Electric-field-enhanced selective separation of products of an enzymatic reaction in a membrane micro-contactor.

Processes employed in separations of products of enzyme reactions are often driven by diffusion, and their efficiency can be limited. Here, we exploit the effect of a direct current (DC) electric field that intensifies mass transfer through a semipermeable membrane for fast, continuous, and selective separation of electrically charged molecules. Specifically, we separate low-molecular-weight reaction products (phenylacetic acid, 6-aminopenicillanic acid) from the original reaction mixture containing a free enzyme (penicillin acylase). The developed microfluidic dialysis-membrane contactor allows a stable counter-current arrangement of the retentate and permeates liquid streams on which DC electric field is perpendicularly applied. The applied electric field significantly accelerates the transport of electrically charged products through the semipermeable membrane yielding high separation efficiencies at short residence times. The residence time of 5 min is sufficient to reach 100% separation yield in the electric field. The same residence time provides only a 50% yield in the diffusion-controlled experiments. We experimentally demonstrated that a combined microreactor-microextractor with a recycle of the soluble penicillin acylase can continuously produce both the reaction products at high concentrations. The developed membrane-contactor is a versatile platform allowing to tune its characteristics, such as selectivity given by the membrane, or the type of the retentate phase, for a specific application.

Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation.

BACKGROUND: Recent studies have demonstrated highly variable blood concentrations of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in critically ill patients with a high incidence of sub-therapeutic levels. Consequently, therapeutic drug monitoring (TDM) of these antibiotics has to be considered, requiring robust and reliable routine analytical methods. The aim of the present work was to develop and validate a multi-analyte ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of the above mentioned antibiotics. METHODS: Sample preparation included a manual protein precipitation step followed by two-dimensional ultra high performance liquid chromatography (2D-UHPLC). Corresponding stable isotope-labeled substances were used as internal standards for all of the analytes, with the exception of tazobactam. The injected sample volume was 7 µL. The run time was 5.0 min. RESULTS: Inaccuracy was ≤8% and imprecision coefficient of variation (CV) was <9% for all analytes. Only minor matrix effects and negligible carry-over was observed. The method was found to be robust during the validation period. CONCLUSIONS: We were able to develop a reliable 2D-UHPLC-MS/MS method addressing analytes with highly heterogeneous physico-chemical properties. The novel assay may be an efficient tool for an optimized process workflow in clinical laboratories for important antibiotics in regards to TDM.

Early discoveries in the penicillin series.

It is now 50 years since the therapeutic potential of penicillin was first demonstrated. This first antibiotic and the series of compounds derived from it have been of immense importance in modern medicine. This article describes the early search for more potent penicillin derivatives, culminating with the discovery of an intermediate of penicillin biosynthesis, 6-beta-amino penicillanic acid (6-APA). A companion article in next month's TIBS will chart the subsequent exploitation of 6-APA and the preparation of a range of clinically important semi-synthetic penicillins.

Adsorption of components of enzymatic synthesis of ampicillin on different hydrophobic resins.

This work compared the performance of three hydrophobic resins for the adsorption of ampicillin (AMP), D-phenylglycine (PG), D-phenylglycine methyl ester (PGME), and 6- aminopenicillanic acid (6-APA). The influence of pH on adsorption efficiencies was assessed in the range of 4.5-8.5, at 4 and 25 degrees C. The values at 4 degrees C were slightly higher than those at 25 degrees C. The adsorption efficiency of AMP and 6-APA decreased at higher pHs, for the three resins. An opposite behavior was found for PGME, and the pH did not affect PG adsorption efficiency. Isotherm models were fitted to experimental equilibrium data and the best models were discriminated.

Enzymatic hydrolysis of penicillin and in situ product separation in thermally induced reversible phase-separation of ionic liquids/water mixture.

