The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology -RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L −1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40 • C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90 • C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

Application of Doehlert experimental design in the optimization of experimental variables for the Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) cell lysis Aplicação do modelo Doehlert na otimização das variáveis experimentais para a lise de Pseudozyma sp. (CCMB 306) e Pseudozyma sp. (CCMB 300)Amanda Reges de SENA 1,3 , Gildomar Lima VALASQUES JÚNIOR 1,2 , Ingara Keisle São Paulo BARRETTO 1 , Sandra Aparecida ASSIS 1 * IntroductionThe cell wall of yeast is basically made of glucan and mannan-protein. Lytic enzymes such as chitinases, proteases and β-1,3-glucanases are usually necessary. There are numerous applications for biotechnologies: preparation of protoplasts, extraction of pigments, mass treatment of residual yeast cell fermentation industries for preparation of animal feed, in studies of the mechanism of cell wall synthesis for control of pathogenic yeast, etc. (HUNTER;ASENJO, 1988;SATO, 2005;SALAZAR; ASENJO, 2007).These enzymes are capable of lysing the cell wall of Saccharomyces cereviseae, Candida sp. and other genera of yeasts, which extends their use allowing the selective manufacture of products, regardless of scale, and can be performed under temperature and pH conditions that do not involve the denaturation of cellular products of interest (FLEURI; SATO, 2005). Mathematical models have been increasingly used to help explain responses of biochemical reactions. This technique is often used for optimization and/or verification of the influence of medium components and cultivation parameters for enzyme production (FLEURI; SATO, 2008).The production and optimization of the parameters that affect the enzymatic synthesis should always be investigated because the optimal conditions vary among different organisms and different enzymes (BRAVO et al., 2000). In many cases, the interaction of parameters that influence fermentation processes can be evaluated with a reduced number of tests using an experimental design (THÉODORE; PANDA, 1995). Several experimental design models could be used to reduce the number of experiments under different conditions. Among them are the Plackett-Burman, Doehlert, Central Composite, and Box-Behnken (LI et al., 2007).The choice of Doehlert design is justified by a number of advantages such as (1) its spherical experimental domain with an uniformity in space filling, (2) its ability to explore the whole of the domain, and (3) its potential for sequentially where the experiments can be reused when the boundaries have not been well chosen at first (BENSALAH et al., 2010). Doehlert designs are easily applied to optimized variables and offer advantages over the Central Composite (CCD) and Box-Behnken designs, used in response surface analysis. They need fewer experiments, ResumoO presente trabalho visou verificar a influência do pH e temperatura na lise de leveduras utilizando planejamento experimental. No estudo, foi utilizado o extrato enzimático, contendo β-1,3-glucanase e quitinase líticas, obtidas do micro-organismo Moniliophthora perniciosa. O delineamento experiment...

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Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na, Ca, K at 5 × 10 mol/L. The half-life at 30 °C was 3465.7 min. The activation energy was 40.32 kJ/mol. The Gibbs free energy, enthalpy and entropy at 30 °C were 85.40, 48.10 and -0.12 kJ/mol K, respectively. Our results suggest that the tannase found in the crude extract of S. cerevisiae is an attractive enzyme for industrial applications, such as for beverage manufacturing and gallic acid production, due its catalytic and thermodynamic properties (heat-stable and resistant to metal ions).

Acremonium sp. L1-4B isolated from lichen in Antarctica was used to produce extracellular proteases through submerged fermentation using cactus pear extract (Opuntia ficus-indica Mill.). A 2 3 factorial design was applied to optimize the protease production using three independent variables, namely temperature, pH and concentration of yeast extract, was also used a Central Composite Design (CCD) under Response Surface Methodology (RSM). All variables and interactions analyzed in the factorial design were significant or marginally significant, a Central Composite Design was developed, and the Response Surface Methodology towards the highest point it was established. The experimental model was validated under 14°C, pH 7.54, and 0.55% yeast extract, showing a protease activity of 447.65 7 2.6 U/mL by a prediction model of 445.48 U/mL. The enzyme showed a molecular weight of 59 kDa; it was inhibited in the presence of PMSF (serine protease); it presented optimal conditions at pH 8.0 and 50°C; it remained stable at pH in the 3.0-9.0 range and between 10 and 40°C; it showed a tolerance to 3000 mM NaCl as well as to surfactants, hydrogen peroxide and urea at 5%. This paper presents a proposal for an economically attractive production methodology using cactus pear as a primary source of carbon. In addition, the protease secreted by Acremonium sp. L1-4B presented a combination of biochemical characteristics that grants a promising variability of biotechnological applications.

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SummaryTannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable eff ects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L.) Skeels) leaves, and identifi ed as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the eff ect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. K m was found to be 7.18·10 -4 mol/L and v max =250.00 U/mL. The tannase activity was the highest in the presence of Ca 2+ at a concentration of 5·10 -3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3 % of its original activity a er 6 h. The study contributes to the identifi cation of microbial species that produce tannase, with potential application in biotechnology.

Tangerine juice was treated with crude extract containing cellulase from Pseudozyma sp. obtained by liquid fermentation. The thermal stability of cellulase was investigated by incubating crude extract at different temperatures and times. The pulp, obtained from tangerine, was pasteurized at 85 °C for 5 min and then used in a clarification process with a Doehlert experimental design. The results showed that the cellulase obtained from Pseudozyma sp. is thermostable at temperatures of 60, 70 and 90 °C and retained 98%, 88% and 80% of activity, respectively, after a 1-h incubation time. The optimum conditions for clarification were verified by varying the enzyme extract concentration (%, v v -1 ) and the time (minutes) in a shaker at 150 rpm, at 50 °C. The optimum condition for clarification was obtained in the 80th min with a 1.25% enzymatic extract concentration (v v -1 ), resulting in a reduction of tangerine juice viscosity by 65%. The analysis of physical and chemical parameters of tangerine juice after clarification showed that the enzyme extract improved the process responsible for the clarification of tangerine juice. The results are promising since this is a methodology that can be used in the citrus juice industry.