Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography.

<p>The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.</p

CD spectra of the Rv0045c protein at different pH and temperatures.

<p>The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).</p

Effects of temperature and pH on enzyme activity of the Rv0045c protein.

<p>The enzyme activities were measured using <i>p-butyrate caprylate (C₆)</i> as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).</p

MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein.

<p>The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.</p

Relative enzyme activity of the Rv0045c protein toward <i>p</i>-nitrophenyl derivatives at pH 7.0 and 37°C.

<p><i>ND</i>: Not detectable</p>a<p>The specific activity toward <i>p</i>-nitrophenyl caproate (C₆) corresponding to 3.5 U/mg protein/min was defined as 100%. And one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min.</p

SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein.

<p>Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni²⁺-affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.</p