Produ????o de Glicose Oxidase (E.C. 1.1.3.4) por fungos isolados da Floresta Amaz??nica

• Author(s): Sousa, Diego Rayan Teixeira de
• Date: 2017
• Origin: Oasisbr
• Subject(s): Microrganismo; Enzima; Bioprocesso; CI??NCIAS BIOL??GICAS
• Document Type: Doctoral thesis
• Language: Portuguese

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FAPEAM - Funda????o de Amparo ?? Pesquisa do Estado do Amazonas

Glucose oxidase (Gox) has several industrial applications. It is believed that there are diversified fungal species that have the production capacity of this enzyme and most are unexplored. The objective of this work was to investigate a production of Glucose Oxidase (EC 1.1.3.4) by fungi isolated from soil samples from the Amazon Forest. Soil fungi were isolated from the Adolpho Ducke Forest Reserve, located in the city of Manaus-Amazonas. To select good GOx producers, such as submitted in submerged bioprocess. The isolates highlighted in the production of the enzyme were submitted to kinetic tests where the enzyme production, biomass and substrate consumption by isolates were evaluated. Univariate and multivariate assays performed to optimize the production process. An enzymatic production for the semi-continuous process. The crude enzyme was purified by precipitation processes, dialysis ion exchange chromatography. The potential of this enzyme in the elaboration of a Glucose quantification kit through calibration curves, the regression coefficient, was investigated as biochemical characteristics and GOx (optimum temperature, optimum pH, stability and ionic effect in the enzymatic activity. However, only seven were identified as potential producers of this enzyme, being five of the genus Aspergillus and two of the genus Penicillium, with values ranging from 14.93 U/mL ??? 35.54 U/mL. Kinetics showed that the Aspergillus LMM01 isolate was the best producer of the enzyme, and this was used isolated in the subsequent experiments. Experiments with variants demonstrated with the greatest effect on the production of GOx were PH and agitation. A result for GOx (7.74 U/mL) was obtained, for 7 days, use the semi-continuous. After purification process, an activity and the enzyme improved 938%, a temperature of 40 ?? C and pH 6.0 were the optimum for a catalytic action of the enzyme and stability presented as its main industrial needs. Fe+++, Pb++, Ag+, Hg++ and Cu++ ions showed significant inhibition of GOx activity. In the present study, a Pearson correlation of the developed Kit and the commercial Kit was strong positive, with value of r = 0.9978. The results are compatible with a literature where the genera Aspergillus and Penicillium stand out as the main industrial producers of GOx.

A glicose oxidase (GOx) possui diversas aplica????es industriais. Acredita-se que existem diversificadas esp??cies f??ngicas que possuem capacidade de produzir essa enzima e a maior parte encontra-se inexplorada. Este trabalho teve como finalidade investigar a produ????o de Glicose Oxidase (EC 1.1.3.4) por fungos isolados de amostras de solo da Floresta Amaz??nica. Foram isolados fungos filamentosos de amostras de solo da Reserva Florestal Adolpho Ducke, localizada na cidade de Manaus-Amazonas. Para selecionar bons produtores de GOx, esses foram submetidos em cultivo submerso. Os isolados destacados na produ????o da enzima foram submetidos a ensaios de cin??tica onde foi avaliada a produ????o da mesma, biomassa e o consumo do substrato pelos isolados. Ensaios univariados e multivariados foram realizados para otimizar o processo de produ????o. Uma produ????o enzim??tica foi realizada utilizando processo semi-cont??nuo. A enzima bruta foi purificada pelos processos de precipita????o, di??lise cromatografia de troca i??nica. Foram investigadas as caracter??sticas bioqu??micas da GOx (melhor temperatura, melhor pH, estabilidade e o efeito de ??ons na atividade enzim??tica). Analisou-se o potencial desta enzima na elabora????o de um Kit de quantifica????o de Glicose atrav??s de curvas de calibra????o, o coeficiente de regress??o e coeficiente de correla????o. Quanto aos resultados obtidos, foi detectada a produ????o de GOx em todas as cepas analisadas, entretanto, apenas sete se destacaram como potenciais produtores desta enzima, sendo cinco do g??nero Aspergillus e dois do g??nero Penicillium, com valores que variaram de 14,93 U/mL ??? 35,54 U/mL. A cin??tica demonstrou que o isolado Aspergillus LMM01 foi o melhor produtor da enzima, sendo portanto este isolado, utilizado nos experimentos posteriores. Experi??ncias multivariadas demonstraram que os par??metros com maior efeito na produ????o de GOx foram o pH e a agita????o. Obteve-se um resultado est??vel para GOx (7,74 U/mL), durante 7 dias, utilizando o processo semi-cont??nuo. Ap??s o processo de purifica????o, a atividade da enzima melhorou 938%, a temperatura de 40?? C e o pH 6,0 foram os melhores para a a????o catal??tica da enzima e a estabilidade apresentou-se adequada as suas principais necessidades industriais. Os ??ons Fe+++, Pb++, Ag+, Hg++ e Cu++ exibiram inibi????o significante na atividade de GOx. No presente estudo, a correla????o de Pearson do Kit desenvolvido e o Kit comercial foi forte positiva, com valor de r = 0,9978. Os resultados foram compat??veis com a literatura, onde destacam-se os g??neros Aspergillus e Penicillium como os principais produtores industrias de GOx.