimalarial efficacy against chloroquine (CQ) sensitive strain (3D7) of <i>Plasmodium falciparum</i>. Assay of twenty seven hydroxyethylamine derivatives revealed four (<b>5e</b>, <b>6j</b>, <b>6o</b> and <b>6s</b>) as strongly active, which were further evaluated against CQ resistant strain (7GB) of <i>P</i>. <i>falciparum</i>. Compound <b>5e</b> possessing the piperidinopiperidine moiety exhibited promising antimalarial activity with an IC₅₀ of 1.16 ± 0.04 μM. Further, compounds <b>5e</b>, <b>6j</b>, <b>6o</b> and <b>6s</b> exhibited low cytotoxic effect on MCF-7 cell line. Compound <b>6s</b> possessing <i>C</i>_<i>2</i> symmetry was identified as the least cytotoxic with significant antimalarial activity (IC₅₀: 1.30 ± 0.03 μM). The combined presence of hydroxyethylamine and cyclic amines (piperazines and piperidines) was observed as crucial for the activity. The current studies suggest that hydroxyethylamine based molecules act as potent antimalarial agent and may be helpful in drug development.</p></div

Coupling of <i>N</i>-phthaloyl-L-amino acids with 5f afforded C₂-symmetric molecules (6r-6u).

<p>Coupling of <i>N</i>-phthaloyl-L-amino acids with 5f afforded C₂-symmetric molecules (6r-6u).</p

Variation of potency of positional tautomers against <i>P</i>. <i>falciparum</i>.

<p>Variation of potency of positional tautomers against <i>P</i>. <i>falciparum</i>.</p