Sistema de duas fases aquosas NaPA/PEG aplicado na purificação de proteases produzidas por fungos filamentosos

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas, 2014.O desenvolvimento de metologias que permitam a purificação de biomoléculas de interesse industral é alvo de estudo devido sua importância comercial. Foi avaliada a purificação de proteases produzidas pelos fungos isolados do Cerrado, Penicillium fellutanum e Penicillium restrictum por extração líquido-líquido em sistema de duas fases aquosas (ATPS), composto por polietilenoglicol (PEG) e poliacrilato de sódio (NaPA). As duas fases no ATPS NaPA/PEG foram formadas através da mistura dos polímeros com um sal (NaCl) e caldo fermentado de P. fellutanum ou P. restrictum. Para os sistemas formados com o caldo fermentado de P. restrictum foram estudados o efeito da massa molar de PEG (2000, 4000 e 6000 g.mol-1), concentração de PEG (4, 6, 8 e 10% p/p), concentração de NaPA (4, 6, 8, 10, 15 e 20% p/p) e concentração do caldo fermentado (25, 35 e 45% p/p) na partição da enzima a 25 °C. Os valores do coeficiente de partição (K) obtidos variavam de 0,06 a 37,73, mostrando a versatilidade do método para a purificação da biomolécula sob investigação. Na maioria dos sistemas analisados conseguiu-se a partição da biomolécula de interesse para a fase oposta àquela das proteínas totais, proporcionando assim efetiva purificação da enzima. O maior K (partição preferencial na fase rica PEG) foi obtido usando-se: concentração de NaPA igual a 20% p/p, concentração de PEG 2000gmol-1 igual a 4% p/p e concentração de caldo fermentado igual a 45% p/p. Para grande número dos sistemas analisados foram obtidos elevados valores referentes ao balanço de massa (BM) indicando a estabilidade da enzima em relação aos componentes do sistema. Diversos sistemas analisados apresentaram elevados níveis de rendimentos (η) – alguns acima de 90%. Para os sistemas formados com o caldo fermentado de P. fellutanum foram analisados o efeito da massa molar de PEG (2000, 4000 e 6000 g.mol-1), a concentração de PEG (3, 6, 8 e 10 % p/p) e a concentração de NaPA (6, 8, e 10 % p/p) sobre o coeficiente de partição (K) a 25°C. Também foi analisada a influência da concentração de Na2SO4 (5, 10 e 15% p/p) na reextração da enzima. Os valores do coeficiente de partição K obtidos variaram de 1,21 até 77,51. A partição na fase superior foi maior em sistemas com maior concentração de NaPA, maior massa molar de PEG e menor concentração de PEG. Usando a estratégia de reextração foi possível direcionar a partição da protease para a fase oposta às demais proteínas, proporcionando assim uma etapa de prépurificação da biomolécula. Os resultados obtidos através dos APTS NaPA/PEG e posterior reextração com Na2SO4 demonstraram as potencialidades do método no processo de prépurificação de proteases a partir dos caldos fermentados de P. restrictum e P. fellutanum. ____________________________________________________________________________________ ABSTRACTThe development of metologias allowing purification of biomolecules of industrial interest is target of study because of its commercial importance. The partition of proteases produced by fungi of the Brazilian Cerrado, Penicillium fellutanum and Penicillium restrictum, in aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and sodium polyacrylate (NaPA) was evaluated. The two phases in ATPS NaPA/PEG were formed by mixing the polymers with a salt (NaCl) and fermented broth of P. fellutanum or P. restrictum. For those systems formed with fermented broth of P. restrictum were studied the effect of molecular size of PEG (2000, 4000 and 6000 g.mol-1), PEG concentration (4, 6, 8 and 10% w/w), concentration NaPA (4, 6, 8, 10, 15 and 20% w/w) and fermented broth concentration (25, 35 and 45% w/w) in partitioning of enzyme at 25°C. The values of partition coefficient (K) obtained varied from 0.064 to 37.73, showing the versatility of the method for the purification of the biomolecule under investigation. In most systems examined it was achieved the partition of biomolecule in the opposite phase to that of total proteins and thus provide effective enzyme purification. The highest K (preferential partitioning in PEG-rich phase) was obtained using: 20% w/w of NaPA concentration, 4% w/w of PEG 2000 g.mol-1 and 45% w/w of broth concentration. For large number of systems analyzed, high values of BM were obtained indicating the stability of the enzyme in relation to system components. Several systems analyzed showed high levels of yield (η) - some over 90%. For those systems formed with the fermented broth of P. fellutanum were analysed the effect of molecular size of PEG (2000, 4000 and 6000 g.mol-1), PEG concentration (3, 6, 8 and 10% w/w) and NaPA concentration (6, 8, and 10% w/w) over the partition coefficient (K) at 25°C. It was also analyzed the influence of Na2SO4 concentration (5, 10 and 15% w/w) in the re-extraction of the enzyme. The values of partition coefficient K obtained ranged from 1.21 to 77.51. The partition in the upper layer was greater in systems with higher NaPA concentration, higher PEG molecular weight and lower PEG concentration. By using the re-extraction strategy it was possible to partition the target protease to the opposite phase to the other proteins, thereby providing a pre-purification step of the biomolecule. The results obtained by ATPS NaPA/PEG/NaCl and subsequent re-extraction with Na2SO4 demonstrated the potential of this method in the pre-purification of proteases from the fermentation broth of P. restrictum and P. fellutanum

