Lipases fatty acid reactions

Rangheard M-S, Langrand G, Triantaphylides C, Baratti J. Multi-competitive enzymatic reactions in organic media a simple test for the determination of lipase fatty acid specificity. Biochim Biophys Acta 1989 1004 20-28. 

The metabolic breakdown of triacylglycerols begins with their hydrolysis to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism of action is shown in Figure 29.2. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain -OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. 

Figure 25-8. Control of adipose tissue lipolysis. (TSH, thyroid-stimulating hormone FFA, free fatty acids.) Note the cascade sequence of reactions affording amplification at each step. The lipolytic stimulus is "switched off" by removal of the stimulating hormone the action of lipase phosphatase the inhibition of the lipase and adenylyl cyclase by high concentrations of FFA the inhibition of adenylyl cyclase by adenosine and the removal of cAMP by the action of phosphodiesterase. ACTFI,TSFI, and glucagon may not activate adenylyl cyclase in vivo, since the concentration of each hormone required in vitro is much higher than is found in the circulation. Positive ( ) and negative ( ) regulatory effects are represented by broken lines and substrate flow by solid lines.

Cherry and Crandall in 1932 (86) used olive oil as substrate with gum acacia as the emufsTfier. This method has served as the basis for a number of modifications that increased the stability of the emulsion, decreased incubation time and gave better precision. When a serum sample is incubated with a stabilized olive oil emulsion, lipase acts at the interface of substrate and water to hydrolyze olive oil into fatty acid plus diglycerides, and to a small extent to monoglycerides and glycerol. The bile salt sodium deoxycholate activates the reaction. These methods measure the liberated fatty acids by titration with a standardized NaOH solution. An indicator such as phenolphatalein, thymolphthalein or methyl red or a pH meter are used to detect the end point. 

Lipase is an enzyme which catalyzes the hydrolysis of fatty acid esters normally in an aqueous environment in living systems. However, hpases are sometimes stable in organic solvents and can be used as catalyst for esterifications and transesterifications. By utihzing such catalytic specificities of lipase, functional aliphatic polyesters have been synthesized by various polymerization modes. Typical reaction types of hpase-catalyzed polymerization leading to polyesters are summarized in Scheme 1. Lipase-catalyzed polymerizations also produced polycarbonates and polyphosphates. 

Lipase (EC 3.1.1.3, triacylglycerol acylhydrolase), both substrate (ester) and product (fatty acid), are poorly water soluble. The reaction occurs at the organic-aqueous interface [8,43,83,84]. 

The lipase-catalyzed fatty acid ester hydrolysis and the lipoxygenation of free polyunsaturated fatty acids are involved in the same lipid degradation pathway. They are respectively the first and second reaction in the lipoxygenase pathway (Fig. 3) [87-91]. The pathway produces volatile products of considerable importance in food technology including Cg[92, 93] or Cg- 94—96 aldehydes and alcohols from polyunsaturated fatty... 

Recently [63], we studied the behavior of two-enzyme system catalyzing two consecutive reactions in a macroheterogeneous medium (modified Lewis cell). The system consisted of lipase-catalyzed hydrolysis of trilinolein and subsequent lipoxygenation of liberated fatty acids (Fig. 3). Our approach compared the kinetic behavior of coupled enzymes in the Lewis cell with the sequential study of separated phenomena presented before ... 

It, thus, appears that the capacity to catalyze reactions of transesterification and esterification is a characteristic of various hydrolases (Chapt. 3). Apart from the carboxylesterases discussed here, lipoprotein lipase has the capacity to synthesize fatty acid ethyl esters from ethanol and triglycerides, or even fatty acids [127]. Ethanol, 2-chloroethanol, and other primary alcohols serve to esterify endogenous fatty acids and a number of xenobiotic acids [128-130]. In this context, it is interesting to note that the same human liver carboxylesterase was able to catalyze the hydrolysis of cocaine to benzoylecgonine, the transesterification of cocaine, and the ethyl esterification of fatty acids [131]. 

Lipases are enzymes of the hydrolase family and, in nature, hydrolyze fatty acid esters in aqueous environment. It is worth recalling that the hydrolysis of esters is a reversible reaction. Chemists thus often use lipases to catalyze the reverse reaction, i.e., the esterification and the ROP of lactones. In 1993, the groups of Kobayashi [91] and Knani [92] reported independently the hpase-catalyzed ROP of sCL and 8-valerolactone. The aliphatic polyesters were functionalized by a carboxylic group at one chain-end and a hydroxyl group at the other chain-end. Accordingly, the polymerization was initiated and terminated by water present in the reaction media. 

The natural substrates of lipases are triglycerides and, in an aqueous environment, lipases catalyze their hydrolysis into fatty acids and glycerol. In anhydrous media, lipases can be active in the reverse reaction [19]. In fact, in the acylation step, acids, lactones, (cyclic) carbonates [20, 21], cyclic amides [22, 23], (cyclic) thioesters [24, 25], and cyclic phosphates [26] have been found to act as suitable acyl donors. In the deacylation step, apart from water, lipases also accept alcohols [27], amines [28, 29], and thiols [30] as nucleophiles although the specificity of lipases is lower for amines and thiols than for water and alcohols [31]. 

