Production of Commercially Suitable

4.5.1. Enzymatic cross-linking of proteins or polysaccharides

There is an increasing interest in food industry for finding new functional ingredients. For example, the modification of proteins to improve or change their functions has become very popular. One of the potential methods for the modification of proteins is cross-linking. This application may improve the functions of proteins related to gel formation, foaming, emulsification, etc.

The modification of proteins by PPO is done by mixing enzyme, target protein(s) and suitable phenolic compounds. The oxidation of phenolic compounds by PPO produces o-quinones that may undergo a spontaneous Michael-like addition with amino-sulphydryl or pyrrolidine side chain of proteins. Thus, with this reaction the proteins are cross-linked by the oxidized phenolic compounds (protein-oxidized phenolic compound-protein). In some proteins, the protein-protein cross-linking with PPO may also occur without using any phenolic compounds. In such proteins, the tyrosine groups in protein oxidized by PPO provide the desired cross-linking. Thalmann and Lötzbeyer (2002), by use of mushroom tyrosinase, studied the cross-linking of lysozyme and whey proteins such as -lactalbumin, -lactoglobulin in the presence or absence of a low molecular weight phenolic compound, caffeic acid. In the presence of phenolic compound, these workers reported the successful modification of all indicated proteins with cross-linking. However, in the absence of phenolic compound only -lactalbumin showed cross-linking.

In the presence of suitable phenolic compounds, the cross-linking of some polysaccharides is also possible (polysaccharide-oxidized phenolic compound-polysaccharide). For example, Micard and Thibault (1999) reported the use of laccase and ferulic acid for cross-linking of sugar beet pectin. The gel obtained from this modified pectin is thermo-irreversible and maintains its structure during heating operation (Minussi et al. 2002).

4.5.2. Production of flavonoid-derived colorants and antioxidants
Some plant secondary metabolites, including the flavonoids, show antioxidant activity. Also, there are some natural plant methabolites with stable and attractive color. These natural compounds may be used in place of synthetic antioxidants and colorants. One of these plant based flavonoids is phloridzin which found in apples. The bioconversion of phloridzin to a number of derivatives provides some novel compounds with higher antioxidant activities. Ridgway et al (1997) extracted and used apple PPO successfully for biosynthesis of antioxidant compounds 3-hydroxyphloridzin and 3-hydroxyphloretin from phloridzin and yellow/orange colored dimerized oxidation products of phloridzin that may be used as food colorants. Ridgway and Tucker (1999) also developed a procedure for the partial purification of commercially suitable PPO from apple leaf. However, since PPO in apples can not oxidize the p-diphenols (laccase activity) and lacks monophenolase activity it may only be used for some specific applications. By use of tyrosinases it is also possible to obtain some plant pigments such as red-violet betalains and gold colored aurones (Strack and Schiliemann 2001) and biosynthesis of antioxidant compounds such as hydroxytyrosol (Espin et al. 2001).
4.5.3.The removal of haze forming polyphenols from beverages
The most frequently encountered problem in wine, beer, and fruit juices is the haze formation, resulted from protein-polyphenol interaction. Thus, different methods have been used in order to remove the haze forming phenolic compounds from the beverages. For example, the haze forming polyphenols may effectively be removed from the beverages by use of polyvinylpolypyrolidone (PVPP) columns. However, because of its health problems on workers and low biodegradability in the wastewaters it is difficult to handle PVPP in factories (Minussi et al. 2002).

The use of in situ PPO or commercial PPO (Laccase) to selectively oxidize haze-forming polyphenols is an alternative to the application of PVPP columns. It was demonstrated that the phenolic compounds oxidized by the PPO and polymerized may be flocked and removed from the beverages (Minussi et al. 2002). The removal of oxygen during enzymatic oxidation enhances the storage stability of beers (Minussi et al. 2002). In contrast, the oxidative changes caused by the enzyme affect the aromatic quality of apple juices adversely (Cemeroğlu and Karadeniz 2001).

4.5.4. Oxygen scavenging and removal of undesirable phenolics from food
Because of their high oil content, foods such as salad dressings and mayonnaise are susceptible to oxidation. The oxidation of fatty acids in these foods by the dissolved oxygen causes the formation of undesirable volatile compounds. Thus, the removal of dissolved oxygen in these foods by PPO improves their quality (Minussi et al. 2002).

