Peroxidase (E.C.126.96.36.199) is an oxidoreductase enzyme of oxidation that catalyzes various electron donor substrates with the loss of H2O2 of aromatic compounds such as phenols hydroquinones and hydroquidniod amines . In addition, peroxidase is a very important enzymes in the industry, cosmetics and medicals.
Peroxidase enzyme can oxidize phenolic compounds in plants under a condition with hydrogen peroxide to make the fruits or vegetables change in their flavor and taste. The functioning process of peroxidase enzyme can be an indicator of ripeness and deterioration of the fruits, but this enzyme can be reactivated again during storage.
The reaction can be shown at below.
1. Oxidative dehydrogenation
2SH + H2O2 → 2S• + 2 H2O
2. Oxidative halogenation
SH + H2O2 + H(+) + X(-) → SX + 2H2O
X = Cl, Br, I
3. H2O2 dismutation
2H2O2 → 2H2O + O2
4. Oxygen-transfer reaction
SH + H2O2 → SOH + H2O
Peroxidase enzyme is an enzyme that can tolerate heat well and serve as a catalyst with four conditions characterized by types of substrates as follows:
1. Peroxidation is the main reaction of peroxidase in vitro with phenolic substrates, such as para-cresol, guaiacol, risorsinal and aniline.
H2O2 + 2AH2 = 2H2O + polymerized product (HAAH)
2. Oxidation is a reaction that occurs with the presence of oxygen molecules and substrates, such as dihydroxyfumaric acid, ascorbic acid and hydroquinone acid.
3. Catalysis a reaction that occurs in the absence of hydrogen donor (AH2) and peroxidase enzyme can act as catalase enzyme by changing H2O2 to H2O and O2, but this reaction is at least 1000 times slower that peroxidation and oxidation.
4. Hydroxylation is a reaction that occurs in the presence of hydrogen donor (AH2), such as dihydroxyfumaric acid and O2 and peroxidase enzyme can accelerate the hydroxylation to various aromatic compounds, such as para-cresol, tyrosine, phenylalanine, benzoic acid and salicylic acid.
Structure of Peroxidase Enzyme
Peroxidase enzyme (POD) is an enzyme generally found in higher-class plants as well as in animals and microorganisms. It is classified as belonging to the group of oxidoreductase enzyme and contains iron as a component in its molecular structure.
Peroxidase enzyme is a hemoprotein with a heme or belongs to the prosthetic group with strongly binding with the enzyme by covalent bonding. It is a tetrapyrrole-ring enzyme and is classified as flat protoporphyrin IX. The structure of this ring is composed of one atom of iron. Generally, the iron has six positions that can have a bond. Four positions of the iron are bonded to a nitrogen atom of the porphyrin ring at the fifth and sixth bonds perpendicular to the plane of the heme. The fifth position is bonded to the nitrogen atom of histidine amino of protein and peroxidase enzyme can exhibit various activities. By exchanging different groups at the sixth position of the iron, peroxidase enzyme can use H2O2 as electron acceptor and oxidize compounds that provide electrons to become a product according to the following equation:
2HA + H2O2 = 2H2O + 2A (A providing colors)
Reaction mechanism of peroxidase enzyme is as below:
E + H2O2 = compound I + H2O ………. (1)
Compound I + AH2 = compound II + AH ………. (2)
Compound II+ AH2 = E + AH + H2O .………. (3)
E is the ferric enzyme in the resting phase.
AH2 is the substrate in the reduced condition.
AH is the oxidized substrate.
The name of peroxidase enzyme depends on the substrates used; for example, if guaiacol in phenolic compounds is used as substrate, peroxidase enzyme is referred to as guaiacol peroxidase. Peroxidase enzyme can be found in both lower-class organisms, such as fungi, protozoa and one-celled algae, and high-class plants and mammals. Peroxidase enzyme can be divided into three groups based on the amino acid sequence and ability to capture metal ions, including intracellular peroxidase, extracellular fungal peroxidase, and secretory peroxidase from higher-class plants (Welinder, 1992)(1).