Hydrolases, which catalyze the hydrolysis of various bonds. The best-known subcategory of hydrolases are the lipases, which hydrolyze ester bonds. In the example of human pancreatic lipase, which is the main enzyme responsible for breaking down fats in the human digestive system, a lipase acts to convert triglyceride substrates found in oils from food to monoglycerides and free fatty acids. In the chemical industry, lipases are also used, for instance, to catalyze the —C N —CONH2 reaction, for the synthesis of acrylamide from acrylonitril, or nicotinic acid from 3-pyridylnitrile. 

Naoe et al. [239] used the sugar ester DK-F-110, a mixture of sucrose esters of fatty acids, as a nonionic surfactant along with isopropyl alcohol and hexane in a reverse micellar system to extract cytochrome C. This surfactant has a critical micellar concentration of 0.5 g/1 and HLB of 11. Aqueous phase pH was found to have a major role in the forward extraction and optimum extraction was achieved at pH 8.0. However, for optimum back extraction, addition of isopropyl alcohol at 20 vol.% was found to be very essential. Further, the esterification reaction rate of Rhizopus delemar lipase was found to be maximum in DK-F-110 systems and also higher than those obtained in AOT and lecithin-RMs at a water concentration of 0.25 mol l h... 

Esters are common components in cosmetics and skin-care products. They can be synthesized from fatty acids and alcohols using either chemical or enzymatic reactions. The chemical reactions are normally catalysed by acid catalysts. Enzymatic synthesis is carried out under milder conditions and therefore it provides products of very high purity. A range of esters such as isopropyl palmitate and isopropyl myristate are now produced industrially using enzymatic synthesis. The reactions are carried out in solvent-free systems using an immobilised lipase as catalyst. In order to get high yields in the reactions, water is removed continuously. 

In order to enhance the potential of synthetic reactions of lipids and the transesterification in organic solvents, a fungal lipase from Phycomyces nites was chemically modified. The promotion of dispersibility in orgaiuc solvents resulted in a much higher reactivity. Chemically modified lipases showed higher reactivity than unmodified lipase when they were utilized for the transesterification of triglycerides and other lipids. The initial rate of transesterification in organic solvents by modified lipase was 40 times faster than that of unmodified lipase. Chemically modified lipase was also found applicable for the syndesis of other fatty acids esters. 

It is clear that the water activity is of crucial importance for the equilibrium yield in a reversed hydrolysis reaction. As expected, the equilibrium yield increases with decreasing water activity. This has been shown, for example, for the condensation of glucose and octanol [62], esterification of lysophospholipids with fatty acids [29, 63], and in normal lipase-catalyzed esterification reactions [64, 65]. The same situation is observed in ionic liquids [66]. 

Fatty acids of sugars are potentially useful and fully green nonionic surfactants, but the lipase-mediated esterification of carbohydrates is hampered by the low solubility of carbohydrates in reaction media that support lipase catalysis in general. Because the monoacylated product (Figure 10.8) is more soluble in traditional solvents than is the starting compound, the former tends to undergo further acylation into a diester. In contrast, the CaLB-catalyzed esterification of glucose with vinyl acetate in the ionic liquid [EMIm][BF4] was completely selective. The reaction became much faster and somewhat less selective when conducted in... 

Enzymatic acylation reactions offer considerable promise in the synthesis of specific ester derivatives of sucrose. For example, reaction of sucrose with an activated alkyl ester in IV,IV-dimethylformaniide in the presence of subtilisin gave T-O-butyrylsucrose, which on further treatment with an activated fatty acid ester in acetone in the presence of lipase C. viscosum produced the T,6-diester derivative (71,72). 

Cheese is ripened for 6 months to 1 year or longer at 5° to 15°C and 70-75% relative humidity. Cheese ripening is a complex process involving a combination of chemical, biochemical, and physical reactions. Proteolytic enzymes, e.g., rennet and lactic starter culture enzymes, hydrolyze caseins to produce flavor compounds and proper body. Lipase and lactase enzymes also hydrolyze their respective substrates to produce a large number of characteristic flavor compounds (Reiter and Sharpe 1971 Harper 1959 Law 1981 Schmidt etal. 1976), including free fatty acids, methanethiol, methanol, dimethyl sulfide, diacetyl, acetone, and others (Moskowitz 1980). 

Create a reaction progress curve by plotting the quantity of fatty acid liberated over the time of reaction (Fig. C3.1.1). Determine the activity (initial velocity, v0) of the lipase from the slope of the linear portion (see Critical Parameters) using the following equation ... 

Conceptually, assays for lipase activity using the colorimetric method (copper-soap procedure Basic Protocol 2) are similar to titrimetry in that liberated fatty acids are being measured however, the colorimetric method is more specific for fatty acids (Lowry and Tinsley, 1976). Quenched subsamples of emulsified acylglycerol/lipase reaction mixtures are combined with the biphasic mixture of cupric acetate/pyridine and benzene. Cupric salts of the fatty acids are formed (molar stoichiometry of fatty acid to Cu2+ of 4 2) and these soaps, which are blue in color, are partitioned into benzene to allow for quantification by measuring absorbance of the clear benzene phase at 715 nm. 

When identical lipase reaction subsamples are measured for fatty acid levels by both titrimetry (Basic Protocol 1) and colorimetry (Basic Protocol 2), estimates by the colorimetric procedure are only 60% of those obtained by titrimetry. However, the nature of this rela-... 

Potentiometry is another useful method for determining enzyme activity in cases where the reaction liberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base required is measured. An example of the use of this method is the determination of lipase activity. The enzyme hydrolyzes triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

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