In cocoa beans, the enzymatic oxidation of phenolic compounds responsible from astringency and bitterness is an important PPO application. Selamat et al (2002) studied the enrichment of fermented cocoa beans with mushroom PPO showed that the concentration of undesirable phenolic compounds in cocoa beans may be reduced effectively to acceptable levels. Also, laccase from Coriolus versicolor studied for the treatment of cocoa bean removed the phenolic compounds responsible from bitterness and other unpleasant tastes successfully (Minussi et al. 2002).

The undesirable phenolic compounds exist also in canola meal, a by product obtained after oil extraction from canola seeds. Although, it is a good source of protein with a favorable amino acid composition, its high phenolics content prevents the use of canola meal as human food or animal feed. Thus, there are some studies to remove undesirable phenolic compounds, especially sinapic acid, from canola meal by Trametes versicolor PPO (Lacki and Dunjvak 1998). The ability of mushroom PPO to oxidize sinapic acid in canola meal was also reported by Choi and Sapers (1994). Thus, PPO from different sources may be suitable to treat the canola meal.


4.5.5. Removal of undesirable phenolics from wastewaters
Aromatic compounds including phenolics and aromatic amines present in wastewaters of chemical, textile, wood preservation and food industries are very harmful for the environment. Thus, there are some studies to oxidize and remove these undesirable phenolic compounds from wastewaters by peroxidases (Minussi et al. 2002, Duran and Esposito 2000, Ikehata and Nicell 2000). As alternative to peroxidases, the immobilization of tyrosinases and/or laccases on different supports and treatment of wastewaters is also studied by different workers. For example, Coriolopsis gallica (white-rot fungus) laccase was studied to degrade tannins in waste waters from beer factories and the effectiveness of Pleunotus ostreatus laccase was also tested for the treatment of wastewater from olive oil production (olive mill wastewater). This treatment reduced the phenolic content of olive mill wastewater almost 90 %. However, no toxicity reduction was observed in the treated wastes (Minussi et al. 2002).
4.5.6. Analytical and clinical applications of PPO
4.5.6.1. Production of biosensors
Biosensors are specific devices that detect, transmit, and record information regarding a physiological or biochemical change in a medium. In practice, it is a probe integrating a biological component with an electric transducer thereby converting a biochemical signal into a quantifiable electrical response. Due to their biological specificity, enzymes are widely used as biological sensing elements (Minussi et al. 2002).

It was reported that a number of biosensors have been developed containing laccase for immunoassays and determination of phenolic compounds and tyrosinase for the detection and quantification of phenolic compounds (Minussi et al. 2002, Rubianes and Rivas 2000, Climent et al. 2001). For example, tyrosinase and catechol oxidase containing biosensors were also developed for detection of phenolic compounds in olive oil, green tea, grape and olive extracts and beer. A PPO biosensor for detection of pesticides in spiked apples and a PPO-alkaline phosphatase combinational biosensor for detection of phosphatase in drinking water were also developed (Mello and Kubota 2002).

4.5.6.2. Clinical applications
One of the most interesting features of mushroom tyrosinase is its ability to inhibit the attachment of some bacteria. For example, Cowan et al (2000) demonstrated that by oxidizing the critical tyrosine residues of glucan binding lectin and glucosyltransferases, tyrosinase prevents the attachment of Streptococcus sobrinus, a bacteria responsible from the formation of oral cavities, to glucans deposited on the tooth surface. Kolganova et al (2002) also showed that the tyrosinase reduces the adhesion of some viruses and pathogenic bacteria to buccal epithelial cells, while unaffecting the attachment of probiotic bacteria. These findings are quite interesting and may open the way of using mushroom PPO in foods such as gums and confectionaries to increase the tooth health of people.

Several other clinical applications such as using tyrosinase as a catalyst to produce L-DOPA, a drug for the treatment of Parkinson`s disease (Sharma et al. 2003), a marker of vitiligo, an autoimmune disease and a tumor suppressing and prodrug therapy agent (Seo et al. 2003) also attracts considerable interest.