Imobilização de células permeabilizadas de Debaryomyces hansenii UFV-1 em alginato de cálcio e sua aplicação na hidrólise de galactoligossacarídeos

• Autor(a) principal: Souza Junior, Waldeck Campanha de
• Data de Publicação: 2006
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2464
• topic: Debaryomyces hansenii; Permeabilização; Imobilização; [Alfa] galactosidade; Galactoligossacarídeos; Debaryomyces hansenii; Permeabilization; Immobilization; [alfa]galactosidase; Galactoligoosaccharides; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

The goal of this work was to immobilize, in calcium alginate, permeabilized cells of the Debaryomyces hansenii UFV-1 yeast containing a [alfa]galactosidase, to characterize the enzyme present in the free and immobilized cells and to evaluate the potential use of immobilized cells in the hydrolysis of the raffinose oligosaccharides (RO) present in the soymilk. The D. hansenii UFV-1 yeast was cultivated in a mineral medium with yeast extract (MME), containing galactose as carbon source, for 36 hours, at 30 ºC. After this period, the medium was centrifuged, the supernatant was discarded and the D. hansenii UFV-1 cells containing the α-galactosidase enzyme were permeabilized with ethanol 50% and later immobilized in calcium alginate. The permeabilized cells presented decrease of 20,5 times in the [alfa]galactosidase activity after the immobilization in calcium alginate. The biggest activities of [alfa]galactosidase to the permeabilized cells was determined in pH 4,5, and 60ºC, while to the enzyme present in the permeabilized and immobilized cells the biggest activities were determined in pH 4,0, and 70ºC. The enzyme present in the permeabilized cells kept 82% of its original activity after pre-incubation at 60ºC, for 5 h. The pre-incubation of immobilized cells at 60 and 70ºC proportioned an increase in the [alfa]galactosidase activity, which showed 157% of its original activity when it was pre-incubated for 72 h, at 70ºC. The KM app value of the [alfa]galactosidase present in the permeabilized cells, for the substrate pNP[alfa]Gal, was 0,32 mM, while, to the enzyme present in the immobilized cells the KM app was 0,82 mM. The enzyme present in the permeabilized cells demonstrated capacity of raffinose hydrolysis, reducing 96,34% of this sugar concentration after the incubation for 20 h, at 50ºC. The [alfa]galactosidase present in the permeabilized and immobilized cells decreased only 75,54% of the raffinose content, in the same conditions. In spite of the lower hydrolysis rate of the raffinose, in solution, by the immobilized cells, these were reused twice, becoming three uses without an expressive lost in the [alfa]galactosidase activity. The use of the immobilized cells, containing the [alfa]galactosidase, in the treatment of RO in soymilk, proportioned decrease of 100% of the raffinose and stachyose rates after incubation at 60ºC for 6 h. These results had shown that the immobilized cells of D. hansenii had presented potential for the industrial use, in the processing of RO present in soy derived products.