Microemulsion lipase-catalyzed hydrolysis

Holmes et al. (1998) performed two enzymatic reactions, the lipase-catalyzed hydrolysis of y>-nitrophenol butyrate and lipoxygenase-catalyzed peroxidation of linoleic acid, in w/c microemulsions stabilized by a fluorinated two-chained sulfosuccinate surfactant (di-HCF4). The activity of both enzymes in the w/c microemulsion environment was found to be essentially equivalent to that in a water/heptane microemulsion stabilized by Aerosol OT, a surfactant with the same headgroup as di-HCF4. The buffer 2-(A-morpholino)ethanesulfonic acid (MES) was used to fix the pH in the range 5-6. 

Holmes et al. reported the first enzyme catalyzed reactions in water-in-CO2 microemulsions (67). Two reactions, a lipase-catalyzed hydrolysis and a lipoxygenase-catalyzed peroxidation, were demonstrated in water-in-C02 microemulsions using the surfactant di(l/7,l/7,5/7-octafluoro- -pentyl) sodium sulfosuccinate (di-HCF4). A major concern of enzymatic reactions in CO2 is the pH of the aqueous phase, which is approximately 3 when there is contact with CO2 at elevated pressures. Holmes et al. examined the ability of various buffers to maintain the pH of the aqueous solution in contact with CO2. The biological buffer 2-(A-morpholino)ethanesulfonic acid sodium salt (MES) was the most effective, able to maintain a pH of 5, depending on the pressure, temperature, and buffer concentration. The activity of the enzymes in the water-in-C02 microemulsions was comparable to that in a water-in-heptane microemulsion stabilized by the surfactant AOT, which contains the same head group as di-HCF4. 

The choice of surfactant is of importance for the rate of many enzymatic reactions in microemulsions. For instance, it has been found that whereas lipase-catalyzed hydrolysis of triglycerides is rapid in microemulsions based on AOT, it is extremely sluggish when... 

In a 1994 work, the effect of the surfactant on lipase-catalyzed hydrolysis of palm oil in microemulsion was further investigated [62]. Three surfactants were used one anionic, one nonionic, and one cationic. As shown in Fig. 10, all three compounds were double-tailed, with similar hydrophilic-lipophilic balance, giving large regions of L2 microemulsions with isooctane and water at 37°C. 

A hydrophilic substrate, acetylsalicylic acid, was subjected to lipase catalyzed hydrolysis in a W/O microemulsion [77]. For comparison, the reaction was also carried out in aqueous buffer. Since hydrolysis of acetylsalicylic acid proceeds spontaneously without added catalyst (intramolecular catalysis), reactions without lipase were performed as controls. It was found that addition of lipase did not affect the rate of reaction in aqueous buffer. However, the reaction in miroemulsion was catalyzed by the lipase, and the rate was linearly dependent on lipase concentration. This is a further illustration of the fact that microemulsions, with their large oil/water interfaces, are suitable media for lipase-catalyzed reactions. The same reactions were also performed using a-chymotrypsin as catalyst. This enzyme, which also catalyzes ester hydrolysis but which, unlike lipase, functions independently of a hydrophobic surface, was not more active in microemulsion than in the buffer solution. 

Another workup approach has been to use the inherent phase behavior of oil-water-surfactant systems to separate product from remaining reactants and from surfactant. A Winsor III system made with a branched-tail phosphonate surfactant was used as reaction medium for lipase-catalyzed hydrolysis of trimyristin. The enzyme resided almost exclusively in the middle-phase microemulsion together with the surfactant. The products formed, 2-myristoylglycerol and sodium myristate, partitioned into the excess hydrocarbon and water phases, respectively, and could easily be recovered [129]. A similar procedure was used for cholesterol oxidation using cholesterol oxidase as catalyst [130]. 

The addition of alcohol, as cosurfactant, to the [Cgmim][TfjN]/AOT/water system leads to stable w/IL microemulsions. DLS and protein solubilization experiments confirm the existence of an aqueous nanoenvironment in the IL phase of [C mirnTf N]/ AOT/l-hexanol/water microemulsions [67]. The kinetics of the enzymatic reactions were performed in this quaternary system. Specifically, lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-NPB) was used as a model reaction [68]. In a similar way, the hpase-catalyzed hydrolysis of p-NPB was investigated to evaluate the catalytic efficiency in water/AOT/Triton X-100/[C mim][PFJ [69]. A large single-phase microemulsion region can be obtained from the combination of two surfactants in IL. 

