POLYPHENOLOXIDASE FROM ATEMOYA FRUIT (ANNONA CHERIMOLA MILL. × ANNONA SQUAMOSA L.)

Abstract

ABSTRACT Polyphenoloxidase (PPO) from atemoya fruit was extracted and partially purified by phase partitioning in Triton X-114 (TX-114), ammonium sulfate precipitation and separation on Sephadex G-100. Polyacrylamide gel electrophoresis of the fraction eluted from Sephadex revealed only one band of enzyme activity with a molecular weight of 82 kDa. The achieved purification was 7.12-fold with a recovery of 3.2%. The optimum pH and temperatures were determined for 4-methylcatechol and catechol, and activation energies (Ea) determined. The Km was 5.52 and 79.3 mmol/L for 4-methylcatechol and catechol, respectively. The enzyme showed no activity with L-dihydroxyphenilalanine, pyrogallol, catechin and P-cresol as substrates. Inhibitory effects of phenolic and non-phenolic compounds were tested and the Ki values and type of inhibition determined. Enzyme showed to be relatively unstable at 55–70C and Ea for heat inactivation was determined. NaCl and KCl showed to be protective agents of atemoya PPO against thermal denaturation. PRACTICAL APPLICATIONS Atemoya (Annona cherimola Mill. × Annona squamosa L.) is a fruit which has excellent sensorial characteristics and that comes reaching high commercial value. However, these fruits are an excellent example of fruit susceptible to enzymatic browning, and this is a problem encountered during handling and processing. The quality of fruits, especially atemoya, depends on controlling the enzymatic browning. Addition of chemicals, pH adjustment, refrigeration, thermal treatments and exclusion of oxygen are among the most effective methods for the inhibition of browning, and their application to a product will depend of the characteristic properties of the enzymes related to browning. The main enzyme involved in browning is polyphenoloxidase (PPO), however, research on fruit enzymes is necessary and there is little information on the properties of PPO from atemoya fruit. Accordingly a wide knowledge of the biochemical properties of fruit PPO can provide support for planning and strategies to reduce browning.