Lactoperoxidase

In the preservation of raw milk, the antimicrobial lactoperoxidase (LP) system present in milk is of interest. This LP system offers an alternative, especially in countries where it is not possible to cool the milk to protect it from spoilage. 

The system consists of LP (EC 1.11.1.7) and the substrates thiocyanate and H2O2. The enzyme, a glycoprotein (carbohydrate content 10%), consists of 612 amino acid residues (Mr 78,000, IP 9.6) and Fe-protoporphyrin IX, which as the prosthetic group carries out the catalysis, as described in 2.3.2.2. Thiocyanate takes part in this reaction process as the electron donor AH. Lactoperoxidase is one of the heat stable enzymes of milk, especially when the structure is fixed by Ca ions. It is still active after 30 min at 63 °C (neutral pH) and after 15 s at 72 °C, but it is inactive after only 2.5 s at 80 °C. Acidification (pH 5.3) accelerates the inactivation by liberating the Ca ions. After 

The thiocyanate concentration in milk depends on the feed, e. g., on the occurrence of glucosinolates (cf. 17.1.2.9.3). H2O2 is not a component of milk, but is supplied by bacteria, e. g., lactic acid bacteria. 

Hypothiocyanite is the main product formed from the hydrogen donor thiocyanate in LP catalysis. 

This compound is a bactericidal agent because it can oxidize the SH groups of structure-forming proteins and enzymes in bacteria. The LP system is used to prolong the shelf life of raw milk and pasteurized milk. H2O2 is produced here by the addition of glucose/glucose oxidase (cf. 2.7.2.1.1) and the concentration of thiocyanate is increased by addition. 

activation of the indigenous enzyme for cold pasteurization of milk or protection of the mammary gland against mastitis and 

addition of isolated LPO to calf or piglet milk replacers to protect against enteritis, especially when the use of antibiotics in animal feed is not permitted. 

which is positively charged at neutral pH, can be isolated from milk or whey by ion-exchange chromatography which has been scaled up for industrial application. These methods isolate LPO together with lactotrans-ferrin (Lf) which is also cationic at neutral pH. LPO and Lf can be resolved by chromatography on CM-Toyopearl or by hydrophobic interaction chromatography on Butyl Toyopearl 650 M (see Fox and Mulvihill, 1992). 

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Fonteh F A, Grandison A S and Lewis M J (2002), Variations of lactoperoxidase activity and thiocyanate content in cows and goats milk throughout lactation , Journal of Dairy Research, 69, 401 -09. 

Seifu E, Buys E M and Donkin E F (2005), Significance of the lactoperoxidase system in the dairy industry and its potential applications a review , Trends in Food Science and Technology, 16, 137-154. 

Figure 12.6 The immobilized glucose oxidase/lactoperoxidase system radioiodinates proteins through the intermediate formation of hydrogen peroxide from the oxidation of glucose. H2O2 then reacts with iodide anions to form reactive iodine (I2). This efficiently drives the formation of the highly reactive H2OI+ species that is capable of iodinating tyrosine or histidine residues (see Figure 12.2).

Ford, D.J., Radin, R., and Pesce, A.J. (1978) Characterization of glutaraldehyde coupled alkaline phosphatase-antibody and lactoperoxidase-antibody conjugates. Immunochemistry 15, 237. 

It has been proposed [91] that nitric dioxide radical formation during the oxidation of nitrite by HRP or lactoperoxidase (LPO) can contribute to tyrosine nitration and be involved in cell and tissue injuries. This proposal was supported in the later work [92] where it has been shown that N02 formed in peroxide-catalyzed reactions is able to enter cells and induce tyrosyl nitration. Reszka et al. [93] demonstrated that N02 mediated the oxidation of biological electron donors and antioxidants (NADH, NADPH, cysteine, glutathione, ascorbate, and Trolox C) catalyzed by lactoperoxidase in the presence of nitrite. 

Nitric oxide and nitrite react with other peroxidase enzymes such as horseradish peroxidase (HRP) (138a,139), lactoperoxidase (138a) and eosinophil peroxidase (140) similarly. The rate constants for reaction of NO with compounds I and II in HRP were found to be 7.0 x 105M 1s 1 and 1.3 x 106M 1s 1, respectively (139). Catalytic consumption of NO as measured by an NO sensitive electrode in the presence of HRP compounds I and II is shown in Fig. 5 where accelerated consumption of NO is achieved even in deoxygenated solutions (140). 

Peroxidases have also been utilized for preparative-scale oxidations of aromatic hydrocarbons. Procedures have been optimized for hydroxylation of l-tyrosine, D-(-)-p-hydroxyphenylglycine, and L-phenylalanine by oxygen, di-hydroxyfumaric acid, and horseradish peroxidase (89). Lactoperoxidase from bovine milk and yeast cytochrome c peroxidase will also catalyze such hydroxylation reactions (89). 

Gorlewska-Roberts KM, Teitel CH, Lay JO, Jr., et al. Lactoperoxidase-catalyzed activation of carcinogenic aromatic and heterocyclic amines. Chem Res Toxicol 2004 17(12) 1659-1966. 

S. Nakamura, K. Yokota, and I. Yamazaki, Sustained oscillations in a lactoperoxidase, NADPH... 

CPO has been applied almost exclusively as biocatalyst for the hydroxyhalo-genation. Recently, lactoperoxidase-catalyzed bromination of laurediols was reported [141,142]. In this reaction, cyclic ethers were unspecifically formed by intramolecular trapping of the carbocation with hydroxy groups (Eq. 8). 

Pruitt KM, Tenovuo JO (1985) The lactoperoxidase system. Chemistry and biological significance. Marcel Dekker, New York Schonbein CF (1856) Verh Nat Ges Basel 1 467 WiUstatter R, StoU A (1919) Ann Chem 416 21... 

HORSERADISH PEROXIDASE LACTOPEROXIDASE LIGNAN PEROXIDASE LYSYL OXIDASE MANGANESE PEROXIDASE MYELOPEROXIDASE OVOPEROXIDASE PEROXIDASE PYRUVATE OXIDASE XANTHINE OXIDASE Hydrogen selenide,... 

Since hemoproteins such as lactoperoxidase and catalase are inhibited more rapidly than the sulfhydryl oxidation occurs, it is unlikely that the rapid activation of guanylate cyclase occurs by sulfhydryl oxidation [132]. Prolonged incubation of the papain or dehydrogenase enzymes with substrate and nitroprusside yielded a turbidity which indicated denaturation of the enzyme to an insoluble form, possibly by the formation of disulfide bridges via the dimerization of thiyl radicals [132]. 

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