An Important Milk Enzyme: Lactoperoxidase

It has been acknowledged since ancient times that milk and dairy products have a vital role in nutrition and contribute considerably to human health. Because of its content, Because of its content, it has many important effects on the life that include immuno‐ globulins, enzymes, hormones, growth factors, antibacterial agents, fat acids, vita‐ mins, and minerals. Approximately 70 indigenous enzymes have been reported in normal bovine milk, including lactoperoxidase. Lactoperoxidase LPO is a natural constituent of milk, saliva, and tears. It also exists in all mammary milk. LPO is an iron heme group basic glycoprotein, with a molecular weight of approximately 78 kDa. The LPO enzyme catalyzes the H 2 O 2 +SCN - → OSCN - + H 2 O reaction. Hydrogen peroxide and hypothiocyanate are indispensable for antimicrobial activity. The biological significance of LPO is involved in the natural host defense system against pathogenic microorganisms. effects.


Peroxidases
Peroxidases (POD: H 2 O 2 -Oxydoreductase E.C.1.11.1.7) are oxidoreductase enzymes, which catalyze the reactive oxygen species generated during metabolism, and are converted into harmless molecules [1]. These exhibit antioxidant characteristics and catalyze the oxidation of organic and inorganic substrates with hydrogen peroxide being the electron acceptor [2]. Those enzymes are present in eukaryotes, prokaryotes, and photosynthetic cells [3,4].
Peroxidases are frequently used in the studies of metabolic reactions, enzymatic functions, protein structures [15] and in clinical diagnoses, microanalytic applications, and the food and drug industry [16,17]. Mammalian POD enzymes are localized in milk, saliva, and tears as lactoperoxidases (LPO) [18] and in leukocytes and platelets as myeloperoxidases [6].
Prosthetic groups of peroxidase are protoheme and are connected to the apoprotein loosely in contrast to many hemoprote [14]. Firstly, protein portion is synthesized in the organisms bearing peroxidases. However, enzymes are not functionally active. The enzyme gets activated by both apoprotein and hem groups [3,19]. The general formula of the reaction catalyzed by peroxidases is shown below [3]. 2 2

ROOH + AH ROH + H O + A ®
The H 2 O 2 formed during the metabolism having oxidizing property must be quickly removed. The catalase and peroxidase enzymes exhibiting antioxidant properties play this role in cells [20]. The amount of hydrogen peroxide in the cells is removed by catalase in peroxisomes. In other parts of the cells, the peroxidase enzymes utilize various aromatic components as the substrate to [21] neutralize H 2 O 2 [22].
The characteristics of peroxidase enzyme that is isolated from milk are similar to animal and human peroxidases [23]. It displays 55, 54, and 45% similarity with myeloperoxidase (MPO), eosinophilperoxidase (EPO), and thyroidperoxidase (TPO), respectively. Following xanthine oxidase, lactoperoxidase is the most common enzyme in milk and it is also commonly present in whey which is the liquid remaining after milk has been curdled and strained. Each lactoperoxidase enzyme contains an iron molecule. The conformation of the protein is stabilized by a chelated calcium ion [24][25][26].
The main function of the LPO enzyme is to catalyze the oxidation of thiocyanate with H 2 O 2 to hypothiocyanite having antimicrobial activity [27,28].
The combined action of these three components, which constitute the 'lactoperoxidase system', was defined by Reiter and coworkers [29][30][31]. The biological significance of the system is protection of the lactating mammary gland and the intestinal tract of newborn infants, thereby providing a natural host defence system against invading microorganisms [23]. The LPO catalyzes the oxidation of some halides (I 2 and Br 2 but not Cl 2 ) to yield the most oxidizable form of I -, that is, I 2 [26].
The LPO-H 2 O 2 -SCNsystem shows bacteriostatic effect and thus prevents bacterial growth and development, whereas the LPO-H 2 O 2 -I 2 system shows bactericidal potency thus killing bacteria. Both SCNand I 2 have shown strong bactericidal effect when present within the system [32,33].

