Biochemistry and Molecular Biology), three general principles are used in the ... constituted by 4 integers, the first referring to one of the six major classes of ...
Alim. Nutr., Araraquara v.18, n.1, p.101-109, jan./mar. 2007
ISSN 0103-4235
OXIDOREDUCTASES FOR BIOANALYSIS AND FOOD CONTROLS Luciana Amade CAMARGO * Maristela de Freitas Sanches PERES ** Edwil Aparecida de Lucca GATTÁS**
ABSTRACT: Most well-known enzymatic methods have been employed in the bioanalysis of food, required by law in a number of countries. Food is a complex sample and hence difficult to analyze. In food analysis, there are many different types of sample, such as: clear or turbid, colorless or colored, neutral or acid, liquids, pastes and solids, that can be analyzed by enzymatic methods. The oxidase peroxidase system may be used for specific analyses. In the reaction catalyzed by the oxidase, hydrogen is transferred from the substrate to oxygen and hydrogen peroxide is formed as an intermediate. Hydrogen peroxide then reacts with a leuco dye in the presence of the enzyme peroxidase and a color develops which is measured in the visible spectrum. This article is a review of two enzymes, peroxidase (POD) and glycerol-3-phosphate oxidase (GPO), pointing out their principal sources, characteristics and applications. KEYWORDS: Enzymes; glycerol-3-phosphate oxidase; applications; peroxidase; applications.
ENZYMES Enzyme, a term from the Greek (en: inside + zyme: Leaven), can be defined as a protein complex (heteroprotein or derived protein) responsible for the control of one or more vital functions (metabolic processes of conversion of nutrients into energy and new materials for the cells), by catalyzing biochemical reactions.29,45 Commercial enzymes are compounded products made on a large scale by microbial synthesis, that can easily be denatured; by intense agitation; heat, ultraviolet and ultrasound waves and by substances such as cyanide, and sodium fluoride, heavy metals, acids or bases. 45 As proteins, the enzymes are constituted by long chains of amino acids joined by peptide bonds and structured three-dimensionally; they may be intracellular (endoenzyme) or extracellular (exoenzyme), in this case able to digest
insoluble nutritional material such as starch, cellulose and proteins. 32,35,45 Structurally, the enzymes are holoenzymes, consisting of a protein (apoenzyme) with an active site, where a cofactor (organic prosthetic group active metal ion) assists them in their catalytic activities. 29, 45 The specificity with which each enzyme binds to its main reactant (substrate) defines its "biological activity", and is a consequence of the three-dimensional arrangement of the amino acids involved in substrate-enzyme binding which generally complement the molecule of the substrate, providing an ideal space and electrical environment for the bound substrate. 29, 32 Like all catalysts an enzyme accelerates the speed of a biochemical reaction, effectively without participating as a reactant or product of this process. It can operate in small amounts and be recovered after the reaction. The enzyme reacts with its substrate to form intermediate compounds that later are converted products and released, allowing its regeneration (Table 1). 45 The enzymes have applications in several sectors: 13, 31 Food: help to improve human and animal food and allow the extraction of oils without the use of dangerous solvents; Drink: they increase productivity in the extraction from the pulp of fruits, improve the color, transparency and aroma of wines and participate in the process of maturation of beers; Leather: they provide softness and flexibility, eliminating the need for chemical solvents during cleaning; Washing powders: they remove stains from fabrics, without fibers danification and they renew the colors of the textiles; Baking: they delay the process of aging in breads; Pharmaceutical Industry: they extract sugar for syrups from other sources (corn starch), without requiring special equipment and strong acids; Textile Industry: they give the "Stonewashed" effect to jeans;
* Pos-graduation - Program of Food Nutrition - Faculty of Pharmaceutical Science - UNESP 14801-902 - Araraquara - São Paulo - Brazil. ** Departament of Food Nutrition - Faculty of Pharmaceutical Science - UNESP - 14801-902 Araraquara - São Paulo - Brazil.
