Purificação de uma beta-galactosidase produzida por aspergillus foetidus através de técnicas cromatográficas

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.A intolerância à lactose é caracterizada pela desordem na absorção do carboidrato lactose, que acomete milhares de pessoas em todo o mundo e está associada à deficiência na produção ou ausência da enzima β-galactosidase intestinal. Dentro deste contexto, a hidrólise enzimática deste dissacarídeo por β-galactosidase desempenha papel importante no processamento de produtos lácteos com baixo teor de lactose para o consumo por indivíduos intolerantes. A literatura descreve que, os microrganismos, como fungos filamentosos, são capazes de produzir enzimas de interesse industrial, como por exemplo β-galctosidases. Desta forma, o objetivo deste trabalho, foi avaliar a produção em meio líquido de β-galactosidase extracelular por fungos filamentosos isolados do solo do cerrado brasileiro, utilizando resíduos de soja como fonte de carbono. Inicialmente foi realizada uma triagem com 25 linhagens de fungos filamentosos quanto ao potencial de produção de β-galactosidase. Após a seleção do Aspergillus foetidus, como o melhor fungo produtor, a produção enzimática foi otimizada variando o meio de cultivo e em seguida, realizou-se um planejamento experimental com análise de superfície resposta com as variáveis independentes pH, temperatura e agitação para avaliar a influência destes parâmetros físico-químicos na produção da enzima. A melhor atividade de β-galactosidase (26,4 U/mL) foi encontrada no sistema contendo casca de soja 2% (p/v) em pH inicial 7,0 a 28°C com 120 rpm. A curva de indução indicou o 7° dia de cultivo como o período de melhor atividade. A β-galactosidase foi parcialmente purificada utilizando cromatografia de gel filtração e troca iônica. A fração mais pura da enzima foi então, caracterizada visando sua aplicação industrial alimentícia e farmacêutica sendo sua atividade máxima a 50°C e pH 3,0. A enzima hidrolisou 63% da lactose presente no leite (Molico®), e quando testada sua estabilidade em condições de simulação gástrica manteve-se ativa por 2 horas a 37°C. Estes resultados apontam para a parcial purificação de uma β-galactosidase ácida e estável de Aspergillus foetidus com potencial aplicação industrial a partir de resíduo de baixo custo.Lactose intolerance is characterized by a disorder in absorption of the carbohydrate lactose, which affects thousands of people worldwide and is associated with a deficiency or absence of intestinal β-galactosidase enzyme. In this context, the enzymatic hydrolysis of this disaccharide by β-galactosidase plays an important role in the processing of dairy products with low lactose content for consumption by intolerant individuals. The literature describes that microorganisms such as filamentous fungi can produce enzymes of industrial interest, such as β-galactosidases. Thus the objective of this study was to evaluate the production of extracellular β-galactosidase by filamentous fungi isolated from the soil of the Brazilian savanna in liquid medium using soybean residue as a carbon source. Initially a screening of 25 filamentous fungi strains grown in soybean residue medium was performed assessing β-galactosidase production potential. After selecting Aspergillus foetidus as the best producer, enzyme production was optimized by varying the culture medium followed by an experimental design with response surface analysis with the independent variables pH, temperature and stirring to assess the influence of these physico-chemical parameters in enzyme production. The best β-galactosidase activity (26.4 U/mL) was found in the system containing soybean residues 2% (w/v) at initial pH 7.0 at 28°C with 120 rpm. The induction curve indicated the 7th culture day as the period of highest activity. β-galactosidase was half purified using gel filtration and ion exchange chromatography. The purified enzyme was then characterized aiming its food and pharmaceutical industrial uses with maximal activity at 50°C and pH 3.0. The enzyme hydrolyzed 63% of the lactose present in milk (Molico®), and when tested its stability in gastric simulated conditions it remained active for 2 hours at 37°C. These results demonstrate the half purification of an acidic and stable β-galactosidase from Aspergillus foetidus with potential industrial application from low-cost residue

Optimization and partial purification of beta‑galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue

β-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular β-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was optimized by a factorial design. Maximal β-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R2 is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal β-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin’s presence, 80% of activity was preserved after 2 h. These results reveal a A. niger β-galactosidase obtained from residue with favorable characteristics for food industries