Polyphenol Oxidases

Bunch of over ripe bananas on white background
Photo by Olaf Speier, c/o 123-rf.com

Polyphenol oxidases (PPOs) are widely distributed in both plants and animals. They are a major cause of enzymic browning in fruits and vegetables. Browning reactions generally reduces the value of fruit and vegetables both nutritionally and commercially.

PPO is a copper-containing enzyme, which catalyses the o-hydroxylation of monophenols to o-diphenols and the oxidation of o-dihydroxyphenols to o-quinones. The enzyme uses molecular oxygen. The first type of reaction illustrates its monophenolase activity whilst the second reaction illustrates its diphenolase activity (Chararra et al., 2001).

The enzyme has two numbers: (EC 1.14.18.1 and EC 1.10.3.1). 

The enzyme has two conserved copper‐binding domains, CuA and CuB, responsible for copper coordination and interaction with molecular oxygen and phenolic substrates. The enzyme also possesses a transit peptide targeted to interact with the thylakoid lumen (Steffens et al., 1994). 

The corresponding o-quinones subsequently polymerise to brown pigments. The enzyme is related to lipoxygenase and peroxidase which are also found in the same animals and plants.

The action of PPO leads to the change in the colour and flavour of fruits and vegetables during harvest, storage and processing, which reduces the commercial value of the fruits, vegetables and their products.

Sources Of PPO

PPO has been widely studied in various plants such as pear (Halim & Montgomery, 1978), blueberry (Farid et al., 1997), plum (Siddig et al., 1992), grape (Cash et al., 1976), medlar
(Barbaros et al., 2002), banana (Yang et al., 2000), tea leaf (Halder et al., 1998) and apple (Shannon & Pratt, 1967).

The enzyme is mainly found in the pericarp of fruits although it can be extracted from the peel and skin. There is a tendency for the enzyme to be found in higher concentrations in the skin than the pulp as has been reported a number of fruits especially mango (Cheema et al., 2015). the extraction and purification of the enzyme for research purposes was recently reviewed (Panadare and Rathod, 2018).

The pH optimum is around 6 with a temperature optimum at 20 °C for the PPO from plum (Siddig et al., 1992).  A grape PPO had optimal activity at pH 3.4 but there was at least 90% activity between pH3 and 4 and then a very sharp drop above pH 4 (Ünal et al., 2007). The PPO from blueberry has a temperature optima at 35 C and pH optima between 6.12 and 6.3 (Siddiq & Dolan, 2017).

The preferred substrates are polyphenols. Among substrates, 4‐methylcatechol was oxidized more rapidly, although catechol, 3-methylcatechol, dopamine, pyrogallol and caffeic acid were also good substrates.

The enzyme from plum for example has a Km of 20 raM of catechol and Vmaxof 5.41 × 10−1 O.D./ min at pH 6.0 (Siddig et al., 1992).

The enzyme is effectively inhibited by  sodium metabisulphite (Na2S2O5), sodium ascorbate, kojic acid, L‐cysteine, L-glutathione and ascorbic acid. Inhibition is usually of a non-competitive type. L-cysteine is claimed to completely inhibit the PPO from Whangkeumbae pear (Zhou et al., 2018).

Purification Of PPO

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE−Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. More sophisticated methods use three phase partitioning (TPP), aqueous two phase extraction (ATPE), reverse micellar extraction (RME) and chromatographic techniques (Panadare & Rathod, 2018). 

Assay Of PPO Activity

The activity of PPO is often measured by spectrophotometry using catechol as the substrate. The increase in absorbance at 410nm is recorded. A sample cuvette containing 2.0 mL of 20 mm catechol (prepared in 0.2 m sodium acetate buffer, pH 4.0), 0.9 mL of 0.2 m sodium acetate buffer pH 4.0 and 0.1 mL of enzyme solution. Guiacol is also another suitable substrate for this assay.

Enzyme Behaviour

PPO has an unusual and interesting characteristic not usually found with other enzymes. It can form an inactive or latent state (Mayer & Harel, 1979). A number of agents  and processing treatments can convert PPO from its latent form to a more active type. The PPO exists in its latent form when bound to cell membranes but is often activated when fruit is processed (Yoruk & Marshall, 2003).

The PPO in strawberry puree is highly thermostable which makes it difficult to produce a non-browned product. Even treatment with steam for 30 minutes has little impact (Terefe et al., 2010). A related enzyme peroxidase is however thermosensitive with inactivation in less than 5 minutes at 70 °C . The PPO in blueberry is completely inactivated in 20 minutes at 85°C.

Inactivation Of PPO

Blanching has long been the accepted method for inactivating any enzymes in food. Hot water blanching is probably the most commercially acceptable because it is so simple and has considerable economic advantages. The disadvantage however is the loss of nutrients as a result of such processing – sugars, vitamins, proteins and water-soluble minerals can all be degraded or leached away when bidding to destroy enzyme activity (Lee, 1958). Some have speculated on whether removing copper from the seed for example might help suppress PPO activity.

