Lipases And Esterases Ec 311

Lipases catalyze the development of hydrolytic rancidity which is a serious defect in milk and some milk products, and, consequently, lipases and lipolysis in milk have been studied extensively. Milk contains three types of esterase: (a) A-type carboxylic ester hydrolase (arylesterase; EC 3.1.1.2), which hydrolyzes aromatic esters, e.g., phenylacetate. It shows little activity on tributyrin, and is not inhibited by organo-phosphates. (b) B-type esterase (glycerol tricarboxyl esterase, aliphatic esterase, lipase; EC 3.1.1.3). Such enzymes are most active on aliphatic esters although they show some activity on aromatic esters; they are inhibited by organophosphates. (c) C-type esterase (cholinesterase; EC 3.1.1.7; EC 3.1.1.8). These enzymes are most active on choline esters but hydrolyze some aromatic and aliphatic esters slowly; they are inhibited by organophosphates.

In normal milk, the ratio of A: B: C types of esterase activity is about 3:10:1 but the level of A-esterase activity increases considerably on mastic infection. A and C esterases are of little technological significance in milk. Lipases hydrolyze ester bonds in emulsified esters, i.e., at a water/oil interface, although some may have limited activity on soluble esters. They are usually activated by blood serum albumin and Ca2+ which bind free fatty acids, which are inhibitory.

Milk lipase was first isolated and characterized by Fox and Tarassuk (41) and Patel et al. (42). The enzyme was optimally active at pH 9.2 and 37°C and was found to be a serine enzyme (inactivated by orga-nophosphates). A lipoprotein lipase (LPL; activated by lipoprotein cofactors) was demonstrated in milk by Korn in 1962 (42a) and isolated by Egelrud and Olivecrona (43). The lipase isolated by Fox and Tarassuk (41) is, in fact, an LPL which is the principal, probably the only, indigenous lipase in bovine milk. It has been the focus of considerable research and has been characterized at the molecular, genetic, enzymatic, and physiological levels (44).

Under optimum conditions, the kcat for milk LPL is

3000 s_1 and milk contains sufficient enzymes (1-2mg/L; i.e., 10-20nM) to theoretically cause rancidity in 10 sec. However, this does not occur in practice because the triglycerides are protected by the MFGM while the lipase is naturally associated with the casein micelles. Also, environmental conditions, e.g., pH, are not optimal. However, if the MFGM is damaged by agitation (e.g., by milking machines, bulk tanks, pumps, etc.), homogenization or temperature fluctuations, lipolysis occurs rapidly and rancidity ensues. Milk LPL appears to be derived from blood plasma and hence any condition that increases the permeability of mammary cell membranes, e.g., physiological stress, mastitic infection, or late lactation, increases the level of LPL in milk and hence the risk of lipolysis. Some individual cows produce milk which becomes rancid spontaneously, i.e., without apparent activation. Apparently, spontaneous rancidity occurs when milk contains a high level of lipoprotein (co-lipase) from blood serum which activates the LPL. Normal milk will become spontaneously rancid if blood serum is added, suggesting that "spontaneous milks'' contain a higher than normal level of blood serum. Dilution of spontaneous milk with normal milk prevents spontaneous rancidity, which, consequently, is not normally a problem with bulk herd milks. Presumably, dilution with normal milk decreases the lipoprotein content of the bulk milk to below the threshold necessary for lipase activation. Natural variations in the level of free fatty acids in normal milk and the susceptibility of normal milks to induced lipo-lysis may be due to variations in the level of blood serum components in milk.

In addition to LPL, human milk contains a bile salts-activated lipase, which probably contributes to the metabolism of lipids by breastfed babies who have limited pancreatic lipase activity. Bovine milk and milks from other dairy animals do not contain this enzyme.

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