101
Table 1 - Enzymes X Chemical Catalysts. Enzymes
Chemical Catalysts
Nature
Nature
Complex Structure
Simple Structure
Low stability
High stability Production
Production
Batch Process
Continuous Process
High Cost (Isolation and Purification)
Moderate Cost
Catalytic Reaction
Catalytic Reaction
High specificity to the substrate
Low specificity to the substrate
Presence of cofactor
Absence of cofactor
Conditions of reaction: mild (Temperature Conditions of reaction: severe (high lower than 100oC; atmospheric pressure;
temperature; high pressure; extreme pH)
neutral pH) Low energy cost
High energy cost
High catalytic activity
Low catalytic activity
Low energy of activation
High energy of activation
High speed
Low speed
Few by-products
Many by-products
Hard/ expensive separation of products
Simple separation of products
Paper: they reduce the amount of chlorine necessary for bleaching. According to IUBMB (International Union of Biochemistry and Molecular Biology), three general principles are used in the classification and nomenclature of enzymes:17 1º) Name a simple catalytic entity for a defined chemical reaction: name + suffix - ase. 2º) Name enzymes in accordance with the reaction they catalyze (specific property that distinguishes one enzyme from the rest): name of substrate + type of catalyzed reaction + suffix - ase. 3º) Groups of name enzymes according to the type of reaction catalyzed. This, together with the name (s) of the substrate (s), provides a way of naming individual enzymes: they are thus grouped within a hierarchical numbering system, each one having an Enzyme Commission (EC) number. This number is a code, assigned to each enzyme, constituted by 4 integers, the first referring to one of the six major classes of enzymes; the second to a subclass, that demonstrates the type of reaction or chemical bond formed on modified by the enzyme; the third, the sub-subclass and, finally, the fourth being the serial number of the enzyme in its 102
sub-subclass, which determines the substrate of the catalytic reaction. In this system, the 6 main divisions or classes 3,17,35,45 established by the EC, are presented in Table 2. The objective of this review is to comment on two oxidoreductases (glycerol 3-phosphate oxidase and peroxidase) and describe their principal sources, characteristics and applications.
OXIDOREDUCTASES Definition Oxidoreductase is a potentially interesting class of enzyme for several chemical processes, such as are used in the pharmaceutical and food industries. They are responsible for catalyzing reactions of oxidation or reduction of substrates, reactions in which electrons are transferred between chemical species. The substrate undergoing oxidation (reducing agent) is considered as a hydrogen or electron donor, while the one that is reduced (oxidizing agent) is denominated the acceptor (Figure 1). 4,8,17
Nomenclature AH2 + B
A + BH2
Oxidized substrate
Reduced substrate
(reducing agent)
(oxidizing agent)
FIGURE 1 - Redox reaction.
The nomenclature adopted for the oxidoreductases is based on the structure: donor:receptor oxidoreductase (systematic name). The oxidoreductases can be subdivided into 4 main types: 9,17 Dehydrogenases: catalyze hydrogen transfer from the substrate to the nicotinamide adenine dinucleotide cofactor (NAD+); Oxidases: catalyze hydrogen transfer from the substrate to molecular oxygen (acceptor group), reducing it to hydrogen peroxide as a byproduct;
Table 2 - Classification of the enzymes according to the Enzyme Commission (IUBMB). 1. Oxidoreductases (Oxidation and reduction reactions. Transference of electrons)
