MILK ENZYMES ITS SOURCES
AND SIGNIFICANCE-PART I
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.
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.
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).
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.
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.
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.
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.
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 αs2-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.
SOURCE-NDRI ECOURSE
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