CHEMISTRY OF MILK

Module 4. Enzymes in milk

Lesson 15
MILK ENZYMES ITS SOURCES AND SIGNIFICANCE-PART I

15.1 Introduction

Activities of 14important enzymes in bovine milk are listed in by way of emphasizing the differences among species, ratios of activities in bovine and human milks are included. Xanthine oxidase, lactoperoxidase, and ribonuclease are especially prominent in bovine milk; lysozyme predominates quantitatively in human milk.

15.2 Hydrolases

The enzymes which are responsible for the hydrolysis of various milk constituents are included in this group. This enzymes play a significant role in dairy industry especially the lipases which are responsible for the development of off flavours in milk and milk products. Similarly some of these hydrolases are useful in the detection of efficiency of milk processing. Enzymes which have the bacteriostatic action are also included in this group of enzymes.

15.2.1 Lipase (EC 3.1.1.34)

The principal lipolytic enzyme of bovine milk is a lipoprotein in the normal function of such enzymes is to liberate fatty acids from lipoproteins and chylomicrons of the blood; the fatty acids are then resorbed by the secretory cells of the mammary gland. A low molecular weight apoprotein cofactor is necessary for the enzyme to attack its acylglycerol substrate when the latter is present as or covered with lipoproteins. Blood serum contains such cofactors, and the lipoprotein lipase is said to be serum-stimulated. In bovine milk the lipoprotein lipase (about 2 mg per liter)is bound largely to casein micelles. It does not attack the glycerides of the fat globules unless the membranes of the latter are damaged (e.g.homogenization) or if lipoproteins containing the apoprotein cofactor are added. The latter effect may arise from leakage of blood serum into milk if the tight junctions between secretory cells are impaired. This enzyme is inactivated at pH 4.6 for one hour. Lipoprotein lipase has been isolated from bovine skim milk using affinity chromatography on Se­pharose to which heparin has been linked covalently. Its molecular weight is about 50,000, and it exists as a non- covalently linked dimer under phys­iological conditions; it contains about 8% carbohydrate. Preparations with activities capable of releasing 300-700 µmol of fatty acids (from a triglyceride emulsion at pH 8.5) per min per mg protein have been achieved.

Cow's colostrum contains little of the lipoprotein lipase but has a different lipase that is not bound to casein, does not bind to heparin-Sepharose, is not activated by blood serum, and is stable at pH 4.6 for 1 h. It disappears after the first few milkings following calving. The two lipases do not exhibit immunological cross-reaction. The colostral lipase probably falls under the classification of a triacylglycerol lipase (EC 3.1.1.3) but does not appear to be homologous to the bile-salt stimulated lipase of human milk. Lipolytic activities of skim milk and colostrum are respectively about 600 and 200 µmol min^-1liter^-1(measured with tributyrin emulsion at pH 8.75, 37°C).

15.2.2 Milk alkaline phosphatase (EC3.1.3.1)

This is a phosphomonoesterase. The scientific name for this enzyme is orthophosphomonoester phosphohydrolase Two major isozymes have been identified, α and βphosphatase, mainly located in the milk plasma and fat globule membrane,respectively. The latter, more abundant, isozyme has been highly purified from bovine milk and found to be a dimer of two identical or very similar subunits each of MW ~ 85,000. It contains about five atoms of zinc per dimeric molecule. Its optimal pH for hydrolysis varies from one phosphate ester to another and with the composition of the medium. Monoesters, such as phosphoserine and β-­glycerophosphate are hydrolyzed maximally near pH 9.0

Milk alkaline phosphatase is used as the method of preference for determining whether the milk has been pasteurized adequately. Alkaline phosphatase by heat bur reversibly reactivated when milk is chilled. Inactivation of alkalinephosphatase by pasteurization is an index of destruction of Mycobacterium tuberculosis. It is possible to determine the inactivation of phosphatase enzyme by easy chemical methods.