A*
B
A
B*
2. Transferases (Transfer a group from one substrate to another.)
A
B
A
B
3. Hydrolases (Hydrolysis: including hydrolytic cleavage of into monomers)
H 2O
4. Lyases (Removal of groups from the substrates by elimination, with the concomitant formation of double
+
bonds or sings, or by adding new groups across double bonds). 5. Isomerases (Isomerisation: intramolecular rearrangements producing optical and geometric isomers)
6. Ligases (Condensation of two molecules
+
associated with the consumption of a high energy compound such as ATP)
AT
AD
103
Peroxidases: catalyze the oxidation of the substrate by hydrogen peroxide; Oxygenases: catalyze the oxidation of the substrate by molecular oxygen, reducing it to water as the by - product. Applications There are countless examples of oxidoreductase applications dispersed in the literature, which essentially concern fine chemistry and pharmacy in the production of chiral compounds (synthesis of amino acids, preparation of hydroxyacids and therapeutic compounds, insecticides, herbicides, etc.), the perfume, flavouring and food additive industries (conversion between alcohols, aldehydes and ketones) and steroid functional group changes (organic biochemistry).8
GLYCEROL 3-PHOSPHATE OXIDASE Main characteristics The enzyme glycerol-3-phosphate oxidase (EC 1.1.3.21; sn-glycerol-3-phosphate:oxygen 2-oxidoreductase; GPO) is a flavoprotein (cofactor: flavin adenine dinucleotide - FAD) belonging to the group of oxidoreductases that catalyze the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate with the concomitant reduction of oxygen to hydrogen peroxide, according to the following reaction scheme: 20,23,41 sn-glycerol-3-phosphate + O2 + H2O2
GPO
dihydroxyacetone phosphate
The enzyme was discovered in the 1960, isolated from lactic bacteria among other microorganisms (Table 3); there is no record register of its occurrence in humans.10,20,23
The S. faecium enzyme had a molecular weight of 131,000 daltons, divided in 2 subunits of 72,000 daltons, with 2 molecules of linked FAD per molecule of enzyme; best specificity was to glycerol-3-phosphate, and oxygen as the preferred electron acceptor. 10 In relation to the stability of this enzyme, Macková et al.23 who studied a microbial GPO isolated from a mutant strain of Aerococcus viridans, observed that increasing the storage temperature had a destructive effect on this enzyme. For instance, at 4ºC, great loss of activity occurred after a period of 15 days, while at 20ºC and 30ºC, the activity fell largely after 30 and 25 hours, respectively. They found the highest stability at -20ºC and the optimal pH for stability (pH 9.0), in a GPO preparation stored at 4oC in the presence of 0.05% sodium azide. After analyzing of all the results, the authors concluded that the low stability of GPO could be improved by the addition of 0.1M EDTA or by the lyophilization in the presence of dextrin. The addition of catalase (enzyme that decomposes hydrogen peroxide) to the medium used to culture of Aerococcus viridans, consisting, basically of glycerol, sodium pyruvate, peptone, phosphates, yeast extract and salt solution, protects against inactivation of bacterial growth as a consequence of an excess of hydrogen peroxide, allowing a larger microbial production of glycerol-3-phosphate oxidase. 39 Applications GPO has practical applications in several coupled systems for the quantitative determination of magnesium 51, glycerol phosphate20, triacylglycerol12,24,42,48, glycerol 27,28, phosphatidic acid 21 and other phospholipids. It can also be used for the measurement of enzyme activities (glycerol kinase 15 and similar enzymes coupled to other enzymes and a chromogen reagent) in blood serum or other biological materials. 23,39,41 In 1998, Compagnone et al.7 reported the development
Table 3 - Some sources of GPO. Organism
References Šuchová
et
al.,41
Machová
et
al.23,
Aerococcus viridans Streitenberger et al. 39 Lactobacillus
Strittmatter 40
Propionibacterium
Ince et al. 16
Streptococcus faecalis
Jacobs & Vandermark 18 Koditschek & Umbreit
Streptococcus faecium Claiborne 6 Other
104
Šuchová et al.41
22
; Esders & Michrina,10
of an amperometric system - FIA (flow injection analysis) for glycerol determination during alcoholic fermentation, based on the following reactions catalyzed by glycerol kinase (GK) and GPO: Glycerol + ATP (Mg2+)
GK
Glycerol-3-phosphate + ADP
Glycerol-3-phosphate + O2+ H2O phosphate + H2O2
GPO
Dihydroxyacetone
