Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

A biotechnology perspective of fungal proteases

Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

Screening of wild type Streptomyces isolates able to overproduce clavulanic acid

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

Biological and physicochemical stability of ceftazidime and aminophylline on glucose parenteral solution

Ceftazidime is a broad spectrum antibiotic administered mainly by the parenteral route, and it is especially effective against Pseudomonas aeruginosa. The period of time in which serum levels exceed the Minimum Inhibitory Concentration (MIC) is an important pharmacodynamic parameter for its efficacy. One of the forms to extend this period is to administer the antibiotic by continuous infusion, after prior dilution in a Parenteral Solution (PS). The present work assessed the stability of ceftazidime in 5% glucose PS for 24 hours, combined or not with aminophylline, through High Performance Liquid Chromatography (HPLC). The physicochemical evaluation was accompanied by in vitro antimicrobial activity compared MIC test in the 24-hour period. Escherichia coli and Pseudomonas aeruginosa were the microorganisms chosen for the MIC comparison. The HPLC analysis confirmed ceftazidime and aminophylline individual stability on PS, while the MIC values were slightly higher than the mean described in the literature. When both drugs were associated in the same PS, the ceftazidime concentration by HPLC decreased 25% after 24 hours. Not only did the MIC values show high loss of antibiotic activity within the same period, but also altered MIC values immediately after the preparation, which was not detected by HPLC. Our results indicate that this drug combination is not compatible, even if used right away, and that PS might not be the best vehicle for ceftazidime, emphasizing the importance of the MIC evaluation for drug interactions.

Ceftazidima é um antimicrobiano administrado por via parenteral, que apresenta amplo espectro de ação, principalmente contra Pseudomonas aeruginosa. O tempo em que a concentração sérica de ceftazidima permanece acima da concentração mínima inibitória (MIC) é um importante parâmetro farmacodinâmico para a determinação da eficácia antimicrobiana e pode ser potencializado através da utilização de infusão contínua em soluções parenterais (PS). Este artigo visa a avaliar a estabilidade da ceftazidima em solução de glicose 5%, na presença e na ausência do fármaco aminofilina, através de cromatografia líquida de alta eficiência HPLC e MIC durante o período de 24 horas. Os microorganismos selecionados para a determinação do MIC foram Escherichia coli e Pseudomonas aeruginosa. Os ensaios em cromatógrafo líquido confirmaram a estabilidade dos fármacos ceftazidima e aminofilina quando são individualmente associados em PS, enquanto os valores de MIC ficaram maiores que os valores encontrados na literatura. Quando ambos os fármacos foram associados na mesma solução parenteral a concentração de ceftazidima obtida por HPLC diminuiu 25% depois de 24 horas. Os valores de MIC mostraram maior decaimento da atividade antimicrobiana neste mesmo período e também valores de MIC alterados nas soluções preparadas no tempo zero, decaimento este que não foi detectado em HPLC. Os resultados indicaram incompatibilidade na associação dos fármacos em PS, enfatizando a importância dos resultados de MIC para interações de fármacos.

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

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.

Kinetic analysis of batch ethanol acetylation in isothermal non-stationary multiphase systems by lyophilized mycelium of Aspergillus oryzae

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integral method. Owing to the poor results obtained by this way, we developed an alternative approach, combining initial rates and integral analysis and reducing the number of empirical parameters to be determined by the use of equilibrium data. All the values of the parameters calculated according to this "composite" approach to kinetic analysis well correlate with experimental data.

Micelas reversas de lecitina de soja: uma alternativa para purificação de proteínas / Soybean lecithin reversed micelles: an alternative for protein purification

No presente trabalho, estudaram-se os efeitos de diversos parâmetros sobre a extração das proteínas caseína e albumina de soro bovino empregando micelas reversas de lecitina de soja. Independentemente da condição empregada, a extração da albumina apresentou baixo rendimento (variando de 0 por cento a 4 por cento, aproximadamente), resultado de um significativo efeito de exclusão por tamanho. Com relação à caseína, o rendimento da extração aumentou 23 vezes com o aumento do tempo de agitação, ou seja, com o maior tempo de contato entre a proteína e o sistema de micelas reversas. A adição de 1-hexanol ao sistema, usado como co-solvente, foi efetiva, aumentando a solubilização da caseína em 36 por cento, sendo os rendimentos da extração desta proteína muito influenciados pelo pH. Os valores máximos de eficiência obtidos foram de 20 por cento em pH 7,9, 80 por cento em pH 5,4 e 100 por cento em pH 5,0 (pH próximo ao pI da proteína).