15.2.3 Acid phosphomonoesterase (EC 3.1.3.2)

A second phosphatase present in milk has a pHoptimum at about 4.0. It has been assumed to be more similar to the phosphoprotein phosphatase (EC 3.1.3.16) of bovine spleen. It is primarily in the milk plasma. Its concentration is quite low (compared to alkalinephosphatase), though higher in colostrum.

Both the alkaline and the acid phosphatase can release inorganic phos­phatase from caseins and from soluble esters, and this may occur in milk or fractions thereof under appropriate conditions. The acid phosphatase is the more active of the two at the pH of milk. These two enzymes differ greatly in susceptibility to inactivation by heat.

15.2.4 Ribonuclease (RNASE, EC 3.1.27.5)

Its content of mixed milk was found in one study to be 11 mg liter^-I and 25 mg liter^-I in another. RNase has been isolated from bovine milk by various methods; it appears to be identical to bovine pancreatic RNase in amino acid composition and immunological cross-reaction. Bovine pancreatic RNase has been well characterized. It consists of a single polypeptide chain of 124 residues, with MW 13,690. In spite of the low content of RNase in human milk (3mgliter^-1), it has been isolated from that source by adsorption on a cation exchanger. Both RNase and lysozyme are so adsorbed; they can be differentially eluted.

15.2.5 Lysozyme (EC 3.2.1.24)

It is quantitatively an important fraction of the proteins of human milk (400 mg liter^-1). It is a powerful bactericide as it attacks polysaccharides of the bacterial cell wall, causinglysis of the bac­teria. Although the lysozyme content in bovine milk contains only about 0.1 mg. liter^-1 it could be isolated from it. The lysozyme of human milk appears to be identical to that found in other human secretions; it is a polypeptide of 129 residues, with MW 14,602. α-lactalbumin and lysozyme are considered to be descendants of a common ancestor. Human lysozyme and α-lactalbumin, both obtained from milk, have different residues at 81 of 129 positions, Both have four disulfide bridges identically placed.

15.2.6 Plasmin (EC 3.4.21.7)

The principal milk proteinase belongs to the alkaline serine proteinase class; it is probably identical to the plasmin of blood Blood plasminogen (human) is a polypeptide of790 residues; activation involves proteolytic cleavage of the C-terminal 230 residues and sometimes the N-terminal 76 residues as well. Apparently, this enzyme enters milk from blood mostly in the form of its zymogen, plasminogen. In fresh milk only a small proportion is in the active form; milk may contain a factor that slowly activates the plasminogen. The enzyme is associated with the casein micelles. It attacks peptide bonds at the C-terminal side of Arg and Lys residues and thus is trypsin-like. Cleavage of Lys-Xis faster than that of Arg-X. Optimal activity occurs at slightly alkaline pH and 37°C. The milk proteins most susceptible to plasmin are β- and αs_2-caseins.αs₁-casein also is attacked while κ-casein is relatively resistant, and the whey proteins α-lactalbumin and β-lactoglobulin are not affected. Plasmin action on β-caseinis respon­sible for production of γ-caseins and the proteose-peptone fragments. Plasmin fully survives pasteurization and partially resists UHT treatments.Increased activity has even been observed after heating milk at 72°C for 15 s.

This has been attributed to conversion of plasminogen to plasmin, to inactivation of inhibitors, or to enhancing the accessibility of susceptible linkages in the substrate. The action of plasmin may produce serious defects in UHT milk products, such as development of a bitter flavor (caused by hydrophobicpeptides of low molecular weight) and changes in viscosity and appearance.

A second proteinase, with maximal activity at pH 4.0, also occurs in milk. Its molecular weight is 36,000, it is heat-labile(inactivated at 70°C for 10 min), and it is partly inhibited by SH-blockingagents. It cleaves αs₁-casein faster than β- or γ-caseins (at pH 5, 37°C). Its action on these proteins produces peptides that in electrophoretic behavior resemble those produced by chymosin. It is not yet clear how this enzyme should be classified; it may well be an aspartate proteinase.