Besides the interest of the food industry in the measurement of glycerol, the determination of this compound is also one of the necessary steps in triglyceride determinations. Commonly, a spectrophotometric method involving sequential enzymes coupled to NADH detection at 340nm is used. However, to obtain a cheap, reliable and easy-to-use method, immobilized enzymes associated with a transducer could be used, as in the biosensor developed by Merchie et al.27 composed of glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate oxidase immobilized together in nylon membranes, monitored by amperometric detection of H2O2. The enzyme glycerol-3-phosphate oxidase plays a significant role in metabolism research since its main glycerol-3-phosphate is an important metabolite at the branch point of some pathways (lipid biosynthesis and glycolysis). Therefore, the development of a fast, reproducible and reliable procedure for the analytical determination of this compound became quite useful in bioanalytical laboratories or during its biotechnological production in industry. 20,23,41 Moreover, dihydroxyacetone phosphate, also obtained via several chemical compounds and biocatalytic routes, such as the metabolization of glycerol, a product of triacylglycerol degradation, is an important substrate for the enzymatic synthesis of saccharides. A purely enzymatic route, with the stable intermediate L-glycerol3-phosphate, seems to be the best approach to synthesize this compound in high quality, without the presence of by-products that are hard to separate and highly to aldolases. 14,23
PEROXIDASE Main characteristics Peroxidase is an enzyme (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase; POD) belonging to the class of the oxidoreductases, widely found in living organisms (Table 4), which has multiple physiological roles. It is a hemeprotein (prosthetic group: ferriprotoporphyrin III) that can decompose hydrogen peroxide (main substrate; acceptor) in the presence of a hydrogen donor (reducing agent), according to the following reaction:47 POD
donor + H2O2
oxidized donor + 2H2O
The hydrogen donors can be: phenols (p-cresol, guaiacol, resorcinol), aromatic amines (aniline, benzidine, o-dianisidine) or other organic compounds, substrates that, on the while, are carcinogenic, mutagenic or extremely toxic and that, according to their identity, can influence the products resulting from this oxidation. Thus, a specific donor is preferentially used for a given purpose; for instance, guaiacol in processes that involve thermal treatment. 11,38,47,49 Applications Peroxidase has been widely used and investigated for analytical purposes and, with the development of bioengineering and biotechnology, it has attracted increasing interest for fast and reliable techniques for monitoring substrate concentrations, metabolites and peroxidase inhibitors, as well as for the control of bioreactors. In this connection, several studies have been published that use these methods (colorimetry, chemiluminescence, fluorescence, spectrophotomery and amperometric measurements).49 In one of these articles, Vojinovic et al.49 described the optimization of a coupled system: PSA (phenol-4-
Table 4 - Some sources of Peroxidase. Organism Armoracia rusticana (Horseradish- root)
References Kenten & Mann
19
; Azevedo et al. 2; Sakuyama
et al. 34; Zhong et al. 52; Sariri et al. 36 Carica papaya (Papaya)
Silva et al. 37
Daucus carota (Carrot Chantenay)
Soysal & Söylemez 38
Dioscorea rotundata (White yam)
Chilaka et al. 5
Ipomoea batatas (Sweet potato)
Neves & Lourenço, 1985 30
Malus communis (Wild Apple - peel / pulp)
Valderrama et al. 46
Roystonea regia (Royal palm tree - leaves)
Sakharov et al. 33
105
sulfonicacid)/ 4-AAP (4-aminoantipyrine) /HRP (horseradish/peroxidase), whose reaction is given below. This study discusses the concentrations of PSA and 4-AAP, buffer type and optimal pH, as well as the activity and apparent stability of HRP and the color intensity developed. The possible use of this system in a FIA system to determine hydrogen peroxide concentration and, coupled with oxidases, to monitor the consumption of nutrients (glucose, galactose, lactate, etc.) and metabolites (ethanol) formed during fermentation, were also tested. Enzyme kinetic tests of the redox reactions, using four different reducing substrates of peroxidase (phenol, chlorophenol, p-cresol and PSA), led the authors to conclude that PSA has better solubility, does not produce a precipitate in reaction with hydrogen peroxide and has lower toxicity than the other compounds, besides providing a stable flow injection analysis, with a linear response up to 8.8 mM H2O2, and good sensitivity and reproducibility. POD
donor (phenol + 4-AAP) + H2O2
oxidized donor + 2H2O
