Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol-gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t 1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis-Menten equation. The K m values indicated that the encapsulation process reduced enzyme-substrate affinity and the V max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.

ABSTRACT. Agro-industrial waste has shown great potential for use in solid-state fermentation due to its low cost and promoting the growth of microorganisms. This study evaluated the interaction between temperature and moisture response surface on lipase from the fermentation of pumpkin seed flour, using Aspergillus niger. Fermentations were performed in Petri dishes in an experimental design using a 2 2 matrix with four replications at the center point and four axial points, varying substrate moisture between 30 and 60% and temperature between 30 and 40ºC. The maximum hydrolytic activity (71.88 U g -1 dry weight) was obtained using flour with 30% moisture and 30°C temperature in 120 hours of fermentation. The interaction between fermentation temperature and initial moisture content of the residue was the parameter that most influenced the process at 95% significance level. The response surface analysis showed that the maximum lipase production can be obtained under temperature and moisture conditions of the flour ranging from 28 to 34.5°C and 25 to 32.5%; or between 32 and 40°C and 58 and 65%, respectively. Pumpkin seed flour showed potential for obtaining microbial lipase.Keywords: agro-industrial waste, fermentation, solid state, microbial enzyme.Avaliação entre temperatura e umidade por superfície de resposta sobre lipase da fermentação de sementes de abóbora utilizando Aspergillus niger RESUMO. Os resíduos agroindustriais têm apresentado grande potencial para utilização em fermentação em estado sólido por possuírem baixo custo e favorecerem o crescimento de microrganismos. Foi avaliada a interação entre temperatura e umidade por superfície de resposta sobre lipase obtida da fermentação da farinha de sementes de abóbora utilizando Aspergillus niger. As fermentações foram realizadas em placas de Petri, segundo planejamento experimental empregando a matriz 2 2 com quatro repetições no ponto central e quatro pontos axiais, variando-se a umidade do substrato entre 30 e 60% e a temperatura entre 30 e 40ºC. A máxima atividade hidrolítica (71,88 U g -1 seca) foi obtida quando utilizado farinha contendo 30% de umidade e temperatura de 30°C em 120h de fermentação. A interação entre a temperatura de fermentação e a umidade inicial do resíduo foi o parâmetro que mais influenciou o processo em nível de 95% de significância. A análise de superfície de resposta demonstrou que a produção máxima de lipase pode ser obtida em condições de temperatura e umidade da farinha entre 28 e 34,5°C e 25 e 32,5% ou entre 32 e 40°C e 58 e 65%, respectivamente. A farinha de sementes de abóbora demonstrou potencial para obtenção de lipase microbiana.Palavras-chave: resíduo agroindustrial, processo fermentativo, estado sólido, enzima microbiana.

ABSTRACT. Immobilization potential of lipase from Aspergillus niger on sol-gel matrix was evaluated by physical adsorption and covalent binding and the biochemical characterization of free and immobilized enzyme was performed. Lipase was produced by solid state fermentation of pumpkin seed flour with 30% moisture, at 30°C for 120h. The enzyme was pre-purified with ammonium sulfate and immobilized in the sol-gel matrix by covalent attachment and physical adsorption. A higher yield of immobilization (81.88%) was obtained in the latter. The free enzyme presented higher hydrolytic activity with pH 4.0, at 37°C; moreover, it was more stable with pH between 6.0 and 7.0, at 35°C. The immobilized lipase showed maximum hydrolytic activity with pH 11.0, at 50°C; it was more stable with pH 11.0, at 37°C. Parameters K m and V max were best determined by Hanes-Woolf linearization.Keywords: enzyme, sol-gel, agroindustrial residue.Lipase microbiana obtida pela fermentação de sementes de abóbora: potencial de imobilização em matrizes hidrofóbicas RESUMO. O objetivo deste trabalho foi avaliar o potencial de imobilização da lipase de Aspergillus niger em sílica por adsorção física e ligação covalente, e realizar a caracterização bioquímica da mesma na forma livre e imobilizada. A lipase foi produzida por fermentação em estado sólido da farinha de sementes de abóbora, contendo 30% de umidade, à temperatura de 30°C em 120h. A seguir, a enzima foi pré-purificada com sulfato de amônio e imobilizada em matriz sol-gel por ligação covalente e adsorção física, obtendo-se nesta última maior rendimento de imobilização (81,88%). A enzima livre apresentou maior atividade hidrolítica em pH 4,0 a 37°C, sendo mais estável em valores de pH entre 6,0 e 7,0 e temperatura de 35°C. A lipase imobilizada por adsorção física apresentou máxima atividade hidrolítica em pH 11,0 e temperatura de 50°C sendo mais estável em pH 11,0 e temperatura de 37°C. Os parâmetros K m e V máx foram melhor determinados pela linearização de Hanes-Woolf.Palavras-chave: enzima, sol-gel, resíduo agroindustrial.

The lipase from Aspergillus niger, obtained from solid-state fermentation of pumpkin seed flour, was encapsulated in a sol-gel matrix. The silica obtained by the sol-gel technique and lipase encapsulated were characterised with regard to superficial area, pore volume and size by nitrogen adsorption (BJH method), scanning electron microscopy, mass loss upon heating (TG) and differential scanning calorimetry. The immobilised biocatalyst and the sol-gel matrix showed features typical of mesoporous solids. The presence of the enzyme encapsulated in the solgel matrix was confirmed since the total mass loss of the BI at a temperature of 1,000°C was approximately 60 %, while for the free enzyme the mass loss was 95 %. This new lipase obtained from the fermentation of agroindustrial waste, which when encapsulated in sol-gel has physicalchemical and morphological features similar to those of conventional biocatalysts, maybe exploited for future applications in biocatalytic processes.

No abstract