65 research outputs found

    Reactores enzimáticos

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    Sistemas aquosos bifásicos uma ferramenta sustentável para a extração de ácido clavulânico a partir de diferentes fontes

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    O ácido clavulânico (AC) é um inibidor de β-lactamases que tem vindo a ser largamente utilizado na área médica. Embora seja de extrema importância, o desenvolvimento de processos alternativos de produção e purificação é ainda insignificante, sendo fundamental o estudo de técnicas de extração mais biocompatíveis, como os Sistemas Aquosos Bifásicos (SABs). Assim, este trabalho objetivou o estudo de Sistemas Aquosos Bifásicos baseados em polímeros como uma ferramenta alternativa para a extração de AC. Foram testados dois SPAB compostos por Polietileno Glicol (PEG) com massa molecular (M) de 4000 g/mol e Poliacrilato de Sódio de 8000 g/mol, nos quais foi alterado o eletrólito indutor da formação de fases, em particular, sulfato de sódio (Na2SO4,) e cloreto de sódio (NaCl). Ademais, este trabalho visou também avaliar a eficiência de extração do AC, bem como compreender o efeito dos contaminantes no processo de migração. Para tal, foi avaliada a extração do AC a partir de três fontes distintas: solução pura (99,9%); solução comercial (60%); diretamente a partir do sobrenadante de um meio fermentando de Streptomyces clavuligerus. Os resultados obtidos demonstraram que independentemente da fonte inicial do AC, ambos os SABs poliméricos promoveram uma partição preferencial do AC para a fase rica em PEG, sendo o coeficiente de partição maior nos sistemas com Na2SO4 do que com NaCl. Após identificar a grande capacidade de partição de AC, o SAB com PEG/NaPA/Na2SO4 foi também utilizado para avaliar a partição de proteínas presente no meio fermentado, sendo também obtida uma preferencial partição destas para a fase rica em PEG. Assim, apesar da baixa capacidade de purificação de AC frente a proteínas contaminantes, os SABs estudados demonstraram que podem ser uma técnica alternativa sustentável e bastante econômica para uma etapa inicial de clarificação/concentração de bioprodutos a partir de caldos fermentados

    Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi

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    L-asparaginase is used for the treatment of acute lymphoblastic leukaemia (ALL); however, its formulation presents drawbacks such as a lack of stability and formation of aggregates. Osmolytes are small molecules accumulated by cells in response to environmental stresses and present a protective behaviour, favouring the equilibrium of macromolecules towards the native conformation. Therefore, osmolytes are employed as excipients in pharmaceutical protein formulations. Herein, recombinant L-ASNase II from Erwinia chrysanthemi (ErA II) was analysed with respect to the effect of osmolytes on kinetic and stability of this biopharmaceutical. The aggregation profiles were analysed trough nanotracking particle analysis and dynamic light scattering. The majority of the tested osmolytes increased ErA II specific activity and stability, being more pronounced for sucrose and sorbitol, which increased almost 70% of ErA II activity. The polyol preserved total enzyme activity for 30 days while sucrose preserved 81.1 ± 5.3% total enzyme activity over this period. Each osmolyte resulted in a specific aggregation profile and the presence of sucrose or sorbitol resulted in a lower quantity of aggregates in the range of 100–300 nm. The present findings may contribute to the improvement of adjuvants in L-ASNase formulations and the optimization of other biopharmaceutical formulations.publishe

    Challenges for the self-assembly of poly(Ethylene glycol)-poly(lactic acid) (PEG-PLA) into polymersomes : beyond the theoretical paradigms

