Enzymes lipolysis

Care should be taken to minimize changes in the fatty acid composition of the food before analysis. Lipids are susceptible to enzymic lipolysis and UFA are susceptible to enzymic and nonenzymic oxidation. Therefore, food samples should be kept at low temperamres ( 20°C or lower), preferably under an atmosphere of nitrogen or in an oxygen-impermeable wrap, before extraction. 

Acetyl-CoA carboxylase is an allosteric enzyme and is activated by citrate, which increases in concentration in the well-fed state and is an indicator of a plentiful supply of acetyl-CoA. Citrate converts the enzyme from an inactive dimer to an active polymeric form, having a molecular mass of several milhon. Inactivation is promoted by phosphorylation of the enzyme and by long-chain acyl-CoA molecules, an example of negative feedback inhibition by a product of a reaction. Thus, if acyl-CoA accumulates because it is not esterified quickly enough or because of increased lipolysis or an influx of free fatty acids into the tissue, it will automatically reduce the synthesis of new fatty acid. Acyl-CoA may also inhibit the mitochondrial tricarboxylate transporter, thus preventing activation of the enzyme by egress of citrate from the mitochondria into the cytosol. 

The presence of the second enzyme in the medium accelerates and increases consumption of the first substrate (trilinolein), because lipoxygenase reacts with the second substrate (linoleic acid) produced by lipolysis of trilinolein. 

Cakes are in principle subject to all the threats to a long shelf life that any other bakery product is subject to. The product can dry out, the starch can retrograde or mould can grow. These are in addition to the threats of oxidation, loss of flavour and lipolysis by any enzymes present. 

Fatty acid utilized by muscle may arise from storage triglycerides from either adipose tissue depot or from lipid stores within the muscle itself. Lipolysis of adipose triglyceride in response to hormonal stimulation liberates free fatty acids (see Section 9.6.2) which are transported through the bloodstream to the muscle bound to albumin. Because the enzymes of fatty acid oxidation are located within subcellular organelles (peroxisomes and mitochondria), there is also need for transport of the fatty acid within the muscle cell this is achieved by fatty acid binding proteins (FABPs). Finally, the fatty acid molecules must be translocated across the mitochondrial membranes into the matrix where their catabolism occurs. To achieve this transfer, the fatty acids must first be activated by formation of a coenzyme A derivative, fatty acyl CoA, in a reaction catalysed by acyl CoA synthetase. 

The degradation of fats (lipolysis) is catalyzed in adipocytes by hormone-sensitive lipase [2]—an enzyme that is regulated by various hormones by cAMP-dependent interconversion (see p. 120). The amount of fatty acids released depends on the activity of this lipase in this way, the enzyme regulates the plasma levels of fatty acids. 

Selected entries from Methods in Enzymology [vol, page(s)] Dilution of enzyme samples, 63, 10 lipolysis substrate effect, 64, 361, 362 dilution jump kinetic assay, 74, 14-19, 28 dilution method [for dissociation equilibria, 61, 65-96 continuous dilution cuvette, 61, 78-96 data analysis, 61, 74, 75 equations, 61, 70-74 errors, 61, 76-78 experimental procedures, 61, 69, 70 merits, 61, 75, 76 theory, 61, 68, 69... 

MICELLAR SUBSTRATES. Phospholipids in micelles are frequently found to be more active substrates in lipolysis than those phospholipids residing in a lipid bilayer". Dennis first described the use of Triton X-100 to manipulate the amount of phospholipid per unit surface area of a micelle in a systematic analysis of the interfacial interactions of lipases with lipid micelles. Verger and Jain et al have presented cogent accounts of the kinetics of interfacial catalysis by phospholipases. The complexity of the problem is illustrated in the diagram shown in Fig. 2 showing how the enzyme in the aqueous phase can bind to the interface (designated by the asterisk) and then become activated. Once this is achieved, E catalyzes conversion of S to release P. ... 

This broad class of hydrolases constitutes a special category of enzymes which bind to and conduct their catalytic functions at the interface between the aqueous solution and the surface of membranes, vesicles, or emulsions. In order to explain the kinetics of lipolysis, one must determine the rates and affinities that govern enzyme adsorption to the interface of insoluble lipid structures -. One must also account for the special properties of the lipid surface as well as for the ability of enzymes to scooC along the lipid surface. See specific enzyme Micelle Interfacial Catalysis... 

