Extração, purificação e propriedades da polifenoloxidase da banana nanica Musa cavendishii, L

AUTOR(ES)
DATA DE PUBLICAÇÃO

1978

RESUMO

The polyphenoloxidase of banana "nanica" (Musa cavendishii L.) was extracted from the central part of half mature fruit by homogenising in a blender at room temperature with a buffer solution of 0.2 M phosphate (pH 7.0) containing 1% of insoluble PVP and 0.5% of Triton X-100. The crude extract was precipitated with 2 volumes of acetone, previously cooled down, and the mixture was maintained at a final temperature of -13°C. The precipitate from acetone was re-extracted with a phosphate buffer, pH 7.0, containing 0,5$ of Triton X-100 and stored at -40°C for 7 days. The in- soluble material obtained after this period was eliminated by centrifugation at 12,062 x g for 15 minutes and the extract put through a Sephadex G-100 column at 5°C. After the elution with a phosphate buffer 0-.0] M, pH 7,80, the fraction corresponding to the greatest enzymatic activity (FT;) liophylized and. subjected to electrophoresis in polyacrylamide gel, produced three active bands, which were cut, eluted with water for 24 hours, dialysed against phosphate buffer 0.2 M, pH 7.0, and liophylized. The enzyme showed a degree of purification of the order of 34 times. The precipitate from acetone and the fraction FH analysed in polyacrylamide gel showed the same number of molecular forms, v,hi varied from 3 to 5 on the substrates employed. The polyphenoloxidase of banana"nanicaV an oxidase specific for o-diphenols, showed greater specificity for dopamine, epinefrine and nor-epinefrine. The purified enzyme showed lesser specificity for substrates in D-forms being more specific for L-dopa (KM = 6,0 x 10-3) than for D-dopa (KM = 2,5 x 10-2). For the racemic DL-dopa the specificity of the enzyme was intermediate (KM = 3,3 x 10-2). The isoeletric point of the purified enzyme determined by isoeletric focusing in polyacrylamide gel was 5.2. Ultracentrifugation experiments in sucrose gradient showed a sedimentation coefficient of 3.9 ± 0.1S for polyphenoloxidase corresponding to a molecular weight of 60,000 ± 2,000. An analysis carried out in polyacrylamide gel in a SDS containing medium showed sub-units with a molecular weight of 30,000 1,000. A study of the action of inercaptoethanol on polyphenoloxidase showed that the enzyme was inactivated by 17 mM concentration and recovered 30% of its activity when submitted to dialysis. Electrophoresis in polyacrylamide gel resulted in the recovery of two active molecular forms. Kinetic studies using a semi purified enzyme (FE) showed a non-competitive inhibition by ascorbic acid in a reaction with catechol at concentration of 0-008 mM, 0.04 mM and 8.08 mM. The same type of inhibition was observed when employing sodium metabisulphite in concentrations of 0.03 mM, 0.06 mM and 0.24 mM. The utilization of cysteine in concentration of 0,083 mM and 0.166 mM showed non-conpetitive inhibition in the presence of catechol changing into a competitive like kinetic at a concen tration of 0.4 mM. The inhibiting action of dithiocarbamic acid on polyphenoloxidase was less effective than that of other inhibitor used. Ascorbic acid proved to be the most potent inhibitor in the case of semi-purified enzyme (FE), whereas in the case of acetone precipitated enzyme cysteine showed strong inhibiting effect in the presence of catechol and DL-dopa. With the exception of dithiocarbamic acid all inhibitors produced a delay period in the enzymatic reaction. In the reaction of DL-dopa with the substrate this delay attained times superior to 600 minutes when using 0.4 mM of cysteine in the reaction mixture. Experiments carried out with acetone precipitates and fraction FE, showed that the enzymes submitted to various temperatures showed accentuated thermal stability at 65°C and 7S°C, the FE fraction being apparently less resistant. Preliminary studies gave evidence that the enzyme exists in the form of complexes with phenol compounds, probably with chlorogenic acid. Of the various ions tested as possible cofactors of this enzymatic reaction NH4+ and β-NAD+ acted as activator whereas Fe2+, Al3+, Ca2+, Zn2+ e Fe3+, showed inhibiting activity. On the other hand, Cu1+ , Cu2+ , Mg2+ e Mn2+ showed no interference with the enzyme activity.

ASSUNTO(S)

banana

ACESSO AO ARTIGO

Documentos Relacionados