Lactoperoxidase enzyme
Lactoperoxidase (LPO, E.C. 1.11.1.7) is one of the crucial enzymes in milk with oxidoreductase activity. The peroxidase isolated from milk was given the name lactoperoxidase [23] and was the first enzyme reported to be found in milk [34]. The main function of the enzyme is to catalyze the oxidation of molecules in the presence of hydrogen peroxide and to help production of products with a wide antimicrobial activity. Pseudohalogens, thiocyanates, or halogens should function as second substrates for the enzyme to exhibit such antimicrobial effects [23,29].
The LPO system shows a significant protective effect in bovine milk. The activation of the system depends on the concentration of the two reactants, thiocyanate and hydrogen peroxide. In the presence of hydrogen peroxide, the system catalyzes the transformation of thiocyanate into hypothiocyanate, which has an antibacterial nature [35][36][37]. The end products of these compounds are oxidized and hence are safe for human health.
Many studies show that this system destroys several bacterial and fungal strains [38][39][40][41][42]. Lactoperoxidase has a broad antifungal activity [43,44]. Mastitis is a bacterial inflammation in mammals. The effects of different concentrations of thiocyanate-H 2 O 2 medium on several antibacterial and antifungal strains were studied to solve this dairy industry issue [45,46]. They are capable of reducing bacterial growth by damaging the cell membranes and inhibiting activities of several cytoplasmic enzymes.
The LPO enzyme, a glycoprotein consisting of 8-10% carbohydrate, comprises a chain containing 612 amino acids. It consists of a single polypeptide chain of molecular weight approximately 78 kDa [47]. It is a basic protein containing heme as its prosthetic group with an isoelectric pH value of 9.2 [24][25][26]. Furthermore, it is very active in acidic pH [48]. It is fairly voluminous as a LPO molecule [49]. The Ca 2+ ion stabilizes the enzyme. The Ca 2+ ion disappears under pH 5.0 and thus reduces the stability of the enzyme [26].
The biocidal activity of the LPO results from the products of the chemical reactions that it catalyzes. Hypothiocyanate, which is the main product of the reaction, interacts with the thiol groups of various proteins, which is critical for the survival of pathogens. The impact of LPO on bacteria results from the oxidation of sulfhydryl. The oxidation of the -SH groups makes the bacterial cytoplasmic membrane lose its ability to transport glucose, potassium ions, amino acids, and peptides [45].
The biological significance of this enzyme results from the fact that it has a natural protection system against the invasion of microorganisms. Besides this antiviral effect, it is report-ed that it protects animal cells against various damages and peroxidative effects [23,[29][30][31]. Lactoperoxidase is a significant agent of the defense system against pathogen microorganisms from the digestive system of neonatal babiess. The LPO enzyme functions as a natural compound of the non-immune biological defense system of mammals and it catalyzes the oxidation of the thiocyanate ion into the antibacterial hypothiocyanate [52].
Although peroxidases have similar catalytic mechanisms, they are distinguished for their ability to oxidize halides and pseudohalides. For example, only myeloperoxidase is able to oxidize bromine, iodine, and chlorine at neutral pH. Lactoperoxidase, on the contrary, can only oxidize iodine and thiocyanate but it either cannot or can hardly oxidize brome under the same conditions (Figure 1) [53]. In the first step of this mechanism, the peroxidase enzyme reacts with one equivalent of the peroxide to form an Fe (IV)-containing compound I that is the porphyrin cation radical. In the second step, the cation radical takes a proton of the substrate and reduced (substrate)-Fe (IV) form, the substrate becomes radical to lost a proton. The compound II takes a proton from the substrate and return the first reducing form. Also radicalic substrate which are formed with interacting each other are polymerization [54].
The LPO enzyme is covalently bound to the heme group proteins, with the bond occurring between the hydroxyl group of the heme group and the carboxyl group of the protein [14]. Approximately 10% of the molecule is composed of carbohydrates and the molecule contains five main potential glycosylation regions and 15 semi-cysteine residues [11,23,45]. The heme group at the catalytic center is protoporphyrin IX, which is covalently bound to the polypeptide chain along with the disulfide bridge. The iron compound, which is a part of the heme group, constitutes 0.07% of LPO. The calcium ion is tightly bound to the enzyme, which ensures the molecular conformation and structural integrity of the enzyme [55].

Lactoperoxidase system in milk
The activation of the natural antibacterial system has been adopted for protecting raw milk. This system is defined as the lactoperoxidase/thiocyanate/hydrogen peroxide (LPO) system. The antibacterial effect of the system on milk is based on the oxidation of SCN − ions catalyzed by the lactoperoxidase enzyme in the presence of H 2 O 2 . The short-lived components, OSCNions, generated in this oxidation reaction have bacteriostatic effect [56,57]. The system is recommended in developing countries where there are no sufficient cooling facilities for collection of raw milk and their transportation to processing centers [57].

Antibacterial effect of LPO
LPO oxidized the -SH groups of the enzymes in bacteria such as hexokinase and glyceraldehyde-3-phosphate dehydrogenase and tends to lose biological functions of these enzymes. As a result, the bacterial cytoplasmic membranes are damaged structurally, and glucose, purine, pyrimidine, and amino acid uptake as well as protein, DNA, and RNA synthesis are blocked. Thus, bacteria growth and proliferation is prevented (Figure 2) [58].

Lactoperoxidase system and health
Milk and milk products area rich sources of mineral, protein, vitamin, nutritional elements [56,57]. Lactoperoxidase showed antifungal activity in apple juice and salt solution. In vitro findings also show that the LPO/H 2 O 2 /halide system has a strong virucidal activity against HIV-1 [59].
In conditions of iodine deficiency, the level of SCN ions in milk plays a vital role in thyroid function. The studies showed that when milk containing 19 ml/L thiocyanate ion and iodine at 0.1 mg/l concentration is consumed, the thyroid functions of patients with iodine deficiency did not have any negative symptoms [60].