Purification of bromelain enzyme from Curauá (Ananaserectifolius LB Smith) white variety, by aqueous two-phase system PEG 4000/potassium phosphate

The Curauá (Ananaserectifolius LB Smith) is a species belonging to the family Bromeliaceae. Occurs in the states of Para, Amazonas, Amapá, Acre, Mato Grosso and Goiás, has two varieties, purple and white, and its fibers are used in automotive and textile industries due to its strength, softness and lightweight. Currently, only the fiber is used in industry, the rest is considered waste. However, this residue contains compounds with important properties to be discovered and studied. An enzyme complex found in this residue is bromelain, a group of proteolysis’ enzymes with application in several areas such as in the food, pharmaceutical and cosmetics industries. Today, its use is aiming the pharmaceutical industry, the production of ointments, gels, creams and lotions because they offer a wide range of therapeutic efficacies: antiedemas, anti-inflammatory, antithrombotic and fibrinolytic activities. This work studied the enzyme purification and recovery presented in the leaves of Curauá by means of an ATPS (aqueous two-phase system) PEG 4000/potassium phosphate. The protein content was measured by Bradford reagent and the enzymatic activity was measured by the Biuret reagent. Batch assays were performed aiming the enzyme extraction and recuperation, using the partition coefficient as indicator. It was used pH 7.0, 8.0 and 9.0, varying the proportional composition between the polymeric and the saline phases (tie-lines). The white variety of Curauá was used. The best purification factor was 4.53 and about 113.04 µg/mL of total protein measured by Bradford8395399CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Purificação da enzima bromelina presente no curauá (Ananas erectifolius L.B. Smith) variedade roxa, por sistema bifásico aquoso PEG 4000/fosfato de potássio

Curauá (Ananas erectifolius LB Smith) is a typical plant of northern and central-western Brazil. It produces a highly strong fiber with applications in several areas, mainly in the automotive industry. Belonging to the family of Bromeliaceae, it contains significant levels of bromelain, a group of proteolysis’ enzymes with application in several areas such as in the food, pharmaceutical and cosmetics industries. In this work we evaluated the purification of the enzyme present in the leaves of Curauá by means of an aqueous two-phase system PEG 4000/Potassium phosphate. The enzyme had its activity measured in each phase by the Biuret method. The purification factor (PF) was obtained in order to optimize the conditions for purification of the bromelain. It was used 03 pH 7.0, 8.0 and 9.0 values, varying the proportional composition in between the polymeric and the saline phases (tie-lines). It was used the purple variety of Curauá. The results showed that PEG system 4000/ potassium phosphate polymer with a higher concentration and at pH 7.0 produced better results in enzyme purification.O curauá (Ananas erectifolius L.B. Smith) é uma planta característica do Norte e Centro-oeste do Brasil e produz uma fibra de alta resistência, com aplicações nas diversas áreas, principalmente na indústria automobilística. Pertencente à família das Bromeliaceae, contém níveis significativos de bromelina, conjunto de enzimas proteolíticas com aplicação em diferentes áreas, tais como em indústrias alimentícias, farmacêuticas e de cosméticos. Neste trabalho avaliou-se a purificação da enzima presente nas folhas do curauá (Ananas erectifolius L.B. SMITH), através de sistema bifásico aquoso PEG 4000/Fosfato de potássio e se mediu a atividade enzimática em cada fase, pelo método de Biureto, obtendo-se o fator de purificação (FP), com o objetivo de otimizar as condições de purificação da bromelina. Utilizaram-se 3 valores de pH 7,0; 8,0 e 9,0 variando-se a composição proporcional entre a fase polimérica e a salina (tie-lines), Foi utilizada a variedade roxa do curauá, em que os resultados mostraram que o sistema PEG 4000/Fosfato de potássio com maior concentração polimérica e em pH 7,0, apresentou melhor resultado na purificação enzimática