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    We acknowledge support from the State of São Paulo Research Foundation (FAPESP-Brazil, projects 2013/08617-7 (Thematic project) and 2014/10456-4 (Apolinário, A.C. PhD fellowship) and the National Council for Scientific and Technological Development (CNPq- Brazil, project 303334/2014-2).Polymersomes (PL), vesicles formed by self-assembly of amphiphilic block copolymers, have been described as promising nanosystems for drug delivery, especially of biomolecules. The film hydration method (FH) is widely used for PL preparation, however, it often requires long hydration times and commonly results in broad size distribution. In this work, we describe the challenges of the self-assembly of poly (ethylene glycol)-poly(lactic acid) (PEG-PLA) into PL by FH exploring different hydrophilic volume fraction (f) values of this copolymer, stirring times, temperatures and post-FH steps in an attempt to reduce broad size distribution of the nanostructures. We demonstrate that, alongside f value, the methods employed for hydration and post-film steps influence the PEG-PLA self-assembly into PL. With initial FH, we found high PDI values (>0.4). However, post-hydration centrifugation significantly reduced PDI to 0.280. Moreover, extrusion at higher concentrations resulted in further improvement of the monodispersity of the samples and narrow size distribution. For PL prepared at concentration of 0.1% (m/v), extrusion resulted in the narrower size distributions corresponding to PDI values of 0.345, 0.144 and 0.081 for PEG45-PLA69, PEG114-PLA153 and PEG114-PLA180, respectively. Additionally, we demonstrated that copolymers with smaller f resulted in larger PL and, therefore, higher encapsulation efficiency (EE%) for proteins, since larger vesicles enclose larger aqueous volumes.Publisher PDFPeer reviewe

    Solubilização micelar do ibuprofeno: influência do grupo polar dos tensoativos no grau de solubilização

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    An important property of micelles with particular significance in pharmacy is their ability to increase the solubility of poorly soluble drugs in water, thus increasing their bioavailability. In this work, the solubilization of ibuprofen (IBU) was studied in micellar solutions of three surfactants possessing the same hydrocarbon tail but different hydrophilic head groups, namely sodium dodecyl sulphate (SDS), dodecyltrimethylammonium bromide (DTAB), and n-dodecyl octa(ethylene oxide) (C12EO8). The results showed that, irrespective of the surfactant type, the solubility of IBU increased linearly with increasing surfactant concentration, as a consequence of the association between the drug and the micelles. The 80 mM DTAB and the 80 mM C12EO8 micellar solutions resulted in a 16-fold increase in solubility of IBU when compared to the buffer solution, whereas the 80 mM SDS micellar solution resulted in a 5.5-fold increase in IBU solubility. The highest value of molar solubilization capacity (chi) was obtained with DTAB, chi = 0.97, followed by C12EO8 ,chi = 0.72, and finally SDS, chi = 0.23. However, due to the stronger tendency of the nonionic surfactant in forming micelles in solution, at the same surfactant concentration, we obtained the same solubility of IBU in both DTAB and C12EO8.Uma propriedade importante das micelas, do ponto de vista farmacêutico, refere-se ao potencial destas em solubilizar fármacos pouco solúveis em água, aumentando sua biodisponibilidade. No presente trabalho, estudou-se a solubilização de ibuprofeno (IBU) em soluções micelares constituídas de três tensoativos apresentando a mesma cauda apolar, porém diferentes grupos hidrofílicos. Os tensoativos estudados foram dodecil sulfato de sódio (SDS), brometo de dodeciltrimetilamônio (DTAB) e óxido de n-dodecil octaetileno (C12EO8). De acordo com os resultados obtidos, a solubilidade do IBU aumentou linearmente com o aumento da concentração de todos os tensoativos estudados, devido às interações entre as micelas e o fármaco. O fármaco IBU apresentou um aumento de 16 vezes em sua solubilidade na presença de DTAB 80 mM e de C12EO8 80 mM. Por outro lado, na presença de SDS 80 mM a solubilidade do IBU aumentou apenas 5,5 vezes. O maior valor para o parâmetro capacidade molar de solubilização (chi) foi observado com o tensoativo DTAB, chi = 0,97, seguido pelo C12EO8, chi = 0,72 e, finalmente, o SDS, chi = 0,23. Entretanto, devido à grande tendência do C12EO8 em formar micelas, o perfil de solubilidade do IBU foi semelhante em DTAB e C12EO8

    Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

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    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed

    Characterization of xylose reductase extracted by CTAB-reversed micelles from Candida guilliermondii homogenate

