Lipases esterification reaction

Stamatis, H., Kolisis, F.N., Xenakis, A., Bornscheuer, U., Scheper, T., Menge, U. 1993. Pseudomonas cepacia lipase Esterification reactions in AOT microemulsion systems. Biotechnol. Lett. 15, 703-708. 

One of the most important characteristics of IL is its wide temperature range for the liquid phase with no vapor pressure, so next we tested the lipase-catalyzed reaction under reduced pressure. It is known that usual methyl esters are not suitable for lipase-catalyzed transesterification as acyl donors because reverse reaction with produced methanol takes place. However, we can avoid such difficulty when the reaction is carried out under reduced pressure even if methyl esters are used as the acyl donor, because the produced methanol is removed immediately from the reaction mixture and thus the reaction equilibrium goes through to produce the desired product. To realize this idea, proper choice of the acyl donor ester was very important. The desired reaction was accomplished using methyl phenylth-ioacetate as acyl donor. Various methyl esters can also be used as acyl donor for these reactions methyl nonanoate was also recommended and efficient optical resolution was accomplished. Using our system, we demonstrated the completely recyclable use of lipase. The transesterification took place smoothly under reduced pressure at 10 Torr at 40°C when 0.5 equivalent of methyl phenylthioacetate was used as acyl donor, and we were able to obtain this compound in optically pure form. Five repetitions of this process showed no drop in the reaction rate (Fig. 4). Recently Kato reported nice additional examples of lipase-catalyzed reaction based on the same idea that CAL-B-catalyzed esterification or amidation of carboxylic acid was accomplished under reduced pressure conditions. ... 

Lipase-catalyzed reaction is useful for polyester synthesis and IE was employed successfully as solvent. Uyama and Kobayashi demonstrated an efficient polyester synthesis lipase-catalyzed esterification of agipic acid with butan-1,4-diol proceeded smoothly in [bmim][BF4] solvent, particularly under reduced pressure conditions (Fig. 8). Further Russel " and Nara independently reported efficient examples of the lipase-catalyzed polyester synthesis in an IE solvent system. 

Nevertheless, in some cases, this criterion is not sufficient for the choice of the solvent. For instance, Kuo and Parkin [78], demonstrated that hydrophobicity of solvent in the presence of lipase also affect selectivity and partition of reactants in esterification reactions. On the other hand, in the presence of certain solvents, even in low concentration, enzyme can be activated [13]. 

Several mechanisms have been proposed for lipase-catalyzed reactions. Kinetic studies of hydrolysis [14,15] and esterification [50] catalyzed by Pseudomonas cepecia lipase, demonstrate that the enzyme has a ping-pong mechanism. 

Lipases are able to catalyze many hydrolytic and esterification reactions in the presence of different substrates. The type of substrate is a key factor affecting the activity and productivity of lipase-catalyzed reactions. 

In a lipase-catalyzed reaction, the acyl group of the ester is transferred to the hydroxyl group of the serine residue to form the acylated enzyme. The acyl group is then transferred to an external nucleophile with the return of the enzyme to its preacylated state to restart the catalytic cycle. A variety of nucleophiles can participate in this process. For example, reaction in the presence of water results in hydrolysis, reaction in alcohol results in esterification or transesterification, and reaction in amine results in amination. Kirchner et al.3 reported that it was possible to use hydrolytic enzymes under conditions of limited moisture to catalyze the formation of esters, and this is now becoming very popular for the resolution of alcohols.4... 

Catalytic sol-gel lipase immobilizates were rapidly commercialized (by Fluka) after their invention in 1995 because of their remarkably stable activity in esterification reactions (and also in the kinetic resolution of chiral alcohols and amines) along with unique stability (residual activity of 70% even after 20 reaction cycles is common). The original procedure for the encapsulation produced by the fluoride-catalysed hydrolysis of mixtures of RSi(OCH3)3 and Si(OCH3)4 has been improved... 

The one-pot dynamic kinetic resolution (DKR) of ( )-l-phenylethanol lipase esterification in the presence of zeolite beta followed by saponification leads to (R)-l phenylethanol in 70 % isolated yield at a multi-gram scale. The DKR consists of two parallel reactions kinetic resolution by transesterification with an immobilized biocatalyst (lipase B from Candida antarctica) and in situ racemization over a zeolite beta (Si/Al = 150). With vinyl octanoate as the acyl donor, the desired ester of (R)-l-phenylethanol was obtained with a yield of 80 % and an ee of 98 %. The chiral secondary alcohol can be regenerated from the ester without loss of optical purity. The advantages of this method are that it uses a single liquid phase and both catalysts are solids which can be easily removed by filtration. This makes the method suitable for scale-up. The examples given here describe the multi-gram synthesis of (R)-l-phenylethyl octanoate and the hydrolysis of the ester to obtain pure (R)-l-phenylethanol. 