Peroxidase has frequently been used as an indicator of effectiveness in the blanching treatment of fruits and vegetables, due to its characteristic being a highly thermostable enzyme. Thus, the loss of activity of the peroxidase in a blanched food product indicates a corresponding loss of activity of other degrading enzymes. However, the occurrence of residual levels of peroxidase can be tolerated in many vegetables, having no adverse effects on the quality of the product.11 It is known that thermal treatments (high temperature for short time, HTST; pasteurization and commercial sterilization) used commercially in the process of extraction of fruits and vegetables are not effective for irreversible inactivation of peroxidase. 46,52 For examples, Valderrama et al.46 studied the behavior of the enzymatic activity of the peroxidase originating from the peel and pulp of Gala and Fuji apples (Brazilian varieties) when submitted to raised temperatures (60, 65, 70 and 75oC) for various periods of time (0, 1, 2, 3, 4, 6, 8, and 10 min). They confirmed their previous suspicion, of a large concentration of the enzyme in the peels of both apples and little inactivation of the enzyme (maximum inactivation of 85% for extracts of the pulp of Gala apple and of the peels of Fuji apple), even after 10 minutes of treatment at 75oC. Other authors, such as Weber et al.50 and Sakharov et al.33 have demonstrated similar effects during the study of the severity of hydrothermal treatments in steam at atmospheric pressure, applied to the peroxidase of products derived from oat (residual activity of 10%: 25 min at 60oC and 29.14 min at 100 oC) and in the study of the thermal stability of peroxidase from leaves of the royal palm tree (Roystonea regia; catalytic activity was preserved after 1 hour of incubation at temperatures above 70ºC and pH 8.0, the pH of maximum stability, only decreasing at 90ºC). 106
In 2005, Soysal & Söylemez 38 studied the activity and kinetics of carrot peroxidase with several hydrogen donors (pyrogallol, guaiacol and o-dianisidine), as well as testing thermal inactivation of this enzyme by heating the carrots and by irradiation with microwaves. The authors concluded that o-dianisidine was the substrate with which the enzyme showed most affinity for hydrogen peroxide and that the microwave treatment (70-700W) was superior to thermal treatment (at 35 - 75oC) when processing carrots, in terms of the period of time necessary for the inactivation of the enzyme and the amount of vitamin C retained in each process. The peroxidase, as well as polyphenol oxidase (PPO), can contribute to the emergence of extraneous flavors in the processed product. During the period of ripening of fruits, amount of soluble the peroxidase increases46, so that, an increase of its activity is observed after the climacteric phase; this was demonstrated in papayas by Silva et al. 37 Peroxidase has served as a parameter of metabolic activity during vegetable growth, because it participates in a great number of oxidative and biodegradation reactions, such as: color change, degradation of chlorophyll or auxins, oxidation of phenols, oxidation of the indole acetic acid, lignin biosynthesis, etc. Many of these modifications, influenced by the environment, irrigation, soil composition and, above all, care during and post harvest, may provoke an adverse consumer response, due to alterations in the appearance, taste, and aroma of the product that could be associated with a degradation of the quality of the food, with respect to its flavor, color, texture and nutritional value. Therefore, the control of peroxidase activity is of the greatest importance to food technology, this being one of the enzymes (alongside PPO) responsible for the darkening of fruits, vegetables and their processed products. 5,33,44,46,47 Chilaka et al. 5 studied the relationship of the inactivation of POD and PPO to the discoloration of processed yam. They incubated an enzymatic extract from yam at 30, 40, 50, 60, 70 and 80oC in sodium acetate buffer (pH 5.4) for various intervals of time. The results demonstrated the completely inactivation of PPO after 30min of incubation at 70oC and the presence of POD after 60min at 80oC; the latter was inactivated only after 2 hours. Even then, POD activity was regenerated 36 hours after the thermal inactivation treatment. This is a common observation among plants that contain peroxidase and it has been associated with quality deterioration of food during storage, as well as color changes during and after the processing. According to the authors, this problem can be solved by adding thiourea. However, Antoniolli et al.1 did not observe the same effect in pineapples that had been processed minimally in CaCl2. They reported a linear decrease until the ninth day in the peroxidase activity. The authors mentioned that the treatment with CaCl2 eventually had a negative effect on the coloration of the pineapple pulp, causing it to darken, with the elapsing of time, relative to the control. The increasing demand by consumers for minimally processed food of high quality has stimulated the
