CHEMISTRY OF MILK

Module 4. Enzymes of milk

Lesson 16
MILK ENZYMES ITS SOURCES AND SIGNIFICANCE-PART II

16.1 Introduction

Among the various milk enzymes oxidases play a very important role especially the reactions involving oxidation and reduction. These oxidases influence these reactions influence the OR potential of the milk. Knowledge about these enzymes will help in proper utilization of the milk and its processing.

16.2 Oxidases

Oxidases are a group of enzymes which catalyzes an oxidation-reduction reaction involving molecular oxygen (O₂) as the electron acceptor. In these reactions, oxygen is reduced to water (H₂O) or hydrogen peroxide (H₂O₂). Some of the enzymes which are included in this group are helpful in increasing the shelf life of the raw milk utilizing the activity of an oxidase for the oxidation of thiocynate. Presence of some of the oxidases like catalase will help in the detection of diseased condition of the udder.

16.2.1 Xanthine oxidase (XO, EC 1.2.3.2)

This enzyme is very prominent in bovine milk. Most of it is associated with the fat globule membrane. Because of its relatively high XO content, milk is a source of choice for isolating this enzyme for investigation. Xanthineoxidase is much less prominent in milks of most other species; goat's milk has only about one-tenth the activity of cow's milk. The activity of XO in cow's milk depends to some extent on the Molybdenum content of the feed consumed.

The molecular weight of xanthine oxidase is about 2,83,000; it is a dimer of two identical subunits, each carrying one flavin-adenine dinucleotide,one Mo, four Fe, and four acid-labile Satoms. The specific activity of the pure enzyme is 5 µmol.min^-1 ·mg^-I.It can oxidize 12 moles of xanthine per mole per second (assuming two active centers per dimer). Although hypoxanthine and xanthine are its normal substrates in the pathway of purine metabolism, it catalyzes oxidation of many other substrates, including various aldehydes, Under some conditions XO catalyzes a two-electron reduction of O₂ to H₂O₂;but at high pH, high O₂₂^- is favoured. concentration, and low xanthine concentration, the one-electron reduction to superoxide ion, O

As isolated from milk, XO utilizes molecular O₂ as an electron acceptor, but if some of its disulfides are reduced with dithiothreitol it becomes a NAD⁺ -dependent dehydrogenase. Reoxidizing the thiols to disulfides with the milk enzyme sulfhydryl oxidaserestores the specificity of the enzyme for O₂ as electron acceptor.

The activity of XO in fresh milk is increased about fourfold by storing at 4°C, by heating at 70°C for 5 min, by homogenization, or by incubation with commercial proteinase or lipase preparations. Such treatments also transfer much of the enzyme from fat globules to plasma.

16.2.2 Sulfhydryl oxidase

(EC number not assigned): This enzyme is present in bovine milk , catalyzesoxidation of thiols to disulfides using molecular O₂ as the electron acceptor. It is an aerobic oxidase, reducing the O₂ to H₂O₂ratherthan to H₂O.

2 RSH+O₂ → RSSR + H₂O₂

(It is completely different from thiol oxidase (EC 1.8.3.2) is not known to occur in milk, which forms H₂O in the oxidation) Sulfhydryl oxidase ca­talyzes oxidation of thiols in both small compounds and proteins. It is a large aggregate of subunits of MW 89,000. It contains about1.1 % carbohy­drate residues by weight and 0.5 atom Fe per subunit. At optimal pH (7.0) and temperature (35°C) its K_m for glutathione as substrateis 90 µM. Prep­arations with specific activities of more than 50 µmol min^-1 mg ^{- I} have been obtained. Milk may contain about 3 mg of this enzyme per liter. Sulfhydryl oxidase is closely associated with γ-glutamyl transferase (EC 2.3.2.2) in skim milk, and it has been suggested that the two activities reside in the same molecule. Such suggestions have been proved wrong, however, because the two activities can be separated. Sulfhydryl oxidaseim mobilized on glass beads has been used to oxidize thiols in UHT milk in an attempt to reduce cooked flavour.