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    A xilose redutase (XR) (E.C.1.1.1.21), produzida por Candida guilliermondii cultivada em hidrolisado de bagaço de cana de açúcar, foi separada diretamente do homogenato livre de células através da técnica de micelas reversas feitas com cetil trimetil brometo de amônio (CTAB). Obteve-se um rendimento de recuperação da enzima de 100% e um fator de enriquecimento de 5,6 vezes. As condições de extração foram: pH=7,0, condutividade elétrica = 14 mS/cm, T= 5 ºC, 5% (w/w) de hexanol, 22% (w/w) de butanol e 0.15M CTAB. A XR após a extração manteve-se estável no intervalo de pH entre 6.0 e 6.5, sendo a constante de inativação térmica cerca de 6,5 vezes maior do que aquela antes da extração. Os valores de Vmax da XR frente à xilose e NADPH antes e após a extração por micelas reversas diferiram cerca de 6%, enquanto que as diferenças nos valores de K M foram mais pronunciadas. O (K M)xilose para a XR após a extração foi cerca de 35% maior do que antes da extração, enquanto que (K M)NADPH foi 30% menor após do que antes da extração. As variações nos valores de K M indicam, indiretamente, que a afinidade da XR simultaneamente diminui para a xilose e aumenta para o NADPH. Este resultado poderia explicar a razão pela qual os valores de Vmax antes e após a extração terem sido praticamente iguais.Xylosereductase (XR) (E.C.1.1.1.21), produced by Candida guilliermondii, grown in sugar cane bagasse hydrolysate, was separated directly from the cell homogenate by reversed micelles of cetyl trimethyl ammonium bromide (CTAB), attaining a recovery yield of 100% and enrichment factor of 5.6 fold. The extraction conditions were: pH=7.0, electrical conductivity= 14 mS/cm, T=5 ºC, 5% (w/w) of hexanol, 22% (w/w) of butanol and 0.15 M CTAB. The XR after extraction was stable in pH interval of 6.0-6.5 and its heat inactivation constant was about 6.5 fold higher than that before extraction. The Vmax values against both xylose and NADPH for XR before and after extraction by reversed-micelles differed about 6%, whereas the difference on K M values were more pronounced. The (K M)xylose for XR after extraction was about 35% higher than before extraction, meanwhile (K M)NADPH was about 30% lower after than before extraction. As the K M variations indirectly signaled, the XR affinity simultaneously diminishes for xylose and increases for NADPH. This could probably explain why the Vmax values for XR before and after extraction were quite similar

    Optimization of β-xylosidase extraction process using reversed micellar systems

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    β-xylosidase, produced by Penicillium janthinellum, was extracted by using the reversed micellar system of the CTAB (cetyl trimethyl ammonium bromide) cationic surfactant containing isooctane, hexanol and butanol. The combined effects of CTAB and butanol concentrations on enzyme extraction were studied by using the surface response methodology. As a function of the results, it was proposed a mathematical model to describe the process of β-xylosidase extraction. This model predicted a maximal enzyme extraction yield of 35.05±6.4% under the following conditions: pH = 8.0, CTAB = 0.20 M, hexanol = 5% (v/v), and butanol = 20% (v/v). Experimentally, a recovery value of about 38% was attained, and showed that the model is appropriate.A enzima β-xilosidase, produzida pelo fungo Penicillium janthinellum, foi extraída pelo sistema micelar reverso formado pelo agente tensoativo catiônico CTAB em isoctano, hexanol e butanol. Os efeitos combinados da concentração de CTAB e de butanol sobre a extração da enzima foram estudados empregandose a metodologia de superfície de resposta. A partir dos resultados obtidos, foi proposto um modelo matemático para descrever o processo de extração da β-xilosidase na região de trabalho estudada. De acordo com a equação do modelo, podem ser obtidos valores máximos de recuperação de 35,05 ± 6,40% nas seguintes condições: pH 8,0, concentração de CTAB 0,2 M, concentração de hexanol 5% e concentração de butanol 20%. Nestas condições, foram realizados novos experimentos e o valor médio de recuperação obtido foi de 38%

    Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

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    Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.Brazilian Research Funding InstitutionsCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES
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