In this work, the effect of different parameters for extraction of casein and bovine serum albumin (BSA) were studied. Such proteins were extracted by soybean lecithin reversed micelles. BSA extraction was not effective, independent of the extraction conditions employed. Owing to its molar mass, the effect of exclusion by size was clearly observed. The casein extraction yield increased about 23-fold as a function of agitation time. In other words, the increase occurred by using higher contact time between protein and reversed micelles. The use of hexanol as a co-solvent was effective, and increased casein extraction to 36 percent. The extraction values were strongly influenced by pH, and the high extraction yield was obtained under the following conditions: 20 percent at pH 7.9, 80 percent at pH 5.4 e 100 percent at pH 5.0 (close to casein isoelectrical point).

Partial purification of glucose-6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems

Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme used in biochemical and medical studies and in several analytical methods that have industrial and commercial application. This work evaluated the extraction of G6PDH in aqueous two-phase system (ATPS) of poly(ethyleneglycol) (PEG)/phosphate buffer, using as enzyme source a medium prepared through commercial baker's yeast disruption. Firstly, the effects of PEG molar mass on the enzyme partition and of homogenization and rest on the system equilibrium were investigated. Afterwards, several ATPS were prepared using statistical analysis (2² factorial design). The results, including kinetic and thermodynamic parameters for the G6PDH activity, showed partial purification of this enzyme in ATPS composed of 17.5 percent (w/w) PEG400 and 15.0 percent (w/w) phosphate. A high enzymatic recovery value (97.7 percent), a high partition coefficient (351), and an acceptable purification factor (2.28 times higher than in cell homogenate) were attained from the top phase. So, it was possible to attain an effective enzyme pre-purification by separating some contaminants with a simple method such as liquid-liquid extraction in aqueous two-phase systems (ATPS).

Glicose-6-fosfato desidrogenase (G6PDH) é uma importante enzima usada em estudos bioquímicos e médicos, bem como em diversos métodos analíticos com aplicação comercial e industrial. Neste trabalho foi avaliado a extração da G6PDH em sistemas de duas fases aquosas (ATPS) constituídos por poli(etilenoglicol) (PEG)/tampão fosfato, usando como fonte de enzima um meio preparado por rompimento de leveduras de panificação comercial. Inicialmente foram investigados os efeitos da massa molar do PEG na partição da enzima e da homogeneização e repouso no equilíbrio do sistema. Na sequência, diversos ATPS foram preparados usando análise estatística (planejamento fatorial 2²). Os resultados, incluindo parâmetros cinéticos e termodinâmicos para a atividade da G6PDH, indicaram parcial purificação desta enzima em ATPS constituídos por 17,5 por cento (p/p) PEG400 e 15,0 por cento (p/p) fosfato. Um alto valor de recuperação enzimática (97,7 por cento), um alto coeficiente de partição (351), e um fator de purificação aceitável (2,28 vezes maior que em homogenato celular) foram obtidos na fase superior do sistema. Assim, foi possível alcançar uma pré-purificação eficaz da enzima separando alguns contaminadores aplicando um método simples tal como a extração líquido-líquido em sistemas bifásicos (ATPS).

Caracterização da xilose redutase extraída por micelas reversas-CTAB a partir de homogenato de Candida guilliermondii / Characterization of xylose reductase extracted by CTAB-reversed micelles from Candida guilliermondii homogenate

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 degrees C, 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 'V IND. max' values against both xylose and NADPH for XR before and after extraction by reversed-micelles differed about 6%, whereas the difference on KM values were more pronounced. The ('K IND. m') 'IND. xylose' for XR after extraction was about 35% higher than before extraction, meanwhile ('K IND. m') 'IND. NADPH' was about 30% lower after than before extraction. As the KM variations indirectly signaled, the XR affinity simultaneously diminishes for xylose and increases for NADPH. This could probably explain why the 'V IND. max' values for XR before and after extraction were quite similar

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 KM foram mais pronunciadas. O (KM)xilose para a XR após a extração foi cerca de 35% maior do que antes da extração, enquanto que (KM)NADPH foi 30% menor após do que antes da extração. As variações nos valores de KM 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

Micellar solubilization of ibuprofen - influence of surfactant head groups on the extent of solubilization

An important property of micells 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 there 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) (`C IND. 12´ E`O IND. 8´). 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...

