Hydrolysis lipase-catalyzed reaction

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. 

The simplest kinetic model applied to describe lipase catalyzed reactions is based on the classic Michaelis-Menten mechanism [10] (Table 3). To test this model Belafi-Bakd et al. [58] studied kinetics of lipase-catalyzed hydrolysis of tri-, di-, and mono-olein separately. All these reactions were found to obey the Michaelis-Menten model. The apparent parameters (K and V ) were determined for global hydrolysis. 

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... 

S-chiral compounds were synthesized through lipase-catalyzed reactions. For example, chiral sulfoxide was synthesized through lipase-catalyzed hydrolysis of the ester to give (R)-ester and (.V)-acid (Figure 18).19... 

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-... 

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.

This article describes our results on lipase-catalyzed enantio-selective hydrolysis of carboxylic acid esters of two industrially important alcohols related to synthetic pyrethroids in two-liquid phase systems. The two alcohols are 4-hydroxy-3-methyl-2-(2 -propynyl)-2-cyclopentenone (HMPC) and the cyanohydrin, a-cyano-3-phenoxybenzy1 alcohol (CPBA). The configurations, and 2y are given for the liberated stereoisomers of the two alcohols in our lipase-catalyzed reactions. 

In order to extend the two-enzyme system to other 2-hydroxy acids, a racemase with a broader activity was found in Lactobacillus paracasei. This was exploited for deracemization of 2-hydroxy-4-phenylbutanoic acid and 3-phenyllactic acid, which are important synthetic intermediates. In addition, in this case the procedure requires a kinetic resolution step and a successive racemization step. O-Acetyl derivatives of the absolute (S)-configuration can be obtained in two successive repeating cycles. Yields are around 60%. Of course the 0-acetyl derivatives of opposite configuration can be obtained when the lipase-catalyzed reaction is apphed in the hydrolysis direction. Obtaining the O-acetyl derivatives of the absolute (R)-configuration requires an additional acetylation step of the initially resolved and racemized (S)-hydroxy acid [12]. 

Modifications. Better lifetime fc Application of such lipases includes resoi acetylation, and hydrolysis of 1 lipase-catalyzed reactions proceed fast enantioselectively (by 3- to 9-fold).-... 

Both enzymatic esterification and hydrolysis are useful tools for resolution of racemic fluorinated building blocks. Among them, lipase-catalyzed reaction is reliable and most... 

The role of water in lipase-catalyzed reactions has been studied extensively, but still remains enigmatic. A minute amount of water is essential for establishing the proper tertiary structure, although a low water concentration enhances the stability of the enzyme. Water also engages in unwanted side-reactions such as hydrolysis and it inhibits the desired reaction by acting as a competitor for the enzyme domain . 

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. 

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. 

Based on the substrates involved in the lipase-catalyzed reactions, they can be classified into different categories esterification, hydrolysis, acidolysis, alcoholysis and interesterification (1). Direct esterification reaction may be enqjloyed for the preparation of stmctured lipids by reacting free fatty acids with glycerol. However, this process is not commonly used in stmctured lipid production. The major problem is that the water molecules are formed as a result of the esterification reaction. The water molecules so produced need to be removed in order to prevent the hydrolysis of the product. Hydrolysis is the... 

Fig. 3 Lipase-catalyzed reaction pathways of D-lactates (a) and L-lactates (b) acyl-enzyme intermediate formation steps a and e, subsequent dimer ftumation steps i), c, /, and g, and hydrolysis steps d and h. O denotes that the step takes place, whereas x denotes that the step does not take place. In steps b, c, d,f, g, and h, the lipase leaving group is omitted...

Kinetic models to describe lipase-catalyzed reaction mechanisms have been proposed, and most have been extensions of the model developed by Michaelis and Menten (1913). However, normal Michaelis-Menten kinetics do not apply to lipase-induced changes, because the substrates (lipids) are not water-soluble and the enzyme operates at an interface (Brockman, 1984). However, rate expressions for the hydrolysis of emulsified lipids catalyzed by immobilized lipases resemble the rate expressions modeled with Michaelis-Menten mechanisms (Benzonana and Desnuelle, 1965). The kinetics and mechanisms of reactions catalyzed by immobilized lipases have been reviewed by Malcata et al. (1990 1992). 

Polyesters have been obtained in organic medium by polyesterification of hydroxy acids,328,329 hydroxy esters,330 stoichiometric mixtures of diols and diacids,331-333 diols and diesters,334-339 and diols and cyclic anhydrides.340 Lipases have also been reported to catalyze ester-ester interchanges in solution or in die bulk at moderate temperature.341 Since lipases obviously catalyze the reverse reaction (i.e., hydrolysis or alcoholysis of polyester), lipase-catalyzed polyesterifications can be regarded as equilibrium polycondensations taking place in mild conditions (Scheme 2.35). 

Orthoformates have been used in the lipase-catalyzed esterification aimed at the kinetic resolution of racemic acids such as flurbiprofen, a nonsteroidal anti-inflammatory drug (Figure 6.18). Orthoformates trap the water as it is formed through hydrolysis, and therefore prevent the reverse reaction, and, at the same time, provide the alcohol for the esteriflcation [65]. 

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

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. 

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 ... 

Fixed-bed reactors employed for lipase-catalyzed hydrolysis and interesterification reactions are highly efficient and have been used on a large scale (Table 5). The two phases may flow through the reactor in the opposite or same directions. If no solvents are used, the effect of viscosity of some substrates (i.e., oil) may be minimized by employing high temperatures which lead to faster rates of inactivation of lipases. 

In addition to the reactions discussed above, there are still other alkyne reactions carried out in aqueous media. Examples include the Pseudomonas cepacia lipase-catalyzed hydrolysis of propargylic acetate in an acetone-water solvent system,137 the ruthenium-catalyzed cycloisomerization-oxidation of propargyl alcohols in DMF-water,138 an intramolecular allylindination of terminal alkyne in THF-water,139 and alkyne polymerization catalyzed by late-transition metals.140... 

Detailed studies on the lipase-catalyzed polymerization of divinyl adipate and 1,4-butanediol were performed [41-44]. Bulk polymerization increased the reaction rate and molecular weight of the polymer however, the hydrolysis of the terminal vinyl ester significantly limited the formation of the polyester with high molecular weight. A mathematical model describing the kinetics of this polymerization was proposed, which effectively predicts the composition (terminal structure) of the polyester. 

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