High temperature -short time (HTST) blanching of apple pieces inactivates PPO. The hotter the water the more effective the treatment. A temperature above 55 ºC is required as below this there is very little deactivation (del Valle et al., 1998).

Another possible treatment is pressure assisted thermal processing (PATP) which was tried on coconut water (Chourio et al., 2018). No enzymatic activity was detected for both enzymes within 300 s at 90 °C/400–600 MPa.

Supercritical carbon dioxide has also been accepted as a process (Gui et al., 2007; Xu et al., 2011) for apple juice. One study found the maximum reduction of PPO activity was 60% when a high carbon dioxide pressure of 30 MPa at 55 °C for 60 min was applied. The apple juice browned but at a slower rate (Gui et al., 2007).

Ultrasound shows possibilities. The inactivation of PPO and peroxidase follows a 1st-order model when ultrasound is applied. It is particularly effective for treating puree and juice (Cao et al., 2018) where the cavitation helps to release PPO from its membrane form which is subsequently denatured by heat.

References

Balasingam, K. & Ferdinand, W. (1970). The purification and properties of a ribonucloenzyme o-diphenol oxidase from potatoes. Biochemistry Journal, 118, pp. 15–23.
 
Barbaros, D., Ahmet, C., Nese, A., Asim, K. & Saadettin, G. (2002). Characterization of polyphenoloxidase from medlar fruits (Mespilus germanica L., Rosaceae). Food Chemistry, 77, pp. 1–7
 
Cano P, Marín MA, Fúster C. 1990. Effects of some thermal treatments on polyphenoloxidase and peroxidase actvities of banana (Musa cavendishii, var enana). J Sci Food Agric 51 pp. 223–31.
Cao, X., Cai, C., Wang, Y., Zheng, X. (2018) The inactivation kinetics of polyphenol oxidase and peroxidase in bayberry juice during thermal and ultrasound treatments. Innov. Food Sci., Emerg. Technol. 45 February pp. 169-178
 
Cash, J.N., Sistrunk, W.A. & Stutte, C.A. (1976). Characteristics of Concord grape polyphenol oxidase involved in juice color loss. Journal of Food Science, 41, pp. 1398–1402
 
Chararra, S., Carcia-Carmona, F. & Cabanes, J. (2001). Hysteresis and positive cooperativity of iceberg lettuce PPO. Biochemical and Biophysical Research Communications, 289, pp. 769–775
Cheema, S., & Sommerhalter, M. (2015). Characterization of polyphenol oxidase activity in Ataulfo mango. Food Chemistry171, pp. 382-387. 
 
Chourio, A.M., salais-Fierro, F., Mehmood, Z., Martinez-Monteagudo, S.I., Saldana, M.D.A. (2018) Inactivation of peroxidase and polyphenoloxidase in coconut water using pressure-assisted thermal processing. Innov. Food Sci., Emerging Technologies. 49 pp. 41-50 https://doi.org/10.1016/j.ifset.2018.07.014
 
Coseteng, M.Y. & Lee, C.Y. (1987). Changes in apple polyphenol oxidase and polyphenol concentrations in relation to degree of browning. Journal of Food Science, 52, pp. 985–989
 
de Jesus Rivas, N., & Whitaker, J. R. (1973). Purification and some properties of two polyphenol oxidases from Bartlett pears. Plant physiology52(5), pp. 501-507.
 
Devece C, Rodriguez‐Lopez JN, Fenoll LG, Tudela J, Catala JM, de Los Reyes E, Garcia‐Canovas F. 1999. Enzyme inactivation analysis for industrial blanching applications: comparison of microwave, conventional, and combination heat treatments on mushroom polyphenoloxidase activity. J Agric Food Chem 47(11):4506–11
 
Dogan, S., & Dogan, M. (2004). Determination of kinetic properties of polyphenol oxidase from Thymus (Thymus longicaulis subsp. chaubardii var. chaubardii). Food chemistry88(1), pp. 69-77. 
 
Dogan, M., Arslan, O. & Dogan, S. (2002). Substrate specificity, heat inactivation and inhibition of polyphenol oxidase from different aubergine cultivars. International Journal of Food Science and Technology, 37, pp. 415–423
Doğan, S., Arslan, O., & Özen, F. (2005). Polyphenol oxidase activity of oregano at different stages. Food Chemistry91(2), pp. 341-345. 
Doğan, S., Turan, P., & Doğan, M. (2006). Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata. Process Biochemistry41(12), pp. 2379-2385.  
 
