Acta Scientiarum http://www.uem.br/acta ISSN printed: 1679-9283 ISSN on-line: 1807-863X Doi: 10.4025/actascibiolsci.v38i2.29220 Acta Scientiarum. Biological Science Maringá, v. 38, n. 2, p. 149-155, Apr.-June., 2016 Permeabilization of Saccharomyces fragilis IZ 275 cells with ethanol to obtain a biocatalyst with lactose hydrolysis capacity Luiz Rodrigo Ito Morioka * , Geyci de Oliveira Colognesi and Hélio Hiroshi Suguimoto Centro de Pesquisa e Pós-Graduação, Universidade Norte do Paraná, Rua Marselha, 591, 86041-120, Londrina, Paraná, Brasil. *Author for correspondence. E-mail: lrodrigomorioka@gmail.com ABSTRACT. The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has beta- galactosidase activity (U mg -1 ). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15ºC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg -1 . The permeabilization of the S. fragilis IZ 275 cells was efficient. Keywords: permeabilizing agent, beta-galactosidase, microbial biotechnology, optimal conditions. Permeabilização de células de Saccharomyces fragilis IZ 275 com etanol para obtenção de biocatalisador com capacidade de hidrólise de lactose RESUMO. A permeabilização foi usada para transformar células de microrganismos em biocatalisadores com alta atividade enzimática. As células de levedura de Saccharomyces fragilis IZ 275 foram permeabilizadas com etanol, como agente permeabilizante. Para otimizar as condições de permeabilização foi utilizado o delineamento de Box-Behnken com 15 ensaios (3 repetições no ponto central). As variáveis independentes e seus níveis foram etanol (29, 32 e 35%), temperatura (15, 20 e 25ºC) e tempo (15, 20 e 25 min.). A função resposta (Y) foi atividade de beta-galactosidase (U mg -1 ). As condições ótimas para a obtenção de uma alta atividade enzimática foram observadas em 35% de concentração de etanol, temperatura de 15°C e tempo de tratamento de 20 minutos. A máxima atividade da enzima beta-galactosidase obtida foi de 10.59 U mg -1 . A permeabilização das células de S. fragilis IZ 275 foi eficiente. Palavras-chave: agente permeabilizante, beta-galactosidase, biotecnologia microbiana, condições ótimas. Introduction Saccharomyces fragilis is described as a homothallic, hemiascomycetous yeast and production of several enzymes among them beta-galactosidase (Llorente et al., 2000; Dagbagli & Goksungur, 2008). The major common feature of S. fragilis is the capacity to assimilate lactose and to use this sugar as a carbon source. The long history of safe association with food products helped S. fragilis achieve GRAS (Generally Regarded As Safe) and QPS (Qualified Presumption of Safety) in the United States and European Union, respectively. This designation means that there are few restrictions on application and largely enhances their potential in the biotechnology sector (Fukuhara, 2006; Schaffrath & Breunig, 2000). S. fragilis, has been more widely adopted by industry, mainly because it possesses traits that are desirable for biotechnology applications. These include the capacity to assimilate sugars, namely lactose and inulin; an extremely rapid growth rate, with typical generation times of about 70 min; thermotolerance, with the ability to growth up to 52ºC; and a high secretory capacity (Fonseca, Heinzle, Wittmann, & Gombert, 2008). Beta-galactosidase is one among other enzymes with industrial potential used in the hydrolysis of lactose in milk and cheese whey, generating food with low levels of lactose, whose result is a better solubility and digestibility of milk and dairy products, making them ideal for consumers intolerant to this sugar (Husain, 2010). To detect enzyme activity, two different substrates are commonly used. X-Gal (5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside) is employed for in