Lipases are enzymes that catalyze the in vivo hydrolysis of lipids such as triacylglycerols. Lipases are not used in biological systems for ester synthesis, presumably because the large amounts of water present preclude ester formation due to the law of mass action which favors hydrolysis. A different pathway (using the coenzyme A thioester of a carboxylic acid and the enzyme synthase [Blei and Odian, 2000]) is present in biological systems for ester formation. However, lipases do catalyze the in vitro esterification reaction and have been used to synthesize polyesters. The reaction between alcohols and carboxylic acids occurs in organic solvents where the absence of water favors esterification. However, water is a by-product and must be removed efficiently to maximize conversions and molecular weights. 

Despite the fact that glycolic acid has been successfully used as an acyl donor in esterification reactions with fatty alcohols, there are few reports dealing with the enzymatic ROP of glycolide [139], On the other hand, cyclic diesters based on ethylene glycol have been polymerized successfully by lipase catalysis and afford AA-BB-typepolyesters [140, 141],... 

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... 

Lipases exhibit high catalytic activity in water, an even higher activity in a two-phase system, such as water/water-immiscible organic solvent, and in water-immiscible organic solvents of low water content86-88,90. This allows for the attainment of favorable equilibria in asymmetric hydrolysis and esterification reactions catalyzed by lipases. They are used to their greatest... 

Relatively few detailed studies of enzyme kinetics in organic media have been carried out. Preferably, full kinetics should be studied, allowing the determination of Km and kcat values, but it is much more common to see just reports on the catalytic activity at fixed substrate concentrations as a function of water activity. That such studies can be misleading was shown in an investigation of lipase-catalyzed esterification [26]. When the reaction rate in the esterification reaction was plotted versus the water activity at three different substrate concentrations, maxima were obtained at three different water activities (Figure 1.4). Such maxima should not be used to claim that the optimal water activity of the enzyme was found. Detailed kinetic studies showed that both the kcat and the Km values (for the alcohol substrate) varied with the water activity. The Km value of the alcohol increased with increasing water... 

When water molecules interact with an enzyme, it is natural that conformational changes can occur, which in turn can cause changes in the selectivity of the enzyme. Since enantioselectivity of enzymes is of major importance for many applications, it is a common task to investigate how to choose reaction conditions providing the maximal enantioselectivity. As might be expected, because water can interact with enzymes in many ways, it is difficult to generalize the effects. In some studies of lipase-catalyzed esterification reactions, no effects of water activity on enantioselectivity were observed [30]. In a similar study, no effects were observed in most cases, while the enantioselectivity of one lipase-catalyzed reaction decreased... 

When substrate activities are used instead of substrate concentrations in studies of enzyme kinetics in organic media, solvent effects due to substrate solvation disappear. Remaining solvent effects should be due to direct interactions between the enzyme and the solvent. In a study of lipase-catalyzed esterification reactions, it was found that Km values based on activities were indeed more similar tban those based on concentrations in different solvents, but still some differences remained [49]. 

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]. 

However, for some specific reactions, solvent-free systems are preferred because of their higher yields. The main area of development should be related to the hydrolysis of glycerides, transesterification, esterification and inter-esterification reactions. As lipases have high and stable activity in SC CO2 (even at the high temperature) an intensive development is expected. 

In the case of lipases and esterases, chiral recognitions are not so strict. Both enantiomers were incorporated to the enzyme to form the substrate-enzyme complex. However, the slow reacting enantiomer lacked the necessary hydrogen-bonding interaction, for example in the hydrolysis of menthol acetate, between the substrate menthol and the enzyme histidine group for the reaction to proceed further (Figure 3(b)).2 3 The explanation was also supported by the observation in the esterification reaction of 1-phenylethanol by lipases.4 Km values of the slow and fast reacting... 

Hydrolytic enzymes such as lipases catalyze hydrolysis of esters in aqueous media, but when used in non-aqueous media such as organic solvents, ionic liquids and supercritical fluids, they catalyze reverse reactions the synthesis of esters. For example, lipases in natural environment catalyze the hydrolysis of fatty acid esters as shown in Figure 6(a). However, when they are used in organic solvents, they catalyze the esterification reaction (Figure 6(b)). 