Lipase-Catalyzed Hydrolysis Goto and coworkers [63] proposed an approach for carrying out enzymatic reactions in water-in-IL microemulsion. 

The lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-NPB) was used as a model reaction. It was found that the hydrolysis rate was faster in the water-in-IL microemnlsions than in the water-in-isooctane microemulsions. Hie intrinsic activity of lipase in the IL microemulsion was about three times higher than that of water/ AOT/isooctane microemulsions of AOT under the given experimental conditions. The enhanced catalytic activity of lipase in water-in-IL microemulsions may be due to (i) aqueous microenvironmental changes, (ii) the partition of the substrate or other molecules involved in the reaction between water and IL phases, and (iii) the existence of 1-hexanol as a cosurfactant. 

Table 15.3 Second-order rate constant for lipase-catalyzed hydrolysis of p-nitrophenyl-/i-hexanoate in different cationic W/0 microemulsions. 

Figure 15.9 Second-order rate constant ((12) for the lipase-catalyzed hydrolysis of p-nitrophenyl-n-hexanoate in cationic W/O microemulsions formulated with cetyltrimethylammonium-based surfactants of different counterions 1-14.

NMR self-diffusion measurements indicated that all microemulsions consisted of closed water droplets and that the structure did not change much during the course of reaction. Hydrolysis was fast in microemulsions based on branched-chain anionic and nonionic surfactants but very slow when a branched cationic or a linear nonionic surfactant was employed (Fig. 11). The cationic surfactant was found to form aggregates with the enzyme. No such interactions were detected with the other surfactants. The straight-chain, but not the branched-chain, alcohol ethoxylate was a substrate for the enzyme. A slow rate of triglyceride hydrolysis for a Ci2E4-based microemulsion compared with formulations based on the anionic surfactant AOT [61,63] and the cationic surfactant cetyltrimethylammonium bromide (CTAB) [63] was observed in other cases also. Evidently, this type of lipase-catalyzed reaction should preferably be performed in a microemulsion based on an anionic or branched nonionic surfactant. Nonlipolytic enzymes such as cholesterol oxidase seem to function well in microemulsions based on straight-chain nonionic surfactants, however [64]. CTAB was reported to cause slow inactivation of different types of enzymes [62,64,65] and also, in the case of Chromobacterium viscosum lipase [66], to provide excellent stability. 

Microemulsion media of low water activity enable hydrolytic enzymes to catalyze condensation as opposed to hydrolysis. This has opened the possibility to perform lipase-catalyzed ester synthesis, an area of considerable practical interest. Lipase-catalyzed transesterification in microemulsion of low water content is another area of industrial relevance, in particular for synthesis of triglycerides with unusual fatty acid composition. 

There are various reports in the literature concerning kinetic studies of the Upase-catalyzed hydrolysis or synthesis of esters in microemulsions [8,9,49,83,84]. A simple MichaeUs-Menten kinetic model was proposed for the hydrolysis of triglycerides [85,86], while the esterifications of aliphatic alcohols with fatty acids follow a ping-pong bi-bi mechanism [87]. According to this mechanism the lipase reacts with the fatty acid to form a noncovalent enzyme-fatty acid complex, which is then transformed to an acyl-enzyme intermediate, while water, the first product, is released this is followed by a nucleophile attack (by the alcoholic substrate) to form another binary complex that finally yields the ester and the free enzyme. The kinetic parameters and determined in these studies represent apparent... 

Properties such as large interfacial area and an ability to solubilize both oil-soluble and water-soluble reactants in a single phase system makes microemulsions ideal as reaction media (Flanagan and Singh, 2006 Gaonkar and Bagwe, 2002). For example, Morgado and co-workers (1996) nsed a continnons reversed micellar system to synthesize lysophospholipids and free fatty acids from lecithin hydrolysis, with applications to the food, pharmaceutical and chemical industries. Hydrolysis was catalyzed by porcine pancreatic phospholipase A. Carvalho and Cabral (2000) reviewed the use of reversed micellar systems as reactors to carry out lipase-catalyzed esterification, biocatalysis, transesterificadon, and hydrolysis reactions. 

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