Duangmal, K. & Owusu Apenten, R.K. (1999). A comparative study of polyphenol oxidases from taro (Colocasia esculenta) and potato (Solanum tuberosumvar. Romano). Food Chemistry, 64, pp. 351–359.
 
Farid, K., Bernard, R., Michel, G. & Maurice, M. (1997). Mechanism of browning in fresh highbush blueberry fruit (Vaccinium corymbosum L): partial purification and characterization of blueberry polyphenol oxidase. Journal of Agriculture and Food Chemistry73, pp. 513–516
 
Golan, A., Goldhirsh, A. & Whitaker, J.R. (1984). Effect of ascorbic acid, sodium bisulfite and thiol compounds on mushroom polyphenol oxidase. Journal of Agricultural Food Chemistry, 32, pp. 1003– 1009.
 
Gui, F., Wu, J., Chen, F. et al. (2007). Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxideFood Chemistry100, pp. 1678–1685 https://doi.org/10.1016/j.foodchem.2005.12.048
Halder, J., Tamuli, P. & Phaduri, A.N. (1998). Isolation and characterization of polyphenol oxidase from Indian tea leaf (Camellia sinensis). Journal of Nutritional Biochemistry, 9, pp. 75–80.
 
Halim, D.H. & Montgomery, M.W. (1978). Polyphenol oxidase of d’Anjou pears (Pyrus communis L.). Journal of Food Science, 43, pp. 603–608
 
Janovitz-Klapp, A.H., Richard, F.C. & Nicolas, J.J. (1990). Inhibition studies on apple polyphenol oxidase. Journal of Agricultural and Food Chemistry, 38, pp. 926–931.
 
Jiang, Y.M. (1999). Purification and some properties of polyphenol oxidase of longan fruit. Food Chemistry, 66, pp. 75–79
 
Lee, F.A. (1958). The blanching process. Advances in Food Research8, pp. 63109.
 
Kahn, V. & Andrawis, A. (1985). Inhibition of mushroom tyrosinase by tropolone. Phytochemistry, 24, pp. 905–908.
 
Kavrayan, D. & Aydemir, T. (2001). Partial purification and characterization of polyphenoloxidase from peppermint (Mentha piperita). Food Chemistry, 74, pp. 147–154
 
Mayer, A. M. (1986). Polyphenol oxidases in plants-recent progress. Phytochemistry26(1), pp. 11-20. 
 
Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry67(21), pp.  2318-2331.
 
Mayer, A. M., & Harel, E. (1979). Polyphenol oxidases in plants. Phytochemistry18(2), pp. 193-215 https://doi.org/10.1016/0031-9422(79)80057-6
 
Montero, P., Avalos, A. & Perez-Mateos, M. (2001). Characterization of polyphenoloxidase of prawns (Penaeus japonicus). Alternatives to inhibition: additives and high-pressure treatment. Food Chemistry75, pp. 317–324
 
Nakamura, K., Amano, Y. & Kagami, M. (1983). Purification and some properties of a polyphenol oxidase from Koshu grapes. American Journal of Enology and Viticulture, 34, 122–127
 
Nicolas, J. J., Richard‐Forget, F. C., Goupy, P. M., Amiot, M. J., & Aubert, S. Y. (1994). Enzymatic browning reactions in apple and apple products. Critical Reviews in Food Science & Nutrition34(2), pp. 109-157.
 
Oktay M, Küfreviolğlu I, Kocaçalişkan I, Şakiroğlu H. 1995. Polyphenol oxidase from Amasya apple. J Food Sci. 60(3) pp. 494–6.
 
Palma‐Orozco G, Ortiz‐Moreno A, Dorantes‐Alvarez L, Sampedro JG, Najera H. 2011. Purification and partial biochemical characterization of polyphenol oxidase from mamey (Pouteria sapota). Phytochemistry 72(1) pp. 82–88
Panadare, D., Rathod, V.K. (2018) Extraction and purification of polyphenol oxidase: A review. Biocat. Agric. Biotechnology  14 pp. 431-437  
 
Pifferi, P.G. & Cultrera, R. (1974). Enzymatic degradation of anthocyanins: the role of sweet cherry polyphenol oxidase. Journal of Food Science, 39, 786 791.   https://doi.org/10.1111/j.1365-2621.1974.tb17980.x
 
Ramirez, E.C., Whitaker, J.R. & Virador, V.M. (2004). Polyphenol oxidase. In: Handbook of Food Enzymology (edited by J.R. Whitaker, A.G.J. Voragen & D.W.S. Wong). Pp. 509–522. New York, NY: Marcel Dekker
 
Rapeanu, G., Loey, A.V., Smout, C. & Hendrickx, M. (2006). Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape. Food Chemistry, 94, pp. 253–261
 
Rodríguez‐López JN, Fenoll LG, Tudela J, Devece C, Sánchez‐Hernández D, de Los Reyes E, García‐Canovas F. 1999. Thermal inactivation of mushroom polyphenoloxidase employing 2450 MHz microwave radiation. J Agric. Food Chem. 47(8) pp. 3028–35.
 
