Produção e caracterização parcial de um composto de baixa massa molecular com atividade fenoloxidasica, de Thermoascus aurantiacus

AUTOR(ES)
DATA DE PUBLICAÇÃO

1995

RESUMO

This work involves the phenoloxidase activity (POx) production by the thermophilic fungus Thermoascus auralltiacus. The best conditions for POx activity production in liquid culture were determined. The addition of lignocellulosic and polyssacharides substrates to culture induced high leveI POx activity. The best substrate was the wheat bran which induced between 1-2 UI/mL, when utilized in concentration of 1.5% (w/v). The type and size of inoculum influenced the activity production, however the agitation and oxygenation of cultures did not affect it significantly. The initial pH between 6-8 supported the higher POx activity production. Different enzymatic activities related to lignin degradation such as laccase, peroxidase, Mn-peroxidase, cellobiose-quinone oxidoreductase and veratryl alcohol oxidase, but no ligninperoxidase, were detected in the cultures containing polyssacharides substrates. After the best conditions to POx activity production by Taurantiacus were established, crude extracts with high POx activity were utilized in the kinetic characterization. The POx activity showed similar characteristics to phenoloxidases laccase type. The crude extracts oxidized various typical substrates of phenoloxidases, in the absence of H202. The higher activities were produced with ABTS, 2,6-DMP and o-dianisidine. Typical inhibitors of oxidases containing metal, such as sodic azide and thioglycollic acid, were the most potent inhibitors of POx activity. The optimal pH for o-dianisidina oxidation was 2.8, but in this pH the stability was rapidly lost after 5 hours of incubation. The optimal temperature was found between 70 and 800C, and the crude extract showed an high thermostability, keeping more than 50% of activity after 5 hours of incubation at 100°C. All the characteristics of the crude extract were similar to phenoloxidases laccase type, except the high optimal temperature and thermostability, never report for phenoloxidases of mesophilic and thermophilic fungi. To elucidate the responsible nature for POx activity, the purification of an active fraction from Taurantiacus was conducted. The unexpected results of the purfication stage redirected this study. Because of that fraction with POx activity crossed over a 1 kDa ultrafiltration membrane, eluted from Sephadex G-10 column in a elution volume correponding with molecular mass of £ 700 Da, as well as presented high thermostability. The possibility of a laccase or some other enzyme to be responsible by POx activity was excluded. The kinetic and structural properties of the fraction partially purified by Sephadex G-10 with high POx activity, were studied. The properties of optimum pH and temperature for activity and stability were similar to those of the crude extract. The POx fraction oxidized the same substrates than the crude extract, but with higher rates. Unlike of the crude extract a decrease of inhibition was observed with POx fraction. The presence of a siderophore type compound (low-molecular mass Fe chelators) responsible by POx activity in the crude extracts as well in the partially purified fraction was then considered, and led to search for these compounds in the T.aurantiacus cultures, using the universal CAS reactive. Through this method the siderophore production by the fungus, in solid and liquid medium, was confirmed. The maximum production of POx activity in liquid medium coincided with a minimum CAS reaction. However, in spite of the positive results in culture medium, the POx fraction partially purified was not reactive with CAS. The structural characterization of partially purified POx fraction revealed a structure hydroxamate type with approximate molecular mass of 530 Da, apparently lacking aminoacids, and some metais (Ca, Mg, Fe) present in the structure. Thus, the existence of a siderophore hydroxamate type, responsible by POx activity, was suggested through of structural characterization. With these results an mechanism of substrate oxidation by POx fraction was proposed, in which Fe would be responsible by the oxidation of different substrates. Preliminary results showed that Fe is involved in the o-dianisidina oxidation as Fe(II). Apparently, the fraction possessed the ability to both chelate Fe(III) and reduce it to Fe(II), when necessary. The extracts with high POx activity fTom T.aurantiacus were used in biotechnological processes such as treatment of Eucalyptus Kraft pulps and effiuent result of Kraft processo The active extracts showed efficience on pulp biobleaching, with a 39% deslignification after 60 minutes at 500C and pH 3, however a viscosity reduction also was observed. The ABTS addition, as a redox mediator, it not increase the deslignification. After 72 hours of treatment with active extracts at 250C and pH 4, the effiuents showed between 60-70% phenol reduction, depending of incubation conditions. Under static conditions a 67% despolimerization and a 5% mineralization of chlorolignins high-molecular mass were observed. Under agitation conditions a 47% mineralization was observed. A colour reduction effiuent of61 or 35% was obtained under agitation or static conditions, respectively

ASSUNTO(S)

thermoascus aurantiacus fungos - biotecnologia

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