Determination of the lactoperoxidase activity with spectrophotometer
The measurement of the activity is based on the oxidation of 2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) chromogenic substrate by H 2 O 2 and observation of the increase in the absorbance caused by the resultant colored compound at 412 nm and pH: 6.0 [61]. For determination of LPO activity with spectrophotometric assay, 2.8 mL of ABTS (1 mM) and 0.1 mL of H 2 O 2 (3.2 mM) were pipetted in the spectrophotometer tube of 3 mL. Enzyme solution of 0.1 mL was added and the tube was turned upside down before it was placed in the spectrophotometer. The increase in the absorbance was observed against blank at 412 nm for 3 min and recorded every 60 sec. Phosphate tampon (0.1 M) at pH: 6.0 was used as blank, instead of the enzyme, while all other solutions were used at the same rates. The following formula was used to determine the activity:

Unit of enzyme activity
One LPO unit (EU) is defined as the amount of LPO that catalyzes the oxidation of l μmol of substrate (ABTS) per min at 20°C [52].

Purification procedures
To date, several chromatographic methods have been reported about purification and characterization of the LPO enzyme from bovine milk [14]. For example, CM-Cellulose [64], CM-Sephadex ion-exchange chromatography [46,64], Sephadex G-100 gel filtration chroma-tography [6,64], hydrophobic affinity chromatography on Phenyl-Sepharose CL-4B [5], and Toyopearl-SP cation exchange chromatography [64] are among the methods used in the purification of LPO enzyme from bovine milk. LPO was purified in one stage using the affinity technique and sulfanilamide was used as the ligand [65].

Kinetic studies
The K m and V max values are suitable parameters for kinetic studies. K m is a substrate concentration at which half of the enzyme active sites are filled. V max is an expression of the catalytic activity of the enzyme.
To find K m and V max values for LPO, activity was measured at 20°C, 412 nm, at pH 6.0, for five different substrate concentrations. For this purpose, generally 0.2-1.5 mL volume from the stock solution of the substrates was used. The total volume was made up to 2.8 mL with a buffer solution and then 0.1 mL enzyme and 0.1 mL H 2 O 2 were added. The K m and V max values were calculated from the Lineweaver-Burk graph [65,66].

Procedure of enzyme inhibition
The K i and IC 50 values show the inhibitory effect on enzyme. The K i and IC 50 values depend on the inhibitory mechanism. IC 50 is the inhibitor concentration required for 50% inhibition and K i value is the constant.
To investigate the inhibitory effects of some inhibitors on LPO and to determine the IC 50 values, the LPO activity was measured in the presence of five different concentrations of inhibitor. For example, the experimental procedure for inhibitor: A control sample without inhibitor was taken as 100% and an activity-[Inhibitor] plot was drawn. To determine the K i , three different concentrations were used for x inhibitor. ABTS was also used as a substrate at five different concentrations. Lineweaver-Burk plots (1/V-1/[S]) were obtained for inhibitor; the K i and the inhibition type were calculated from these plots [65].

Concentration
LPO is the second most abundant whey enzyme in bovine milk, [31,77] and its concentration is approximately 30 mg/L [23]. The peroxidase activity in cow milk is 20 times richer than in human milk and contains 1.

Application fields
There is a growing interest in the purification of LPO with increasing applications. The LPO system's natural biological functions are preferred against antimicrobial chemicals. Lactoperoxidase has many fields of application. It is widely used especially in milk-processing facilities in the milk industry [79]. LPO is used in milk and cheese to reduce the microflora [23]. The LPO enzyme derived from various animal sources has a significant role in the suppression of bacterial growth and helps bacterial inhibition. Inhibition of bacterial growth by the bovine LPO is attributed to the peroxidase system, which contains H 2 O 2 and thiocyanate [9]. The antimicrobial effect of the LPO system occurs naturally in milk. LPO has a bacteriostatic effect on gram-positive and gram-negative bacteria. The antibacterial studies on the LPO enzyme purified from camel milk show that the LPO-thiocyanate and peroxide system leads to significant inhibition of pathogenic bacteria.
Most popular applications of the system include food production for preservation of raw milk, pasteurized milk, and cheese milk during storage and/or transportation to the processing plants. The system can be used to avoid the suppression of acidity in yoghurt. In the absence of refrigeration, LPO system is preferable [80] and the system can be used to extend the shelflife of pasteurized, raw, and cheese milk [79,81]. This is used for the preservation of emulsions and cosmetics.
The LPO system can ensure an extensive spectrum of antimicrobial properties against bacteria and yeasts when it is composed of LPO, H 2 O 2 , SCN -, and I 2 . Hence, it is preferred in oral care products and cosmetic preservation [82,83]. The system is used against fish pathogenic bacteria in aquaculture with strong bacterial effects.