Purificação de penicilina G por adsorção em resinas hidrofóbicas
Barros, André Nogueira Castro de
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The separation of biotechnological products is an area of great economic importance once it represents the majority of the cost of this product. On industrial production of penicillin G (penG), the separation of the antibiotic has been made by extraction on organic solvent. However the decrease on the use of these solvents has become imperative in many chemical processes, caused mainly by environmental issues, which has motivating the pharmaceutical company PRODOTTI S/A, penG producer, on the research of new and less environmental aggressive ways to obtain this substance. Adsorption is one of the methods of concentration/purification most used by the chemical industry and it is based on the attraction exercised over the product (liquid phase) by a solid phase. The project sponsored by UFSCar/Prodotti/FINEP in progress has as one of its aims the study of the technical availability of substitution of extraction of penG by solvents for extraction by adsorption in hydrophobic resins. The penG, which is a weak acid, has therefore a favorable adsorption in low pH values. Nevertheless, penG can degrade itself under such conditions of pH, being required, in this way, adequate conditions for the operation. Therefore, the objective of this assignment was evaluate the influence of pH and temperature on the efficiency on adsorption of penG by hydrophobic resins, seeking conditions for maximum efficiency that do not result in degradation. Initially, it was necessary to develop the methodology that could allowed that analysis of a huge amount of penG samples in culture medium, which means that the penG could be analyzed in the presence of other nutrients and also could be distinguished between the intact and the degradated form. Some methods such as CLAE, iodometric and bioassay were analyzed and the conclusion was the CLAE method was necessary and also was able to quantify the penG s degradation rate. This features allowed the studies of penG s stability when exposed to different value pH and temperatures, the maximum half-life that it was stable in each condition and the time of growth, which means that it was possible to determine the time that the variation in the medium s concentration that did not affect the quantification of penG. Nevertheless, the CLAE method is very expensive to be applied to a large number of samples originated from culture medium. With this in mind, a new methodology was developed based on the complete hydrolysis of penG catalyzed by the penicillin G acilase, and this reaction proved to be precise and reproducible, reasons for being chosen in the later steps of this study. The efficiency of adsorption of penG was evaluated in the resins XAD-4, XAD-7, XAD-761 and activated carbon, and the XAD-4 resin has shown to be the most efficient with little increment of efficiency at pH 4.0 (44%) when compared to pH 6.0 (36%). Therefore, the XAD-4 resin was studied for parameters such as adsorption kinetics and the isotherms of adsorption were determined in different value of pH and temperatures. The Langmuir s isotherm had the best fit into the data collected in all the conditions, with maximum value of qmax = 595.06±51.54 mg penG/g resin, at pH 4 and 4°C. In order to analyze the adsorptions in value of pH lower than 4.0 without the occurrence of penG s degradation that usually occurs at high concentrations of this molecule and at value of pH lower than 4.0, two strategies referring to the adsorption conditions were studied. For the first one, the fermentative broth s pH is adjusted and right after this same broth is maintained in contact with the adsorbent. For the second method, the adsorbent was add to the fermentative broth at a pH of 7.0 and with the adsorption process the pH was reduced until reach the complete adsorption of the penG. The last process showed a better efficiency of adsorption of 0.77g penG/g resin and productivity of 0.31 g penG/g resin/hour at pH 4.0, meanwhile the direct adsorption at pH 4.0 had an efficiency of 0.548g penG/g resin (0.548 g penG/g resin/hour). The second strategy with the gradient is better since it increases the efficiency and decreases the productivity of the process. The results showed that the ideal situation would be to work with pH values even lower than 4, but elevated concentrations of penG will lead to the degradation of the antibiotic in this pH range. The desorption of penG was studied using a technique of experimental planning and analysis such as pH, temperature and composition of mixture water/ethanol. The results showed that to obtain a maximum desorption, the process must be effectuated at 8°C and utilizes as eluent a mixture of 82.5% ethanol and 17.5% water on a pH of 6.2.