Production of Commercially Suitable

POLYPHENOL OXIDASES

4.1. Polyphenol oxidases
Polyphenol oxidases (PPOs) are copper containing oxidoreductases that catalyze the hydroxylation and oxidation of phenolic compounds in the presence of molecular oxygen. According to the type of their substrates PPOs can be divided into three groups namely tyrosinase, catechol oxidase and laccase. Tyrosinase (EC. 1.14.18.1) catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase or cresolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase or catecholase activity). Catechol oxidase (EC. 1.10.3.1) catalyzes only the oxidation of o-diphenols whereas laccase (EC. 1.10.3.2) catalyzes the oxidation of o-diphenols as well as p-diphenols to the corresponding quinones. Both laccase and catechol oxidase cannot catalyze the hydroxylation reactions (Figure 4.1).

A

(1) Cresolase activity (2) Catecholase activity

B

C

Figure 4.1. The reactions of polyphenol oxidases (Cemeroğlu et al. 2001)

(A: Reaction mechanism of tyrosinase; B: Reaction mechanism of catechol oxidase; C: Reaction mechanism of laccase)

The o-quinones formed by PPOs are very reactive and turn spontaneously to tri hydroxy benzenes (THB). The THBs then interact with o-quinones in the medium to form hydroquinones that further polymerize to complex and dark colored pigments referred melanins (Figure 4.2). This reaction is called the enzymatic browning and it is undesirable during processing of fruits and vegetables.

Figure 4.2. The nonenzymatic reactions during formation of dark colored melanins (Cemeroğlu et al. 2001)

4.2. Substrates of PPO
Phenolic substances, the natural substrates of PPO, occur in many fruits and vegetables to provide color pigments and characteristic flavor. Although, fruits and vegetables contain a wide variety of phenolic compounds only a few of them are good substrates of PPOs. The most important natural substrates of PPOs in fruits and vegetables are catechins, cinnamic acid esters (especially chlorogenic acid), 3-4-dihydroxy phenylalanine (DOPA) and tyrosine (Vamos Vigyazo, 1981) (Figure 4.3.).


Catechin Chlorogenic acid

Tyrosine L-DOPA

Figure 4.3. Some of the good substrates of PPO in plants
The substrate specificity of PPO and the available phenolic substrates in different fruits and vegetables vary greatly. For example, in mushrooms and potatoes the precursor of enzymatic browning is tyrosine. However, in peaches and pears the primary phenolic compounds causing enzymatic browning are tannins and chlorogenic acid, respectively. The substrate specificity of PPO may also be different in various sections of the plant. For example, in apple peel and flesh the precursors of enzymatic browning are chlorogenic acid and catechins, respectively (Davidek et al. 1990).
Sources_and_Some_Characteristics_of_PPO'>4.3. Sources and Some Characteristics of PPO
PPOs are widely distributed in nature. These enzymes are abundant in higher plants but found also in animals and some microorganisms, especially in fungi. The properties of PPO from different sources vary considerably. For example, it was reported that the substrate specificity of plant PPO is broader than that for the PPO in animals (Burton, 1994).

In plants, PPOs are located mainly in thylakoid membrane of chloroplasts and mitochondria. The enzymes exist also in the vesicles or other bodies in nongreen plastid types and cytoplasm (Nicolas et al., 1994). In higher plants and fungi there are different isoforms of tyrosinases such as immature, mature latent and active forms. For example, in mushrooms almost ~ 98-99 % of the PPO is present in its latent form (Vamos- Vigyazo, 1981, Nicolas et al. 1994, Minussi et al. 2002, Seo et al. 2003).

In living organisms, PPO has many different roles. For example, in insects they are responsible for the melanization and sclerotization of the exoskeleton. In crustaceans such as octopus, they form the black colorant sepia. In animals and human, the enzyme is responsible for hair and skin coloration whereas in plants it has vital roles in the defense mechanisms against insects and microbial pathogens (Strack and Schliemann 2001).

The activity and biochemical characteristics of PPO change according to the source of enzyme (Table 4.1). However, in most cases optimum pH of enzyme varies between 5.0 and 7.0. The enzyme mostly shows low pH stability between 3.0 and 4.0. Also, the PPO enzymes do not belong to an “extremely heat-stable enzyme” group and short exposures to temperatures between 70 ^o and 90 ^oC are sufficient to inactivate them (Vamos-Vigyazo, 1981).