development of several techniques for preservation of food without thermal treatment, among which the pulsed electric field (PEF) stands out as one of the most promising technical innovations for the maintenance of better flavor, color and nutritional quality. 52 Zhong et al.52 used horseradish peroxidase, widely studied structurally and biochemically, as a model for the investigation of the inactivation of peroxidase by conformational change induced by PEF, applied to a buffered enzyme solution at 40oC. Although peroxidase is one of the enzymes responsible for the loss of quality of food, as it leads to the production of brown polymers (melanins), it is important to point out that the oxidative degradation of phenolic compounds in some products, such as tea, raisins, prunes and palm tree, is a welcome event that intensifies the brownblack coloration characteristic of these products. 44 Recentlly, tea became one of the most consumed beverages in the world, due to evidence of a correlation between its consumption and the prevention of certain diseases (cardiovascular, degenerative and of autoimmune). Thus, Mello et al.25,26 investigated the antioxidant potential of this kind of product, with reference to the phenolic components, by developing a biosensor of peroxidase immobilized on silica-titanium, which eliminated the need for previous treatment of the sample. The readings from this biosensor demonstrated good correlation among types of tea and phenolic components. The new instrument was easy to manipulate provided selective response and optimal analytical characteristics, which may be attributed to the efficient immobilization of the enzymes, and allowed rapid estimation of the antioxidant capacity of plant extracts. As general rule, the prevention of enzymatic browning can be only achieved by a combination of different treatments: refrigeration, modified atmosphere, high temperature and pressure, irradiation, microwaves, sonication, employment of enzyme inhibitors, etc. Moreover, it is clear that enzymatic darkening will be hand to prevent, if one considers the range of factors that act on that process, such as: care during harvest, transport and storage post harvest; enzymes in the plants (phenylalanine ammonia-lyase - PAL, peroxidase - POD, polyphenol oxidase - PPO); endogenous phenolic content (substrates for POD and PPO), etc., besides the high cost of the instrumentation necessary and the possible adverse and undesirable effects that tratments can have on food. It is believed that the best alternative would be the employment of genetic engineering, which would permit the regulation of these enzymes. 44 Finally, it is worth mentioning that horseradish peroxidase has been recommended for the removal of phenols and aromatic amines from aqueous solutions, as well as to decolorize phenolic industrial effluents, since this enzyme oxidizes numerous phenols in the presence of hydrogen peroxide, generating the corresponding phenoxy radicals and forming substances that are much less soluble in water than the original substrate which, after precipitation, can be separated from the solution by filtration or flocculation.
Therefore, Thong et al.43, in order to understand the kinetics of this reaction, studied the removal of phenols and chlorophenols by peroxidase from residual water. The effects of the hydrogen peroxide concentration and the substrate (phenol, 3-chlorophenol, 4-chlorophenol) on the removal of pollutants were also discussed, showing the existence of an optimal concentration of peroxide (0.6mM for 4chlorophenol, for instance), above which the process could suffer a small inhibition. 43
FINAL CONSIDERATIONS The enzymes are concial tool for the progress of biotecnology. Among the oxidoreductases, two enzymes stand out: glycerol 3-phosphate oxidase and peroxidase. The first was discovered recently and has been used mainly in the quantification of metabolites and enzymes in various materials, whereas, the second is found throughout nature and possesses several functions and technological applications.
CAMARGO, L. A.; PERES, M. F. S.; GATTÁS, E. A. L. Oxidorredutases para bioanálises e controle de alimentos. Alim. Nutr., Araraquara, v. , n. , p. , 2006.
RESUMO: A maior parte dos métodos enzimáticos serve para a regulamentação de alimentos em diversos países. O alimento é uma amostra complexa e de difícil análise. As amostras de alimentos analisadas através de metodologias enzimáticas podem se apresentar de diferentes formas, tais como: líquidos límpidos e turvos, sem ou com cor, neutro ou ácido, pastas e sólidos. O sistema enzimático compreendido por oxidase/peroxidase pode ser utilizado para análises especificamente. Nestas reações hidrogênio é transferido do substrato e ocorre a formação de peróxido de hidrogênio como intermediário. Peróxido de hidrogênio reage com um reagente de cor na presença de peroxidase e forma uma substância corada medida em espectro visível. Com base nesta classe enzimática, fez-se, neste artigo, um estudo de revisão a respeito de 2 enzimas: peroxidase (PO) e glicerol-3-fosfato oxidase (GPO), ressaltando suas principais fontes de obtenção, características e aplicações. PALAVRAS-CHAVE: Enzimas; aplicações; glicerol-3fosfato oxidase; aplicações peroxidase.
REFERENCES
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