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.

Fatty acid composition of tomato leaves as biomarkers of metal-contaminated soils.

The aim of this study was to determine whether fatty acid composition of leaves, cotyledons or roots could be used as an indicator of the bioavailability and of the adverse effects of heavy metals on plants. Tomato seedlings were grown on soils obtained by mixing increasing amounts of a highly metal-contaminated soil with an uncontaminated sandy soil, and the fatty acid composition of plant tissues was analyzed. The fatty acid composition of roots and cotyledons of plants grown on contaminated soils was mostly the same as in the control plants. In contrast, significant changes in the fatty acid composition of primary leaves occurred. Our results clearly indicate a relationship between metal accumulation (Cd, Pb, Zn, and Cu) and the fatty acid composition of primary leaves, with the contribution of 18 C atom fatty acids (as 18:3 fatty acid and precursors) being more closely correlated with the availability of heavy metals in soils.

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.

Transfer and subsequent metabolism of lysolipids studied by immobilizing subcellular compartments in alginate beads.

The transfer and subsequent metabolism of lysophosphatidylcholine between subcellular compartments were studied in vitro by embedding membranes in alginate beads. After several experiments to validate the process, it was demonstrated that lysophosphatidylcholine was transferred from microsomes embedded in alginate beads to immobilized chloroplasts, that this transfer involved the partition of this molecule, and that the imported lysophosphatidylcholine was further used as substrate for phosphatidylcholine biosynthesis. More generally, the technique used makes it possible to avoid any cross-contamination between compartments, to evidence a transfer of molecules, and to study the metabolism of the imported molecules in the acceptor compartment.

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton motive force, energy recycling by end product excretion, and metabolic uncoupling during anaerobic growth of Pseudomonas mendocina.

Batch cultures of Pseudomonas mendocina, grown in rich medium with glucose excess, showed metabolic differences dependent upon whether the growth conditions were aerobic or anaerobic, with or without added electron acceptor. Under anaerobic conditions in the absence of nitrate, P. mendocina reached the stationary phase of growth after 2 or 3 days, followed by a stationary phase of 4 to 5 days. Under these conditions, a mixed-type fermentative metabolism (formic, lactic, and acetic acids) appeared. A fivefold-higher specific rate of glucose consumption and eightfold-higher production of organic acids, compared with aerobic cultures, were shown by this microorganism growing anaerobically in the absence of exogenous electron acceptors. The gradients of organic acid produced by P. mendocina under these conditions reached a maximum (lactate, 180 mV; formate, 150 mV; acetate, 215 mV) between days 2 and 3 of culture. The proton motive force (delta p) decreased during growth from -254 to -71 mV. The intracellular pH remained alkaline during the culture, reaching a steady-state value of 7.9. The gradients of organic acids apparently contributed to the generation of a delta p, which, according to the Energy Recycling Model (P. A. M. Michels, J. P. J. Michels, J. Boonstra, and W. N. Konings, FEMS Microbiol. Lett. 5:357-364, 1979), would produce an average energy gain of 1 or 1.5 mol of ATP equivalents per mol of glucose consumed with H+/ATP stoichiometry of 3 or 2, respectively. Low YATP and Yglucose values were observed, suggesting that an uncoupled metabolism exists; i.e., ATP produced by catabolic processes is not directly used for biomass synthesis. This metabolic uncoupling could be induced at least in part by organic acids and the ATP wastage could be induced by a membrane-bound ATPase involved in intracellular pH regulation.