TLC analysis of hydrolysis products of triolein by His6-tagged N-TPL.

<p>Lane1: reaction products after 15 min incubation at 37°C of triolein in the absence of enzyme. Lane2: reaction products after 15 min incubation at 37°C of Triolein in the presence of His6-tagged N-TPL (14 µg). Reaction products were extracted with Chloroform/methanol mixture (2∶1, v/v).</p

Hydrolysis rate of TC3 by His6-tagged N-TPL and TPL as a function of substrate concentration.

<p>The TC3 solutions were systematically prepared in 30 mL of 0.33% GA and 0.15 M NaCl. The CMC of TC3 (12 mM) is indicated by vertical dotted line. Bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.001 versus TPL.</p

Stability of His6-tagged N-TPL and TPL.

<p>Effect of temperature (<b>A</b>) and pH (<b>B</b>). His6-tagged N-TPL was incubated at different temperatures and pH for 5 min and TPL was incubated at different temperatures and pH for 30 min. The activity is measured under optimal conditions (without colipase) and using TC4 as substrate. 100% activity corresponds to AS = 70 U/mg and AS = 9500 U/mg for His6-tagged N-TPL and TPL, respectively. Bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.001 versus TPL.</p

Flow sheet of His6-tagged N-TPL purification.

<p>Flow sheet of His6-tagged N-TPL purification.</p

Time-course of the expression of His6-tagged N-TPL by four isolated clones of recombinant <i>Pichia pastoris</i>.

<p>The culture was carried out in 250 ml Erlen-meyer’s falsks with shaking at 150 rpm and 30°C.</p

REC, TEC and TREC values of TPL obtained using 1,2-<i>sn</i> dicaprin, 2,3-<i>sn</i>-dicaprin and 1,3-<i>sn</i>-dicaprin substrates, spread at the air/water interface forming monomolecular films at 20, 25 and 30 mN.m⁻¹.

a<p>Mean values of either TEC or REC or TREC for the three dicaprin isomers at a given surface pressure.</p>b<p>Overall mean TEC, REC and TREC values obtained with the three dicaprin isomers, tested at three surface pressures.</p

Hydrolysis kinetic of tributyrin by His6-tagged N-TPL in the presence of colipase.

<p>Lipolytic activity was followed at pH 8.5 and at 37°C.</p

Regioselectivity and Stereospecificity of nTPL, rTPL and 6His-rTPL.

<p>The vicinity index (V.I.) and the stereospecificity index (S.I.) of the three TPL forms were calculated using the following definitions: V.I. = [A_1,3−½ (A_2,3+A_1,2)]/[A_1,3+½ (A_2,3+A_1,2)] and S.I. = (A_2,3−A_1,2)/(A_2,3+A_1,2) at two surface pressure values (15 and 25 mN.m⁻¹), where A_1,3, A_1,2 and A_2,3 are lipase activities measured with 1,3-<i>sn</i>-dicaprin; 1,2-<i>sn</i>-dicaprin and 2,3-<i>sn</i>-dicaprin, respectively.</p

Q-PCR analysis of the N-TPL cDNA copies number in the genomic DNA of N3, N4, N5 and N6 clones.

<p>Correlation coefficient: 0.999; Slope: −3.484; PCR Efficiency: 93.6%.</p

Histogram of the maximum activities of the three TPL forms.

<p>(<b>A</b>) maximum activities on monomolecular films of 1,2-<i>sn</i>-dicaprin, 2,3-<i>sn</i>-dicaprin, and 1,3-<i>sn</i>-dicaprin, Data based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104221#pone-0104221-g001" target="_blank">figure 1</a>. Activities are expressed as the number of moles of substrate hydrolysed per unit time (min) and unit surface (cm²) at an arbitrary lipase concentration of 1 M in the aqueous subphase of the reaction compartment of a “zero-order” trough (volume, 130 ml; surface area, 111 cm²). (<b>B</b>) Histogram of kinetic parameters of TPL forms. nTPL (black bars), rTPL (gray bars) and 6His-rTPL (white bars).</p