Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2020-05-12T11:09:47Z No. of bitstreams: 1 2012 - L?via Nolasco Macedo Muruci.pdf: 2196519 bytes, checksum: 2641a71d876714242f07d8b53d9c0831 (MD5) Made available in DSpace on 2020-05-12T11:09:47Z (GMT). No. of bitstreams: 1 2012 - L?via Nolasco Macedo Muruci.pdf: 2196519 bytes, checksum: 2641a71d876714242f07d8b53d9c0831 (MD5) Previous issue date: 2012-12-10 Lipases are an important group of enzymes, mainly because of the great versatility of applications. They are enzymes that, besides catalyzing the hydrolysis of oils and fats, catalyze reverse reactions, such as esterification, transesterification and interesterification. The lipase production by solid state fermentation (SSF) has received great attention, mainly by the possibility of using agroindustrial coproducts. The aim of this work was to produce, concentrate and characterize lipase of Aspergillus niger obtained by (SSF), using alkaline soapstocks and wheat bran. The enzyme production was performed in aerated columns at 32?C for 48 hours. First, in order to select the variables that significantly influenced the production of lipases, an experimental Plackett & Burman planning was conducted. Four process variables were evaluated: inoculum and inducer concentration (corn soapstock and olive oil), volume of solution of ammonium sulfate and aeration. Based on the results obtained, a full factorial design that aimed to improve the production of the enzyme was performed. In this case, two independent variables were studied: aeration, in vvm (0, 0.5, 1) and concentration of corn soapstock % w/w (0, 0.5, 1). Results obtained showed that the highest lipase activity (215 U/gds) and specific activity (11392.1 U/g protein) were obtained in test 4 (1.0% corn soapstock, 1.0 vvm aeration, 60 mL of solution of ammonium sulfate and 106 spores/g medium. Then, the enzyme production ocurred in conditions selected as optimal and other two samples of soapstock were tested: sunflower and canola, besides corn soapstock and in the absence of inducer. It was found that the highest lipase activity (254.4 U/gds) was obtained without the use of soapstock. Therefore, its presence is dispensable for the production of lipase A. niger under the conditions studied. A kinetic study showed that the lipase production occurred associated with fungal growth and an increased productivity (4 U/gds.h) occurred in 48 hours of fermentation in the absence of soapstock. Then, the enzyme concentration was performed by precipitation with ammonium sulfate in four saturation conditions (40, 60, 80 and 90%), and it was observed that best results were obtained with 90% saturation. The enzyme extract was also concentrated by lyophilization. Partial characterization of the enzyme was carried out with concentrated samples. The lipase showed optimum activity at pH between 3.0 and 5.6 and temperatures of 30 to 55?C. It presented thermostability profile similar to the temperatures of 30, 40 and 50?C, retaining about 42% of its activity for up to 30 hours of incubation. While the pH stability profile was also similar to the values tested after 30 h of incubation, the higher residual lipase activities were observed at acidic pH: pH 3.0 (59%), pH 4.0 (55%) and pH 5.0 (62%). The enzyme retained 100% of activity in 150 days stored at -18? C. The evaluation of the specificity in different vegetable oils showed higher hydrolytic activity in coconut oil (201 U/mL). Therefore, it is suggested that the enzyme has an affinity for the medium chain fatty acids. As lipases constituem um importante grupo de enzimas, principalmente por apresentarem grande versatilidade de aplica??es. S?o enzimas que al?m de catalisar a hidr?lise de ?leos e gorduras, catalisam rea??es inversas, como esterifica??o, transesterifica??o e interesterifica??o. A produ??o de lipases por fermenta??o no estado s?lido (FES) tem recebido grande aten??o, principalmente pela possibilidade de utilizar res?duos agroindustriais. O objetivo deste trabalho foi produzir, concentrar e caracterizar lipase de Aspergillus niger obtida por (FES), utilizando borras alcalinas e farelo de trigo. A produ??o da enzima foi conduzida em colunas aeradas a 32?C, por 48 horas. Primeiramente, para selecionar as vari?veis que influenciavam de forma significativa a produ??o de lipases foi conduzido um planejamento experimental de Plackett & Burman. Foram avaliadas quatro vari?veis de processo: concentra??o de in?culo e de indutor (borra de milho e ?leo de oliva), volume de solu??o de sulfato de am?nio e aera??o. Com base nos resultados obtidos, foi realizado o delineamento fatorial completo 22 que visou melhorar a produ??o da enzima. Neste caso, foram estudadas duas vari?veis independentes: aera??o em vvm (0; 0,5; 1) e concentra??o de borra de milho em % p/p (0; 0,5; 1). Resultados obtidos revelaram que a maior atividade lip?sica (215 U/gms) e espec?fica (11392 U/g de prote?na) foram obtidas no ensaio 4 (1,0% de borra de milho, 1,0 vvm de aera??o, 60 mL de solu??o de sulfato de am?nio e 106 esporos/g de meio). Em seguida, a produ??o da enzima ocorreu em condi??es selecionadas como ?timas, testando-se outras duas borras: girassol e canola, al?m da borra de milho e na aus?ncia de indutor. Verificou-se que a maior atividade lip?sica (254 U/gms) foi obtida sem o uso de borra. Portanto, a presen?a das borras ? dispens?vel para a produ??o de lipase de A. niger, nas condi??es estudadas. Um estudo cin?tico demonstrou que a produ??o de lipase ocorreu associada ao crescimento f?ngico, e a maior produtividade (4 U/gms.h) ocorreu em 48 horas de fermenta??o, na aus?ncia de borra. Em seguida, foi realizada a concentra??o enzim?tica por precipita??o com sulfato de am?nio em quatro condi??es de satura??o (40, 60, 80 e 90%), e foram observados melhores resultados com 90% de satura??o. O extrato enzim?tico tamb?m foi concentrado por liofiliza??o. A caracteriza??o parcial da enzima foi realizada com amostras concentradas. A lipase apresentou atividade ?tima a pH entre 3,0 e 5,6 e a temperaturas de 30 a 55?C. Apresentou perfil de termoestabilidade semelhante para as temperaturas de 30, 40 e 50oC, retendo cerca de 42% de sua atividade por at? 30 horas de incuba??o. Embora o perfil de estabilidade ao pH tamb?m tenha sido semelhante para os valores testados, ap?s 30 horas de incuba??o, as maiores atividades lip?sicas residuais foram observadas em pH ?cido: pH 3,0 (59%), pH 4,0 (55%) e pH 5,0 (62%). A enzima manteve 100% da atividade em 150 dias estocada a -18?C. A avalia??o da especificidade sob diferentes ?leos vegetais demonstrou maior atividade hidrol?tica sob o ?leo de coco (201 U/mL). Portanto, sugere-se que a enzima possua afinidade por ?cidos graxos de cadeia m?dia.