Large scale purification of Clostridium perfringens toxins: a review

Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and commonly found in the intestines of animals, including humans. C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (alpha, ß, épsilon, iota). However the toxins (theta, delta, lambda and enterotoxin) are also synthesized by C. perfringens strain. Many attempts to purify the toxins produced by C. perfringens have been proposed. In this review we discuss the purification methods used to isolate toxins from C. perfringens reported in last four decades

Otimização do processo de extração de BETA - xilosidase por sistemas micelares reversos / Optimization of beta xilosidade extration process using reversed micelar systems

A enzima "BETA"-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 empregando-se a metodologia de superfície de resposta. A partir dos resultados obtidos, foi proposto um modelo matemático para descrever o processo de extração da "BETA"-xilosidase na região de trabalho estudada. De acordo com a equação do modelo, podem ser obtidos valores máximos de recuperação de 35,05 ñ 6,40 por cento nas seguintes condições: pH 8,0, concentração de CTAB 0,2...

Influence of pH on the partition of glucose-6-phosphate dehydrogenase and hexokinase in aqueous two-phase system

Glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase (HK) are important enzymes used in biochemical and medical studies and in several analytical methods. Aqueous two-phase system (ATPS) formed by a polymer solution and an electrolyte solution provides a method for the separation and purification of enzymes with several advantages, including biocompatibility and easy scale up of the process. In this work, the effects of different pH values on the storage stability and partitioning behavior (K, partition coefficient) of the enzymes G6PDH and HK from baker's yeast extract were investigated in ATPS. The results, obtained from the 17.5 percent PEG 400 : 15.0 percent phosphate system, showed that when the pH was increased from 5.0 to 8.8, the K HK increased 26-fold and the K G6PDH 2.2-fold. In the 20.0 percent PEG 1500 : 17.5 percent phosphate system, the K HK and K G6PDH increased 13 and 1.2-fold, when the pH value was increased from 3.8 to 8.8, respectively. This leads to the conclusion that the partition coefficient for both enzymes is favored by high pH values. A statistical analysis of the results was conducted to confirm this conclusion

Liquid-liquid extraction of pharmaceuticals by aqueous two-phase systems

Aqueous two-phase systems are generally composed of a water solution of two structurally distinct hydrophilic polymers or of one polymer and certain salts. From analytical to commercial scale, aqueous two-phase systems have their application in the purification, characterization and study of biomaterials. In this review, we present some applications of aqueous two-phase systems in the separation and study of various pharmaceuticals, incluing recombinant proteins, antibodies and antigens, antibiotics, amino acids and oligopeptides, lactic acid, enzymes etc. Some new developments of the aqueous two-phase systems are reviewed and the prospects of this excellent technology are discussed...

Purificação de glicose-6-fosfato desidrogenase (G6PDH) em sistemas de duas fases aquosas utilizando PEG/citrato / Purification of glucose-6-phosphate dehydrogenase (G6PDH) by aqueous two-phase system by using PEG/citrate

Foi estudada, neste trabalho, a purificação por extração líquido-líquido da enzima glicose-6-fosfato desidrogenase (G6PDH), de Saccharomyces cerevisiae, em sistemas de duas fases aquosas, preparados com polietilenoglicol (PEG) e sal citrato. Foram avaliados, também, a estabilidade e o equilíbrio do sistema, além do efeito da concentração de citrato de sódio, concentração e massa molar do PEG na partição desta enzima, com auxílio de um planejamento experimental do tipo fatorial 2ü. Os resultados das extrações mostraram que é possível purificar a G6PDH nesse tipo de sistema, com a condução do processo em duas etapas. A primeira delas, conduzida com 17 porcento (p/p) de PEG 400 e 20 porcento (p/p) de citrato...