Malcata, F.X. and Hill Jr., C.G. (1995) Indnstrial ntihzation of a hollow-fiber membrane reactor for the controlled lipolysis of bntterfat. Enzyme EngineeringXII, edited by M.-D. Legoy and D. N.Thomas. Annals of the New York Academy of Science, Vol. 750, 401-407. 

The increased use of tanks for the storage of raw milk on the farm between pickups has introduced the danger of potential off-flavor development caused by lipases that are produced by certain microorganisms (psychrotrophs) at low temperatures. The exocellular lipases of psychrotrophic bacteria are extremely heat resistant, and although the microorganisms are killed, the enzymes survive pasteurization and sterilization temperatures. Rancidity may become noticeable when cell counts exceed 106 or 107/ml. Downey (1975) has summarized the potential contribution of enzymes to the lipolysis of milk (Table 5.1). 

Most, if not all, milks contain sufficient amounts of lipase to cause rancidity. However, in practice, lipolysis does not occur in milk because the substrate (triglycerides) and enzymes are well partitioned and a multiplicity of factors affect enzyme activity. Unlike most enzymatic reactions, lipolysis takes place at an oil-water interface. This rather unique situation gives rise to variables not ordinarily encountered in enzyme reactions. Factors such as the amount of surface area available, the permeability of the emulsion, the type of glyceride employed, the physical state of the substrate (complete solid, complete liquid, or liquid-solid), and the degree of agitation of the reaction medium must be taken into account for the results to be meaningful. Other variables common to all enzymatic reactions—such as pH, temperature, the presence of inhibitors and activators, the concentration of the enzyme and substrate, light, and the duration of the incubation period—will affect the activity and the subsequent interpretation of the results. 

Table 5.1. Contribution of Enzymes Present to Lipolysis of Milk.

Downey (1980) reasoned that although milk lipoprotein lipase is present in sufficient amounts to cause extensive hydrolysis and potential marked flavor impairment, this does not happen in practice for the following reasons (1) the fat globule membrane separates the milk fat from the enzyme, whose activity is further diminished by (2) its occlusion by casein micelles (Downey and Murphy 1975) and by (3) the possible presence in milk of inhibitors of lipolysis (Deeth and Fitz-Gerald 1975). The presence in milk of activators and their relative concentration may also determine whether milk will be spontaneously rancid or not (Jellema 1975 Driessen and Stadhouders 1974A Murphy et al. 1979 Anderson 1979). 

According to Tarassuk and Frankel (1955), foaming promotes lipolysis by providing (1) greatly increased surface area, (2) selective concentration of enzyme at the air-liquid interface, (3) activation of the substrate by surface denaturation of the membrane materials around the fat globules, and (4) intimate contact of the lipases and the activated substrate. 

Downey, W. K. 1975. Identity of the major lipolytic enzyme activity of bovine milk in relation to spontaneous and induced lipolysis. Int. Dairy Fed. Doc. 86, 80-89. 

Add an appropriate amount of enzyme to initiate lipolysis on the emulsion substrate, start timer, and continue stirring. 

Metabolic Effects. Thyroid hormones affect energy substrate utilization in a number of ways. For instance, these hormones increase intestinal glucose absorption and increase the activity of several enzymes involved in carbohydrate metabolism. Thyroid hormones enhance lipolysis by increasing the response of fat cells to other lipolytic hormones. In general, these and other metabolic effects help to increase the availability of glucose and lipids for increased cellular activity. 

PC requires biotin for activity. Biotin is bound to the enzyme via a peptide-like linkage involving e-NH2 groups of certain lysine residues. This type of biotin complex is biocytin (see Chapter 6). Another compound necessary for PC activity is acetyl-CoA, a positive effector. PC is activated as cellular levels of acetyl-CoA increase, as when extensive lipolysis takes place. Acetyl-CoA is produced in large amounts from fatty acids via the /8-oxidation reaction (see Chapter 19). PC can also be considered an anaplerotic reaction, those reactions that replenish crucial intermediates for metabolic pathways. In this case, oxaloacetate, an important intermediate in the Krebs cycle, is replenished by a reaction catalyzed by PC. 

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