Sanchez-Ferrer, A., Bru, A., Cabanes, J. & Garcia-Carmona, F. (1988). Characterisation of catecholase and cresolase activities of Monastrell grape polyphenol oxidase. 
Phytochemistry, 27, 319–321.
 
Serradell, M.A., Rozenfeld, P.A., Martinez, G.A., Civello, P.M., Chaves, A.R. & Anon, M.C. (2000). Polyphenol oxidase activity from strawberry fruit (Fragaria · ananassa, Duch., cv Selva): characterisation and partial purification. Journal of the Science of Food and Agriculture, 80, 1421–1427.
 
Shi, C., Dai, Y., Xia, B., Xu, X., Xie, Y. & Liu, Q. (2001). The purification and spectral properties of polyphenol oxidase I from Nicotiana tabacum. Plant Molecular Biology Reporter, 19, 381a–381h.
 
Shannon, C.T. & Pratt, D.E. (1967). Apple polyphenol oxidase activity in relation to various phenolic compounds. Journal of Food Science32, pp. 479–483
Siddiq, M., Sinha, N.K., Cash, J.N. (1992)  Characterization of Polyphenoloxidase from Stanley Plums. J. Food Sci.,  https://doi.org/10.1111/j.1365-2621.1992.tb11292.x

Siddiq, M & Dolan, K.D. (2017) Charcterization of polyphenol oxidase from blueberry (Vaccinium corybosum L.). Food Chem., 218 March pp. 216-220 https://doi.org/10.1016/j.foodchem.2016.09.061

Valle, J.M., Aranguiz, V. & Leon, H. (1998). Effects of blanching and calcium infiltration on PPO activity, texture, microstructure and kinetics of osmotic dehydration of apple tissueFood Research International31, pp. 557–569 https://doi.org/10.1016/S0963-9969(99)00029-0

Vámos‐Vigyázó, L., & Haard, N. F. (1981). Polyphenol oxidases and peroxidases in fruits and vegetables. Critical Reviews in Food Science & Nutrition15(1), pp. 49-127
Wang, J., Jiang, W., Wang, B., Liu, S., Gong, Z., & Luo, Y. (2007). Partial properties of polyphenol oxidase in mango (Mangifera indica L. cv.“Tainong”) pulp. Journal of Food Biochemistry31(1), pp. 45-55.  
 
Weemaes, C.A., Ludikhuyze, L.R., Broeck, L., Hendricks, M.E. Tobback, P.P. (1998). Activity, electrophoretic characteristics and heat inactivation of polyphenoloxidases from apples, avocados,  grapes, pears and plums. Lebensmittel-Wissenschaft und-Technologie, 31, pp. 44–49.
 
Wong, T. C., Luh, B. S., & Whitaker, J. R. (1971). Isolation and characterization of polyphenol oxidase isozymes of clingstone peach. Plant Physiology48(1), pp. 19-23
 
Xu, Z., Zhang, L., Wang, Y., Bi, X., Buckow, R. & Liao, X. (2011). Effects of high pressure CO2 treatments on microflora, enzymes and some quality attributes of apple juiceJournal of Food Engineering104, pp. 577–584. https://doiorg/10.1016/j.jfoodeng.2011.01.020  
 
Yang, C.P., Fujita, S., Ashrafuzzaman, M.A., Nakamura, N. Hayashi, N. (2000). Purification and chracterization of polyphenol oxidase from banana (Musa sapientum L.) pulp. Journal of Agricultural Food Chemistry, 48, pp. 2732–2735
 
Yokotsuka, K., Makino, S. & Singleton, V.L. (1988). Polyphenol oxidase from grapes: precipitation, re-solubilization and characterization. American Journal of Enology and Viticulture, 39, pp. 293–302
 
Yoruk, R., & Marshall, M. R. (2003). Physicochemical properties and function of plant polyphenol oxidase: a review 1. Journal of Food Biochemistry27(5), pp. 361-422. https://doi.org/10.1111/j.1745-4514.2003.tb00289.x
 
Zawistowski, J., Bilideris, C.G. & Eskin, N.A.M. (1991). Polyphenol oxidase. In: Oxidative Enzymes in Foods (edited by D.S. Robinson N.A.M. Eskin). Pp. 217–273. New York, NY: Elsevier
 
Zhou, X., Xiao, Y., Meng, X., Liu, B. (2018) Full inhibition of Whangkeumbae pear polyphenol oxidase enzymatic browning reaction by L-cysteine. Food Chem., 266 Nov. pp. 1-8    https://doi.org/10.1016/j.foodchem.2018.05.086 .

Be the first to comment

Leave a Reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.