Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.

Optimization of polymerization parameters for the sorption of oseltamivir onto molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) are tailor-made polymers with high selectivity for a given analyte, or group of structurally related compounds. The influence of the process parameters (the moles of functional monomer and cross-linker, the selection of functional monomer and solvent) on the preparation of oseltamivir (OS)-imprinted polymers was investigated. A mathematical method for uniform design to optimize these selected parameters and to increase the MIP selectivity for template molecules was applied. The optimal conditions to synthesize MIP were 0.69 mmol 30% acrylamide (AA) + 70% 4-Vinylpyridine (4-VP) and 5.0 mmol ethylene glycol dimethacrylate (EGDMA) copolymerized in 5 ml toluene in the presence of 0.1 mmol OS. MIP showed high affinity and selectivity for separation of the template molecule from other compounds. In the present study, we have established an effective LC-MS/MS method to identify and quantify OS with good sensitivity, accuracy and precision.

Behavior of pharmaceuticals in waste water treatment plant in Japan.

The fate of pharmaceuticals in a wastewater treatment plant (WWTP) in Kumamoto, Japan with activated sludge treatment is reported. Selected pharmaceuticals were detected in influent. Results from the present study confirmed that Acetaminophen, Amoxicillin, Ampicillin and Famotidine were removed at a high rate (>90% efficiency). In contrast, removal efficiency of Ketoprofen, Losartan, Oseltamivir, Carbamazepine, and Diclofenac was relatively low (<50%). The selected pharmaceuticals were also detected in raw sludge. In digestive process, Indomethacin, Atenolol, Famotidine, Trimethoprim and Cyclofosamide were removed at a high (>70% efficiency). On the other hand, removal of Carbamazepine, Ketoprofen and Diclofenac was not efficient (<50%).

Determination of oseltamivir (Tamiflu®) and oseltamivir carboxylate in dried blood spots using offline or online extraction.

BACKGROUND: Using dried blood spots (DBS) for quantitation of the antiviral drug oseltamivir (Tamiflu(®)), an ester prodrug, and its active metabolite oseltamivir carboxylate could provide ethical and logistic benefits. Hence, its feasibility was investigated using a previously developed column-switching LC-MS/MS method. RESULTS: Sensitivity, precision and accuracy in DBS were comparable to standard plasma assays. Chemically treated cards provided enhanced ex vivo stability of the ester prodrug in rodent blood. Online extraction was realized using the manual TLC-MS interface or the fully automated Sample Card and Prep system. Rat pharmacokinetic study data showed good correlation between plasma, liquid blood and DBS. CONCLUSION: From a bioanalytical perspective, DBS is potentially suited for Tamiflu analysis in animals and humans. Automation of the process by online DBS extraction promises workload reduction and throughput increase.

Antiviral pandemic risk assessment for urban receiving waters.

An 80% metabolic conversion of the Tamiflu pro-drug (oseltamivir phosphate, OP) to its metabolite oseltamivir carboxylate (OC) and a high excretion rate combined with poor removal at sewage treatment works (STWs) means that potentially high STW OC emissions may occur in receiving waters. A risk assessment approach undertaken within the River Lee catchment in North East London indicates that predicted environmental concentrations for surface waters (PEC(sw)) are likely to be in the general range of 40-80 microg L(-1) during a pandemic sitaution within urban catchments having low dilution capacities. This implies low risk exposure levels which confirms previous studies, but there are considerable uncertainties associated with the methodology as well as risks that might result from persistent, long term chronic exposure to low-level water and sediment concentrations which might be mobilised under subsequent extreme flow conditions.

Antiviral drugs in wastewater and surface waters: a new pharmaceutical class of environmental relevance?

An analytical method was developed for the determination of nine antiviral drugs (acyclovir, abacavir, lamivudine, nevirapine oseltamivir, penciclovir, ribavirin, stavudine, zidovudine) and one active metabolite (oseltamivir carboxylate) in raw and treated wastewater as well as in surface water using LC/ESI tandem MS detection. Relative recoveries exceeded 80% and limits of quantification ranged between 0.2 and 10 ng L(-1). Matrix effects were compensated by the use of stable isotope labeled surrogate standards and optimized chromatographic separation on a Hydro-RP column. All antiviral drugs, except ribavirin, were detected in raw wastewater. A significant reduction in concentrations was observed for acyclovir, lamivudine, and abacavir in treated wastewater indicating a substantial removal, while nevirapine, zidovudine and oseltamivir were found in similar concentrations in raw and treated wastewater. Concentrations in river waters were in the lower ng L(-1)-range, with a maximum of 190 and 170 ng L(-1) for acyclovir and zidovudine, respectively in the Hessian Ried. Estimated total compound loads in the Ruhr river ranged between <0.1 and 16.5 g d(-1) while for the Rhine river values between 370 and 1800 g d(-1) were determined. The ratios of oseltamivir to oseltamivir carboxylate were approximately a factor of 10 higher for the Rhine than for the other rivers and streams indicating a significant contribution from other sources such as discharges from manufacturing facilities.

Fast hydrophilic interaction liquid chromatographic separations on bonded zwitterionic stationary phase.

Separation science is an art of obtaining adequate resolution of the desired compounds in minimum time, and with minimum effort in terms of sample preparation and data evaluation. In LC, where selectivity is a main driving force for separation, the availability of different separation modes capable of operating at high flow rates is a way to make combined optimal use of selectivity, efficiency, and speed. The separation of polar and hydrophilic compounds is problematic in RP LC due to the poor retention. Hydrophilic interaction liquid chromatography (HILIC) is a more straightforward separation mode to address this problem. Herein, it is shown that separations in HILIC mode are equally efficient as for RP, providing a potential for very fast separations on short columns. This is not only facilitated by the low viscosity of the mobile phase compositions used, compared to typical RP eluents, but also due to higher column permeability. To exemplify this, baseline separations of uracil and cytosine are shown in less than 4 s and of Tamiflu and its main metabolite in less than 40 s, both under isocratic conditions. HILIC must therefore be considered having potential for high throughput purposes, and being an attractive candidate as the second separation dimension in 2-D HPLC.