Resource of Peroxidase
Peroxidase has been wildly found in plants, animals, bacteria and fungi.
Peroxidase Enzyme in Plants
Peroxidase enzyme can be found in different parts of plants, such as seeds, roots, stems, barks, leaves and fruits, and in different parts of cells, such as cell walls, cytosol and chloroplast, which may be in the form of an independent solution or may bind to the cell walls with ionic or covalent bonding that is released from cells or within cells. These isozymes of peroxidase enzyme are very specific to different substrates, such as ascorbate pyrogallol o-dianisidine and guaiacol.
Plants will produce peroxidase enzyme in several isozymes for various applications, such as repairing cell walls by speeding up the process of synthesizing a cell wall component, i.e. lignin suberin, to strengthen the wood. Also, the production of this lignin can inhibit the growth of hypha.
Shivakumar et al. (2003)(2) categorized peroxidase enzyme in plants into two categories, including peroxidase enzyme in cell walls that is involved with an oxidative burst reaction leading to hypersensitive cell death and peroxidase enzyme in cytosol that is involved with an oxidative cross-linking reaction in the process of producing lignin in the cells.
Peroxidase enzyme in cytosol will have an increased agility after incubation with the fungus that causes downy mildew. This increased agility is related to lignin and it can thus stop the infection in the cells of plants. Therefore, this process makes the plant resistant against plant diseases. In addition, peroxidase enzyme is also involved in many processes, such as auxin catabolism, disease prevention, and production of lignin and suberin. When considering the pI value of peroxidase enzyme, it can be classified into two groups, i.e. anionic and cationic. Isozymes of peroxidase enzyme in cationic group are related to the process of accelerating the decomposition of indole-3-acetic acid (IAA) in the presence of oxygen, which affects the growth of plant cells.
Peroxidase enzyme that is involved in creating lignin in the cell walls serves to speed up the polymerization of phenolic compounds with hydroxycinnamyl alcohol as its initial substance. Then, they will bind together into phenylpropanoid and produce lignin at the time when the plants are responding to the disease and accumulating the lignin and phenolic compounds, which is related to disease resistance of plants.
An average of 82% primary recovery of the royal palm tree peroxidase was achieved after precipitation for leaf color compound elimination. Further purification by ion-exchange, gel filtration and affinity chromatography, a 12.5% yield and 73-folds of purified enzyme was obtained.
The barley leaf peroxidase has been extracted and precipitated using acetone and ammonium sulfate which achieved 72% and 52% primary recovery.
As for precipitation by ion-exchange and gel filtration chromatography, 39% yield and 1,200 purification folds was achieved, respectively. Purification of Opuntia ficus indica fruit using ammonium precipitation, DEAE cellulose column and hydroxyapatite column, sequentially, achieved 82-folds with 47% enzyme recovery. It showed a single band of 58,000 ± 2,000(M.W.) in SDS-PAGE.
Applications of peroxidase
1. Medical Properties
Peroxidase from horseradish root tubers is commonly used at commercial level. The enzyme is also widely used in clinical biochemistry as a labeling enzyme, i.e., peroxidase enzyme electrode for determination of glucose and urate in serum. In case of diabetic patient, it is used to detect disease states. Moreover, peroxidase has been used in the biotransformation for the synthesis of drugs and chemicals.
2. Treatment of Wastewater
In biotechnology, peroxidase is an interesting enzyme because of its biochemical properties. It has broad substrate specificity and its activity remains over a wide range of pH, temperature and substrate concentration.
The reaction of enzyme occurs under mild condition without having to use strong chemical that cause the environmental impact. The enzyme is mostly used in the industrial manufacture for wastewater degradation.
In the paper industry, the harmful phenolic compound accumulated in the drainage from the processing is considered biohazard. Peroxidase acts as the polymerization catalysts of phenol to reduce its harmfulness. After the polymerization reaction, phenol turns into polyphenol which could be easily eliminated. Moreover, it can degrade the industrially important dye in the fiber industry effectively. In addition, peroxidase is used as an electrode biosensor to determine organomercurry compound and nitric oxide in natural water.