Phosphatase alkaline, milk

As indices of the thermal history of milk alkaline phosphatase, y-glutamyl transpeptidase, lactoperoxidase. 

Isolation and characterization. Alkaline phosphatase is concentrated in the fat globule membrane and hence in cream. It is released into the buttermilk on phase inversion consequently, buttermilk is the starting material for most published methods for the purification of alkaline phosphatase. Later methods have used chromatography on various media to give a homogeneous preparation with 7440-fold purification and 28% yield. The characteristics of milk alkaline phosphatase are summarized in Table 8.2. The enzyme appears to be similar to the alkaline phosphatase of mammary tissue. 

Phosphatase Test. The phosphatase [9001-78-9] test is a chemical method for measuring the efficiency of pasteurization. AH raw milk contains phosphatase and the thermal resistance of this enzyme is greater than that of pathogens over the range of time and temperature of heat treatments recognized for proper pasteurization. Phosphatase tests are based on the principle that alkaline phosphatase is able, under proper conditions of temperature and pH, to Hberate phenol [108-95-2] from a disodium phenyl phosphate substrate. The amount of Hberated phenol, which is proportional to the amount of enzyme present, is determined by the reaction of Hberated phenol with 2,6-dichloroquinone chloroimide and colorimetric measurement of the indophenol blue formed. Under-pasteurization as well as contamination of a properly pasteurized product with raw milk can be detected by this test. 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Acid phosphomonoesterase (EC 3.1.3.2). Milk contains an acid phosphatase which has a pH optimum at 4.0 and is very heat stable (LTLT pasteurization causes only 10-20% inactivation and 30 min at 88°C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7, the enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+ (as is alkaline phosphatase), but it is slightly activated by Mn2+ and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation. 

Luick, J. R. and Mazrimas, J, A. 1966. Biological effects of ionizing radiation on milk synthesis. III. Effects on milk lipase, esterase, alkaline phosphatase, and lactoper-oxidase activities. J. Dairy Sci. 49, 1500-1504. 

Morton, R. K. 1955. Some properties of alkaline phosphatase of cows milk and calf intestinal mucosa. Biochem. J. 60, 573-582. 

Pasteurization inactivates many enzymes, including alkaline phosphatase and lipoprotein lipase. The absence of active alkaline phosphatase in cheese is often used to determine if the milk has been properly pasteurized prior to cheesemaking. Since pasteurization kills most of the lactic acid bacteria in milk, the lactic acid developed during cheese-... 

Incubate in avidm alkaline phosphatase diluted T50, or avidin peroxidase diluted T100 m TBT, containing 5% (w/v) nonfat milk (e.g, Marvel)... 

Most of these observations have since been verified. Phosphorylation by substrate has been shown to occur under acid conditions by using a stopped-flow technique (118, 165) as illustrated in Fig. 4. Under alkaline conditions the phosphoryl enzyme cannot normally be observed or isolated because the rate of dephosphorylation exceeds the maximum rate of phosphorylation (170). One interesting aspect is that the pH-rate profiles for phosphorylation and dephosphorylation are quite different, as is the case for E. coli alkaline phosphatase (171). Barman and Gut-freund studied the formation and breakdown of milk phosphoryl phosphatase using a rapid-quenching technique and concluded that dephosphorylation could not be rate limiting for the hydrolysis of p-nitrophenyl phosphate at pH 7 (S3). 

Serra et al. [105] Alkaline phosphatase Milk (process evaluation pasteurisation) Tyrosinase/physically included in the composite matrix Graphite-Teflon composite electrode/-0.10 V vs. Ag/ AgCl -... 

A 6-month-old infant has been fed unmodified cow s milk supplemented with com flour. He was healthy except that he was severely anemic, and an X-ray of his wrist showed retarded bone development. His hemoglobin was only 4.5 g/dL, and he had an elevated serum alkaline phosphatase. He was treated with iron supplements, folate, and ascorbic acid to no avail. Then serum copper analysis was done, and it showed a level of 9 fig/dL (normal is 85-163 /ig/dL). Thereupon his diet was supplemented with copper sulfate, and he showed dramatic improvement. Address the following questions ... 

Nakanishi, T., Tagata, Y. 1972. The distribution and some properties of esterase, alkaline phosphatase and acid phosphatase in cow s milk. Jap. J. Dairy Sci. 21, A-207-215. 

N16. Nayudu, P. R. V., and Moog, F., Intestinal alkaline phosphatase Regulation by a strain-specific factor in mouse milk. Science 162, 656-657 (1966). 

FIGURE 8.11 First-order reaction rate constants k for heat inactivation, plotted against mass fraction of water w. AP alkaline phosphatase, in skim milk, 80°C. Ec killing of Eschericia coli, in skim milk, 63°C. Ch chymosin, in whey, 80°C. Li lipoxygenase, in sucrose/calcium alginate, 72°C. 

Shamsi, K., Versteeg, C., Sherkat, R, and Wan, J. 2008. Alkaline phosphatase and microbial inactivation by pulsed electric field in bovine milk. Innovative Food Science and Emerging Technologies 9 217-223. 

Dephosphorylation. In vivo, phosphoprotein phosphatases (phos-phoprotein phosphohydrolase EC 3.1.3.16) participate in the regulation of phosphate turnover and in rapid reversal of the phosphorylating reactions, yet there is very little information on this important group of enzymes. Alkaline (104) and acid (105) phosphatases of milk can hydrolyze the phosphate groups of phosphoserine residues in the caseins. E. coli alkaline phosphatase dephosphorylates many phosphoproteins (106,107). A phosphoprotein phosphatase of liver dephosphorylates phosphorylated histones and protamines but has little or no activity on casein or phosvitin (108). The 60-fold purified enzyme had an apparent Km for dephosphorylation of histone I (FI) of 2 X 10"5M and a pH optimum of 7-8. Histone phosphatase activity was detected in all eukaryotic cells examined, but it was not found in the extracts of several prokaryotes. 

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