Enzymatic hydrolysis of penicillin G to produce 6-aminopenicillanic acid, key intermediate for the production of semisynthetic ß-lactam antibiotics, is one of the most relevant example of industrial implementation of biocatalysts. The hydrolysis reaction is traditionally carried out in aqueous buffer at pH 7.5-8. However, the aqueous rout exhibits several drawbacks in enzyme stability and product recovery. In this study, several ionic liquids (ILs) have been used as media for enzymatic hydrolysis of penicillin G. The results indicated that hydrophobic ILs/water two-phase system were good media for the reaction. In addition, a novel aqueous two-phase system based on the lower critical solution temperature type phase changes of amino acid based ILs/water mixture was developed for in situ penicillin G hydrolysis and product separation. For instance, hydrolysis yield of 87.13% was obtained in system containing 30 wt% [TBP][Tf-ILe] with pH control (pH 7.6). Since the phase-separation of this medium system can be reversible switched from single to two phases by slightly changing the solution temperature, enzymatic hydrolytic reaction and product recovery were more efficient than those of aqueous system. In addition, the ILs could be reused for at least 5 cycles without significant loss in hydrolysis efficiency.

A validated LC-MS/MS method for the quantification of piperacillin/tazobactam on dried blood spot.

Background Therapeutic drug monitoring (TDM) of antibiotics in children is of fundamental importance for effective patient management. The use of dried blood spot (DBS) offers a number of advantages over conventional blood collection. The aim of this study is to develop and validate a method to measure piperacillin/tazobactam in DBS. Results The analysis was performed by using LC-MS/MS operating in multiple reaction monitoring mode. The method has been validated by applying EMA guidelines and its suitability for TDM was evaluated by using samples from low birth weight neonates. Conclusion This paper describes a fast and cost-effective micromethod for the simultaneous determination of piperacillin/tazobactam levels on dried blood spot that is suitable for TDM in children.

Isolation and purification of penicillin G acylase obtained from Escherichia coli (NCIM-2400) and immobilisation on Eupergit C for the production of 6 amino penicillanic acid.

Penicillin G-Acylase is produced by submerged cultivation of E. Coli (NCIM-2400) and extracted from the harvested fermented broth, purified (affinity chromatography) and immobilised on Eupergit C (Synthetic polymer in bead form). The immobilised penicillin G acylase properties are studied and compared with soluble penicillin G-acylase. The control parameters for conversion of penicillin G-K to 6 APA are optimised [e.g. substrate (Pen G-K) concentration ratio to immobilised penicillin G-acylase, temperature, pH etc.] in a stirred tank reactor. Our findings suggest that immobilised penicillin G-acylase can be used commercially and the productivity of 1 kg. of immobilised enzyme is around 400 kg of 6 APA under given desired stipulated conditions.

Optimization of 6-aminopenicillanic acid (6-APA) production by using a new immobilized penicillin acylase.

A new immobilized penicillin acylase (ECPVA) was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C. Enzymic hydrolysis of penicillin V catalysed by ECPVA was optimized using a 2(3) factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration. The immobilized enzyme showed an optimal pH value of 9.5-10.5, and an optimal temperature of 60 degrees C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 degrees C.

Comparison of aqueous and nonaqueous carrier electrolytes for the separation of penicillin V and related substances by capillary electrophoresis with UV and mass spectrometric detection.

A method for the determination of penicillin V together with its impurities and by-products formed during biosynthesis, using capillary electrophoresis (CE) with UV and electrospray-mass spectrometric (ESI-MS) detection is presented. Aqueous and nonaqueous electrolytes containing 20 mM ammonium acetate were investigated to determine their suitability for the separation of these analytes. These carrier electrolytes were optimized with respect to the pH and the solvent/s used (water, methanol, acetonitrile, ethanol and isopropanol) and it was shown that although the nonaqueous electrolytes offered unique separation selectivities, the best results in terms of selectivity and sensitivity were obtained for the aqueous system. Finally, the applicability of this method for the analysis of a mixture representative of a real fermentation broth was demonstrated using an aqueous carrier electrolyte with both UV and ESI-MS detection.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.