Production, purificação, characterization and study of the alkaline application of one protease produced by 191 Cellulosimicrobium cellulans. / Produção, purificação, caracterização e estudo da aplicação de uma protease alcalina produzida por Cellulosimicrobium cellulans 191.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Cellulosimicrobium cellulans 191 is a microorganism that produces an enzyme complex capable of disrupting yeast cell walls, which is composed of b-1,3-glucanase, protease and chitinase. This microorganism adheres to yeast cell walls and than lyses them. It was isolated from industrial fermentation sludge. Dried Saccharomyces cerevisiae cells were used in the culture medium as an inductor for protease production. The initial culture medium was composed of 1% of dried cells of S. cerevisiae; 13.6 g/L of KH2PO4; 2.0 g/L of (NH4)2SO4; 4.2 g/L of KOH; 0.2 g/L of MgSO4 .7H2O; 0.001 g/L of Fe2(SO4)3.6H2O; 1 mg/L of biotin and 1mg/L of thiamine. A 28-3 experimental design was performed, the independent variables being the components of the initial culture medium. The components KH2PO4, KOH and the inductor showed a significant effect on the protease activity of the culture medium. A new (22) design was performed using two factors, pH and the percentage of dried yeast cells. The results showed that the culture medium indicated for maximum protease production was composed of 8% of dried yeast cells; 0.2 g/L of MgSO4.7H2O and 2.0 g/L of (NH4)2SO4 in a 0.15M phosphate buffer at pH 8.0. The highest protease production was obtained after 24 hours of fermentation of the microorganism at temperatures between 20 and 27ºC. Protease production was increased about 36 times after optimization of the culture medium and the fermentation conditions. For protease production, Cellulosimicrobium cellulans 191 was grown on the optimized culture medium at 28ºC for 24 hours with agitation at 150 rpm. The cells were collected by centrifugation and 40% of ammonium sulfate added to the supernatant. The precipitate was collected by centrifugation and ammonium sulfate added to the supernatant up to 80% saturation. The precipitate, which contained the protease, was collected by centrifugation, dialyzed and purified by DEAE-Sepharose and Q-Sepharose chromatography. The purification factor was 16.8 and the yield was 7.8%. The molecular mass of the purified protease was estimated at around 55 KDa by gel filtration on Sephacryl S-200. The optimum pH and temperature ranged from 7.0 to 8.0 and from 50 to 64ºC, respectively. When stabilized with DTT, the protease was stable in the pH range from 4.0 to 7.5 for one hour at 83ºC. The kinetic parameters Km and Vmax were 0.027 mg of casein/mL and 6.25 U/mg of enzyme, respectively. The purified protease was activated by 1.0 mM of FeCl3 and partially inhibited by phenylmethylsulfonilfluoride (PMSF). The protease was capable of lysing viable cells of Saccharomyces cerevisiae alone. The application of this alkaline protease in the isolation of yeast cell wall compounds was studied. Glucan is the major component of the Saccharomyces cerevisiae cell wall. Over the last few years, numerous benefits of glucan have been described with respect to human health. Conventional isolation processes involving extreme conditions of pH cause degradation of the polymeric chains. A method combining physical extraction steps and enzymatic treatment using the alkaline protease from Cellulosimicrobium cellulans 191 was proposed in this study, obtaining glucan with a purity of 87.4% and a yield of 33.7%. During this process, the mannoprotein of the yeast cell wall was easily obtained.

ASSUNTO(S)

glucanas protease enzymes enzimas glucanas proteases

ACESSO AO ARTIGO

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