Table 4.1. Some properties of PPOs from different fruits and vegetables

Source	Optimum pH	Optimum temperature (^oC)	Temperature stability ^a	Reference
Banana peel	6.5 (Dopamine)	30 (Dopamine)	90 % (30 min, 60 ^oC) inactivated at 80 ^oC	Yang et al ( 2001)
Banana pulp	6.5 (Dopamine)	30 (Dopamine)	80 % (10 min, 70 ^oC)	Yang et al (2000)
Chineese cabbage	5 (Catechol)	50 (Catechol)	unstable > 50 ^oC	Nagai and Suzuki (2001)
Cabbage	7.6 (Phloroglucinol)	40 (Phloroglucinol)	40 % (10 min, 100 ^oC)	Fujita et al (1995)
Malatya apricot	8.5 (Catechol)	-	50 % (47 min, 60 ^oC) 50 % (16 min, 80 ^oC)	Arslan et al (1998)
Grape	5 (Catechol)	-	inactivated at 70 ^oC	Garcia and Buzaleh (1994)
Amasya apple	7.0 (Catechol) 8.6 (pyrogallol) 6.6 (L-DOPA)	15 (Catechol) 50 (L-DOPA) 70 (Pyrogallol)	-	Oktay et al (1995)
Artichoke	5-7 (Catechol) 8 (L-DOPA) 6.5 (Pyrogallol)	25 (Catechol)	50 % (6 min, 60 ^oC) 50 % (4 min, 70 ^oC)	Aydemir (2004)
Iceberg lettuce	4.4 and >7.5 (-SDS) >5.0 (+SDS)	-	inactivated at 90 ^oC	Chazarra et al (1996)
Coffee	6.0-7.0	25-30	-	Mazzafera and Robinson (2000)

^a % retained activity after the indicated heating

4.4. The Effects of PPO on Food Quality
The enzymatic browning reaction is a widespread phenomenon, mostly undesirable in food technology due to the formation of unpleasant discoloration and development of some off-flavors. Thus, numerous studies have been conducted related to the inhibition and inactivation of PPO by chemicals and thermal processes, respectively. Most of these studies are related with the light colored plant products such as apple (Oktay et al. 1995, Yemenicioğlu et al. 1997), potato (Coetzer et al. 2001, Yemenicioğlu, 2002), and mushroom (Kermasha et al. 1993, Friedman and Bautista 1995, Gomes and Ledward 1996, Jimenez and Garcia-Carmona 1997, Rodziguez-Lopez et al, 1999, Weemaes et al. 1997, Beaulieu et al. 1999, Devece et al. 1999, Fan and Flurkey 2004).

A novel and promising approach for the control of PPO activity in fruits and vegetables without the use of chemicals is the use of molecular biology. By employing an antisense RNA technique Bachem et al (1994) blocked PPO gene expression in potatoes and obtained very low PPO activity-containing cultivars. Coetzer et al (2001) also reduced the PPO activity in potatoes by using sense or antisense RNA techniques and successfully controlled their enzymatic browning. One of the concerns related to these methods is that PPO in plants is involved in disease and pest resistance (Li and Steffens 2002). The inactivation of PPO enzymes in plants may cause a susceptibility increase to diseases and may encourage a wider pesticide use. In fact, hypersusceptibility to pathogens was reported in tomatoes with reduced PPO activity induced by the use of an antisense technique (Thomas-Berberan and Espin 2001). Thus, alternative strategies should be developed to increase the application potential of molecular techniques.

4.5. Industrial Applications of PPO
In industry, the commercial PPOs used are laccase obtained from Trametes versicolor, a white-rot fungus, by fermentation and tyrosinase extracted from mushrooms (Agaricus bisporus). Recently, extensive studies have been conducted related to the use of PPO in food industry and in other branches of industry. Enzymatic cross-linking of proteins and polysacharrides, production of color compounds and antioxidants, clarification of beverages, production of biosensors are some of the example uses of the enzyme. These uses of PPO in industry are discusses with further details below.