16.2.3 Catalase (EC1.11.1.6)

This enzyme catalyzes the decomposition of H₂O₂ to H₂O and O₂. It is found in many tissues and is particularly prominent in liver, eryth­rocytes, and kidneys of animals. In milk its activity parallels leukocyte count and is higher in mastitic milk and colostrum than in normal milk. It increases with the multiplication of bacteria. Cells associate with the fat globules:therefore, catalase accumulates in the cream layer. Crystalline catalases have been prepared from several sources, but because little is present in milk it has not been fully purified therefrom. Milk catalase has a molecular weight of about 210,000, its isoelectric pH ≈ 5.5, and it contains heme iron. Catalase activities on the order of 300 µmol per min per liter have been reported for milk.

16.2.4 Lactoperoxidase (LP, EC 1.11.1.7)

This enzyme catalyzes oxidation by H₂O₂ of a long list of electron-donor compounds,including aromatic amines, phenols, ar­omatic acids, leuko dyes, tyrosine and tryptophan, ascorbate, iodide, nitrite, and thiocyanate. LP may amount to as much as 1% of the total serum proteins of milk (i.e., 60 mg·kg^-I). Its activity in milk increases with advancing lactation to a maximum about 40 days postpartum and thereafter declines somewhat. It is a glycoprotein (MW 77,500)containing 20-26 hexosamine residues (nosialic acid) and one heme per mole. Whether it consists of one or two polypeptide chains has not been settled. LP catalyzes oxidation of thiocyanate (SCN^-) to a product hypothiocyanite (OSCN^-) that inhibits certain bacteria. Thiocyanate is a natural constituent of milk and H₂O₂ is produced by some bacteria themselves. Thus, in milk such bacteria exhibit self inhibition.

16.2.5 Superoxide dismutase (SOD, EC 1.15.1.1)

This enzyme catalyzesthe dismutation of superoxide ion O₂ to H₂O₂and O2. The enzyme consists of two identical subunits of MW 16,000, each containing one Cu and one Zn per mole. The primary structure of SOD from bovine erythrocytes is known completely. It has one free thiol and one disulfide bond.It functions as a non covalently linked dimer, and its specific activity is3300 µmol/ min/ mg. Bovine milk serum contains a superoxide dismutase that is similar if indeed notidentical to that of the erythrocytes. Estimated content in milk range from0.15-2.4 mg/ kg. It is a rather heat-stable enzyme; the activity is not de­creasedby heating milk at 63°C for 30 min. It may well be an important antioxidant protecting milk constituents from oxidation by superoxide ion generated by oxidations catalyzed by xanthine oxidase and lactoperoxidase as well as by riboflavin-sensitized photore actions.

16.3 Transferases

The only transferase discussed here is the galactosyl transferase component of lactose synthase (EC 2.4.1.22). In the absence of α-lactalbumin this en­zyme transfers a galactosyl residue from UDP-galactose to an N-acetyl­glucosamine residue either free or in a protein-bound oligomer. In performing this function the enzyme is sometimes designated glycoprotein β-D-galac­tosyl-transferase (EC 2.4.1.38). α-lactalbumin modifies the transferase so that the K_m for transfer of a gal­actosyl group to D-glucose is much reduced, and a β-1,4-glycosidic linkage is formed. The transferase is found in membranes of Golgi vesicles and there performs the synthesis of lactose as α-lactalbumin transiently combines with it. It enters milk with golgi membrane fragments. Its concentration in bovine milk is approximately 3-4mg/liter. The specific activity of the purified enzyme is 14 µmol/min/mg proteins at 37°C and neutral pH. As isolated from milk, the transferase has a molecular weight of about 42,000. The transferase contains 12-13% carbohydrate consisting of about 8% neutral sugars, 1% glucosa­mine, 1% galactosamine, and 2% sialic acid.