Penicillin acylase purification with the aid of hydrophobic charge induction chromatography.

The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.

Penicillin acylase purification with the aid of pseudo-affinity chromatography.

The introduction of affinity chromatography has opened a new dimension in protein purification. This article reviews the current techniques used in the penicillin acylase purification process, especially pseudo-affinity chromatography. A profile for a suitable ligand is established. An aromatic ring and the presence of one or several amino groups seem essential for proper interaction. Immobilized metal affinity chromatography now seems to be a good competitor.

Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase.

The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu2+ was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH4Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH4Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH4Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97-100%).

Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate.

A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu2+ as metal ion. Two elution methods were performed: 1 M NH4Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90-99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.

Preparation, evaluation and application of new pseudo-affinity chromatographic supports for penicillin acylase purification.

New pseudo-affinity chromatographic supports for penicillin acylase were prepared and evaluated with three different samples: pure penicillin acylase, industrial clarified feedstock and crude extract. The different gels were studied for their purification fold (three to six) and their recovery power (80-100%). The best support was characterized by its dynamic capacity, (20 mg/ml) and its recovery power was tested at five flow-rates (30, 150, 300 and 750 cm/h) to determine the optimal flow-rate (300 cm/h). In addition we used cleaning in place to test the resistance to hard conditions of sanitization by 1 M NaOH (90% of recovery for 12 h of contact). These gels may therefore be used on an industrial scale.

Yeast mitochondrial metabolism: from in vitro to in situ quantitative study.

In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M). At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolites and permeabilization. We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies. It appeared from ethanol metabolism +/- NAD+ or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S. By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M. Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism. When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed. So, the intrinsic uncoupling of proton pumps is not a particularity of M. Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP. Such a mechanism also occurs in yeast for several respiratory substrates: the K0.5 for each substrate toward the respiration rate in PS always exceeds that for M. It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e. regulation of the substrate permeability through porin channels. In the porin-deficient yeast mutant, the K0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS. This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel.

Mechanistic stoichiometry of yeast mitochondrial oxidative phosphorylation.

This study investigates the relationships between the efficiency of oxidative phosphorylation (ATP/O) and respiratory flux in yeast mitochondria. To manipulate the electron flux through the respiratory chain, different substrates leading to NAD(P)H were used. By testing the effect of ADP either on respiratory rate in the presence or absence of oligomycin or on the level of NAD(P)H, on one hand, and the effects of uncouplers on respiration, on the other, we distinguished several categories of substrates: those for which the low respiration rate was mainly controlled by dehydrogenase activities and others for which the respiration was high and controlled downstream from the dehydrogenases. By using these different substrates, we observed that the ATP/O ratio decreased irrespective of the proton-motive force when the electron flux increased, unlike the situation when the respiratory rate was modulated by addition of the respiratory inhibitor. This result suggests that the oxidative phosphorylation efficiency depends on the value of the flux crossing the proton pumps. This relationship between efficiency (ATP/O) and electron flux was linked to a main control upstream from the respiratory chain. Such changes in the ATP/O ratio at least involved changes in the stoichiometry (H+/2e-) of the respiratory chain. Indeed, in non-phosphorylating mitochondria, the ratio of stoichiometries at site 2+3 over site 3 varied according to the proton-motive force. This cannot be explained by a variation in proton leak alone but involved both (i) a variable stoichiometry (H+/2e-) in relation to the electron flux value and (ii) different relationships between the variation in stoichiometry and the flux value at each coupling site.