RESPONSE METHODOLOGY.

OLIVEIRA, B.H*, FONTANA, M., LIMA, V.M.G.

Depto. de Ciências Biológicas, Universidade Estadual Paulista “Júlio de Mesquita Filho” Av. Dom Antônio, nº 2100, Pq. Universitário. Assis – SP. *bio_henri@yahoo.com..br

Keywords: lipase; Fusarium sp.; experimental design; surface response.

Introduction. Lipases (E.C. 3.1.1.3) catalyse the hydrolysis of triacylglycerols of long chain in free fatty acids, mono and diacylglicerols and glycerol. These enzymes are important for presenting versatile character; since, beyond the hydrolysis they participate of synthesis reactions in aquo-restricted environments. Filamentous fungi are the preferred ones as source of lipases, because they produce extracellular enzymes, facilitating the extraction after the fermentative process. When developing industrial fermentation, designing media and optimising fermentation conditions are of critical importance because these factors could strongly interfere with the yield of lipase production. Experimental design techniques present a more balanced alternative to the one-factor-at-a-time approach for fermentation improvement. In the present study, the effect of carbon source and concentration of surfactants on lipase production by Fusarium sp. in batch fermentation was determined using experimental design and surface response methodology.

Material and methods. The concentration of carbon source and presence of surfactants was evaluated by two experimental designs 22 independents, with 4 trials (-1 and +1), two central points and 4 axial points (-1.41 and +1.41), totaling 10 experiments each, using a computer program STATISTICA 8. The independent variables were corn oil (CO) and Tween 80 (TW) or Triton X-100 (TR) using yeast extract (0.5%) as nitrogen source. The results were evaluated by experimental design and surface response methodology.

Results and discussion. First Experimental Design (corn oil and Tween 80): The highest lipolytic activities (2.79 U/mL) were achieved in test 9 (0, -1.41). These results indicated that the addition of Tween 80 is not advantageous to increase the lipolytic activity. From the responses obtained in the first-order 22 experimental design (assays 1 to 10), the main effects from the interaction of CO and TW was not statistically significant at a confidence limit of p<0.05. However, the main effect of TW isolated was statistically significant. Thus, an antagonistic effect was observed, causing an average loss of 1.08 U/mL of activity. Second Experimental Design (corn oil and Triton X-100): The highest lipase activity was obtained in trials 1 (-1, -1), 2 (-1, +1) and 9 (0, -1.41), where values of 4.89, 4.34 and 5.59 U/mL were observed, respectively. From the responses obtained in the first- order 22 experimental design (assays 1 to 10), the main effects from the interaction effect of CO and TR was not statistically significant at a confidence limit of p<0.05. However, the main effect of CO and TR isolated was statistically significant. Thus, a synergetic effect of CO and TR was observed, causing an average increase of 1.65 and 1.64 U/mL of activity, respectively.

With these results it is possible to conclude that the surfactants Tween 80 and Triton X-100 have different effects on lipase production by Fusarium sp., while the first acts as an inhibitor and the second induces the production of the enzyme. Through the estimate was best defined as medium composition corn oil 2.0% (w/v) and Triton X-100 1.7% (w/v). Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2010/07998- 9).

PRODUCTION OF α-AMYLASE BY Bacillus subtilis (UCP 0999) USING MEDIUM CONTAINING ALTERNATIVE SUBSTRATES

SOARES DA SILVA, R. C. F. 1, *, BORBA, P. P.1, ALVES DA SILVA, C. A.2

1*

Mestrado em Desenvolvimento de Processos Ambientais – UNICAP

2*_{Núcleo de Pesquisas de Ciências Ambientais (NPCIAMB), Universidade Católica de Pernambuco “Rua do} Príncipe, 526. Boa Vista - Recife – PE”. *rita.freire.78@hotmail.com

Keywords: amylase, Bacillus subtillis, alternatives substrates.

α-Amylases (E.C. 3.2.1.1.) are starch-degrading enzymes that catalyze the hydrolysis of internal α-1.4-Ο-glycosidic bonds in polysaccharides with the retention of α-anomeric configuration in the products. Most of the α-amylases are metalloenzymes, which require calcium ions (Ca2+) for their activity, structural integrity and stability. Amylases find potential application in a number of industrial processes such as in the food, fermentation, textile and paper industries. The amylolytic enzymes are responsible for 25 to 33% of world production of enzymes. Bacillus species continue to be dominant bacterial workhorses in microbial fermentations. Bacillus subtilis (natto) is the key microbial participant in the ongoing production of the Soya-based traditional natto fermentation, and some Bacillus species are on the Food and Drug Administration's GRAS (generally regarded as safe) list. The capacity of selected Bacillus strains to produce and secrete large quantities (20–25 g/L) of extracellular enzymes has placed them among the most important industrial enzyme producers. The use of alternative means of production have been considered a great economic challenge for the industries that use and produce these enzymes and the synthetic media and the most expensive used in submerged fermentation of amylase. The objective was to evaluate the use of alternative carbon sources of low cost to produce a high value industrial enzyme by B. subtilis. The production by of amylase by

Bacillus subtilis (UCP 0999) replacing the source of starch for water and rice flour banana

peel. The medium of production of amylase: 0.65% peptone, 3.0% yeast extract, 0.5% KH2PO4, 0.25% MgSO4.7H2O, 1.5% NaNO3; pH 7.0. Economic means the soluble starch

was replaced by water of banana peel flour at a concentration of 10 g / L. The production occurred in flasks containing 250 mL of culture medium, 37 °C, orbital shaker, 150 rpm during 96h. The samples were subjected to quantitative assessment of amylase production using the dosage of reducing sugars (DNS). The results revealed that the use of alternative sources of starch in water and rice flour in the banana skin, produced the enzyme when compared to control medium containing soluble starch in 47 UI/mL is the best growth was between 40 and 70 hours in neutral pH, during fermentation kinetics. The showed that half the flour with the banana peel had a high significative score. The results suggest that formation of the enzyme is slow during the logarithmic growth, followed by an increased rate of synthesis to the extent that the growth rate decreases and reaches the stationary phase.

QUANTIFICATION OF TOTAL AND FECAL COLIFORMS AS TOOL FOR