As proteases fazem parte da classe de enzimas com a capacidade de hidrolisar
ligaÃÃes peptÃdicas em proteÃnas e fragmentos de proteÃnas, modificando os substratos com
grande seletividade e especificidade. A fermentaÃÃo semi-sÃlida à um processo fermentativo
no qual o crescimento do microrganismo e a formaÃÃo dos produtos ocorrem na superfÃcie do
substrato sÃlido prÃximo à ausÃncia de Ãgua livre, geralmente utilizando matÃria-prima
natural como fonte de carbono e energia. Realizou-se neste estudo o cultivo semi-sÃlido de
fungos filamentosos, visando à produÃÃo de proteases utilizando como substrato a torta de
canola. Avaliando-se, em um primeiro estudo, o potencial de diferentes linhagens de
Aspergillus para a produÃÃo de proteases. As linhagens estudadas foram Aspergillus niger
(CNPAT 001, IOC 207, IOC 4222, IOC 4220 e IOC 3883) e Aspergillus oryzae IV. A melhor
linhagem obtida para a produÃÃo de protease foi a linhagem de Aspergillus oryzae IV. Em
seguida, estudou-se a influÃncia da adiÃÃo de diferentes volumes de Ãgua a torta, onde a
melhor produÃÃo foi obtida nos meios com proporÃÃo 40 mL de Ãgua para 100 g de torta.
Determinada a melhor umidade do meio, avaliou-se a temperatura de incubaÃÃo, os melhores
resultados obtidos foram em meios incubados a 20ÂC. Na sequÃncia, foram feitos estudos para
determinaÃÃo da melhor concentraÃÃo de inÃculo, as concentraÃÃes testas foram 1Ã104, 1Ã105,
1Ã106 e 1Ã107 esporos por grama de meio, sendo 1Ã107 a melhor concentraÃÃo analisada.
Avaliou-se a influÃncia da suplementaÃÃo do meio fermentativo com fontes de fÃsforo,
carbono e nitrogÃnio. A suplementaÃÃo da torta de canola, com 1% (m/m) de fosfato de sÃdio
monobÃsico nÃo influenciou positivamente na produÃÃo de proteases. A suplementaÃÃo do
meio com diferentes fontes de nitrogÃnio foram estudadas na proporÃÃo de 1,0 % (m/m) em
relaÃÃo ao peso do substrato, sendo o extrato de levedura a melhor fonte avaliada,
apresentando maior produÃÃo de proteases. Em seguida, testou-se o perÃodo de incubaÃÃo do
meio sem suplementaÃÃo de nutrientes, entre 0 e 240 horas de fermentaÃÃo com amostras
sendo retiradas a cada 24 h de processo fermentativo. A maior produÃÃo ocorreu em 96 h com
produÃÃo de 336 U.g-1 de proteases. ApÃs esta etapa, avaliou-se a suplementaÃÃo do meio com
diferentes fontes de carbono em diferentes concentraÃÃes, 1,0; 2,0; 3,0; 4,0; 5,0; 7,5; 10,0;
12,5 15,0 % (m/m) em relaÃÃo ao peso do substrato, a maior produÃÃo de proteases ocorreu
no meio com adiÃÃo de glicose na concentraÃÃo de 7,5 % obtendo 452 U.g-1. Na Ãltima etapa,
fez-se a caracterizaÃÃo do meio ao longo do processo fermentativo, atravÃs de determinaÃÃes
analÃticas de ordem quÃmicas e fÃsico-quÃmicas. A maior produÃÃo de proteases obtida foi de
452 U.g-1 em 96 horas de processo fermentativo, nos meios suplementados com 7,5 % de
glicose, nas seguintes condiÃÃes fermentativas: temperatura de incubaÃÃo do meio a 20ÂC,
torta umedecida com 40 mL de Ãgua por 100 g de substrato e concentraÃÃo de inÃculo de
1Ã107 esporos.g-1. / The proteases belong to the class of enzymes with the capacity to hydrolyze
peptidic connections into protein and protein fragments, modifying the substrate by great
selectivity and specification. The semi-solid fermentation is a fermentation process in which
the growth of microorganisms and the formation of the products occur on the surface of the
solid substrate next to the absence of free water, usually using natural raw materials as a
source of carbon and energy. On this study, it was done the semi solid cultivation of filament
fungi, in order to produce proteases using as a substrate a cake of canola. It was evaluated, in
a first study, the potential of different strains of Aspergillus for the production of proteases.
The strains studied were Aspergillus niger (CNPAT 001, IOC 207, IOC 4222, IOC 4220 and
IOC 3883) and Aspergillus oryzae IV. The best strain obtained for the production of protease
was a strain of Aspergillus oryzae IV. Later on, it was studied the influence of the different
quantities of water and coke canola added; the best production was obtained in proportion
with 40 mL of water to 100 g of cake. After determination to medium of moisture, was
evaluated the temperature of incubation, the best results were obtained in media incubated at
20Â C. Further, studies were done to determine the best concentration of inoculum, the
concentrations tested were 1 Ã 104, 1 Ã 105, 1 Ã 106 and 1 Ã 107 spores per gram of medium,
being 1 Ã 107 to better concentration analyzed. The influence of supplementation of
fermentation medium with sources of phosphorus, carbon and nitrogen was evalueted.
Supplementation of the cake of canola, with 1% (w/w) monobasic sodium phosphate did not
influence positively the production of proteases. Supplementation of medium with different
sources of nitrogen were studied in the proportion of 1.0% (w/w) in the weight of the
substrate, being the yeast extract of the best source evaluated, showing increased production
of proteases. Then, it was tested the incubation period of the medium without
supplementation of nutrients, between 0 and 240 hours of fermentation with samples being
removed every 24 h of fermentation process. The highest production occurred at 96 h with
production of 336 U.g-1 protease. After this stage, was evaluated the supplementation of
medium with different carbon sources at different concentrations, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5,
10.0, 12, 5 15.0 % (w/w) in the weight of the substrate; the increased production of proteases
occurred in the medium with the addition of glucose at a concentration of 7.5 % obtaining 452
Ug-1. In the last stage, was performed the characterization of the medium during the
fermentation process by of a chemical and physicochemical analytical determinations. The
increased production of proteases obtained was 452 U.g-1 in 96 hours of fermentation process,
in media supplemented with 7.5 % glucose, at the fermentation conditions: temperature of
incubation of the medium at 20Â C, damp cake with 40 mL water per 100 g of substrate and
concentration of inoculum of 1 Ã 107 spores.g-1.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:2455 |
| Date | 19 February 2009 |
| Creators | Adriana Crispim de Freitas |
| Contributors | Gustavo Adolfo Saavedra Pinto, Andrea Lopes de Oliveira Ferreira, Antonio SÃlvio do Egito, Claudia Miranda Martins |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Engenharia QuÃmica, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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