Purificação e caracterização bioquimica da polifenoloxidase (PPO)em fruto da anonacea Pinha (Annona squamosa L.)

AUTOR(ES)
DATA DE PUBLICAÇÃO

1999

RESUMO

Polyphenoloxidases (PPO) are widely distributed in nature, being firstly related to the enzymatic browning of vegetables "in natura", causing a loss of color in fruit products and processed or frozen vegetables, decrease in nutritional value and modification of the organoleptic properties, resulting in products with bad appearance, which are rejected by the consumers. On the other hand, PPO has an important role in the flavor and color of black tea, in the decrease of bittemess and astringency of cocoa products and in the formation of aldehydes from amino acids. The objective of the present work was to extract, purifyand study the biochemical characteristics ofthe PPO (EC 1.10.3.2) ofripe custard apple (Annona squamosa L.) pulps, with the purpose of obtaining the necessary information for the conservation of fruits pulps with better organoleptic properties, as well as to study the use of the enzyme in food processing. The optimum pH for activity of the partially purified PPO was 6.5 and for the purified enzyme 7.0, with pH stability between 6.5 and 7.5. The optimum temperature for activity of the partially purified and purified enzyme was 20°C. The purified enzyme showed fast inactivation at temperatures above 50°C, when incubated for 30 minutes with catechol substrate. PPO was purified 411 (Fraction I) and 118 (Fraction II) fold in an ion exchange column of DEAE-Toyopearl 650M, and 566 fold in a gel column of Toyopearl HW 55F. The enzyme of the most active fraction was characterized biochemically. The enzymatic activity of both the partially purified and purified PPO was strongly inhibited by the following reagents at concentrations of 1.5, 5.0 and 10. O mM with respect to the final volume of the reaction mixture, at a temperature of 20°C: glutathione, l3-mercaptoethanol, L-cysteine, ascorbic acid and sodium metabisulfite, and to a lesser extent by KCN and thiourea. Little inhibition was verified by KCI, NaCI, citric acid and EDTA. In a comparative study amongst the inhibition treatments for the partially purified and purified PPO, with concentrations in accordance with the limits permitted by the legislation, the treatment with 10mM ascorbic acid + temperature of 70°C for two minutes was effective and adequate to substitute the use of S02. The partially purified and purified enzyme used the 0diphenols as substrates and no activity towards monophenols was detected. With respect to the kinetic parameters, the purified enzyme presented values for Km and Vmax of 7.14 mM and 302.0 units/min/ml for catechol and 25.0 mM and 180.2 units/min/ml for L-dopa respectively, substrates which demonstrated greater specificity. The molecular weight was estimated as 90.000 daltons using gel filtration on Sephadex G-200 and about 79.000 to 84.000 daltons on SDS-PAGE. In the analysis of copper, the purified enzyme gave a value of l1ppm by weight ofthe liofilized sample. The amino acid composition of the custard apple fruit PPO, presented greater amounts of aspartic acid, glutamic acid and lysine and smaller amounts of methionine, arginine and tyrosine, with an absence of cysteine. Partially purified and purified PPO was stable during the period of 6 months of storage at -10°C.

ASSUNTO(S)

purificação annona squamosa anona

ACESSO AO ARTIGO

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