For the resolution of cyanopentafluorophenylethanol with lipase, (he reaction temperature was decreased to improve the enantioselectivities (Figure 15(b)). The ameso comPounds were conducted to obtain fluorinated amino, . "UIC. 1 > e)). Esterification of meso alcohol gave the corresponding W-ammo acid, whereas the hydrolysis gave the corresponding(5)-product. 

Moreover, lipase-catalyzed reactions of linear and cyclic poly(3-hydroxy-butanoates) were subjected to hydrolysis, transesterification, and intramolecular esterification. A cyclic polymer along with linear polymers was pro-... 

Here, we report the application of this procedure for immobilizing Mucor miehei lipase. A catalytic test was aimed at producing esters by direct esterification reactions with a large range of carboxylic acids (from C4 to C16), and a diversity of alcohols (from C4 to C8). Several reaction model systems are analyzed in order to illustrate the kind of products that can be made by using an experimental preparation of lipase immobilized on POS-PVA particles. 

Esterification reactions were carried out in a closed reactor with 10 mL of dried n-heptane containing suitable amounts of alcohol and acid. A molecular sieve (aluminum sodium silicate, type 13X BHD Chemicals) was used to removal water. The mixture was incubated at 37°C for 24 h with continuous shaking at 150 rpm. The effects of concentration of immobilized lipase (5-50 mg/mL) molar ratio of reactants (0.5-2.0), acid chain length... 

In addition to the chain length, the effect of branching of the carbon chain was studied. Specificity of POS-PVA lipase was studied by monitoring esterification reactions of ft-butanol, sec-butanol, and ferf-butanol with butyric acid, as shown in Table 2. The highest rate of conversion to ester (60%) occurred in the presence of ft-butanol, compared with sec-butanol and ferf-butanol. The branching was found to decrease significantly the esterification yield by a factor of 0.4 for sec-butanol and 0.65 for tert-butanol. Antczak et al. (24) reported a similar conversion pattern. 

FFAs from tuna oil were esterified with 1 to 10 mol MeOH using immobilized C. antarctica lipase. The reaction velocity decreased with increasing the amount of MeOH. However, the degrees of esterification at 24 h (nearly equilibrium state) were 88 and 95% when using 1 and 2 mol of MeOH for FFAs, respectively. Although more than 2 mol of MeOH were used, the degree did not increase owing to the equilibrium of esterification and its reverse reaction (hydrolysis of FAMEs) (Watanabe et al, 2002). 

Figure 2.10. Lipase-catalyzed reaction on a mixture of FAStE and water in the presence or in the absence of MeOH. A, Hydrolysis of FA steryl ester and esterification of sterol with FFA. B, Lipase-catalyzed reactions in a mixture of FAStE, water and MeOH.

Lipases are commonly recognized as biocatalysts in hydrolysis and esterification reactions. The primary advantages of the used reactions are in asymmetric hydrolysis of chiral esters, as well as asymmetric esterification of a wide range of substrate... 

There are many reports of enzymatic catalysis in scC02 performing hydrolysis, oxidations, esterifications, and franr-esterification reactions. For example, the enzymatic kinetic resolution of 1-phenylethanol with vinyl acetate in scC02 using lipase from Candida antarctica B produces (R)-l-phenyethylacetate in >99% ee (i.e., enantiomeric excess, a measure of how much of one enantiomer is present as compared to the other), as shown in Figure 12.20. 

The range of nucleophiles that lipases accept is not confined to water or alcohols. There are numerous examples of amines, hydrazine, phenols," and hydrogen peroxide. Proteases have frequently been used in biocatalytic transformations involving ester hydrolysis and esterification reactions and their different stereoselection often provides a useful complement to the lipases. - ... 

Most of lipase-catalyzed acylations of sugars in organic solvents have been reported as transesterification rather than esterification reactions. The displacement of the equilibrium towards products has been accomplished by using activated acyl donors [58] such as 2,2,2-trichloroethyl esters and, more often, enol esters. The use of enol esters, such as a vinyl or an isopropenyl ester, was, in fact, first reported in lipase-catalyzed reactions with sugars [59]. In the reaction, an unstable enol is liberated which instantaneously tautomerizes to the corresponding aldehyde or ketone, making the reaction irreversible. 

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