Lipase catalysis, hydrolysis

Fig. 3. Examples of hydrolysis reactions performed in microemulsions. Scheme 1 alkaline hydrolysis of 4-nitrophenyldiphenyl phosphate. Scheme 2 catalysis of phosphate ester hydrolysis by a iodosobenzoate. Scheme 3 alkali- and lipase-catalysed hydrolysis of 4-nitro-phenyldecanoate...

So far, only very little attention has been focussed on the use of zeolites in biocatalysis, i.e., as supports for the immobilization of enzymes. Lie and Molin [116] studied the influence of hydrophobicity (dealuminated mordenite) and hydrophilicity (zeolite NaY) of the support on the adsorption of lipase from Candida cylindracea. The adsorption was achieved by precipitation of the enzyme with acetone. Hydrolysis of triacylglycerols and esterification of fatty acids with glycerol were the reactions studied. It was observed that the nature of the zeolite support has a significant influence on enzyme catalysis. Hydrolysis was blocked on the hydrophobic mordenite, but the esterification reaction was mediated. This reaction was, on the other hand, almost completely suppressed on the hydrophilic faujasite. The adsorption of enzymes on supports was also intensively examined with alkaline phosphatase on bentolite-L clay. The pH of the solution turned out to be very important both for the immobilization and for the activity of the enzyme [117]. Acid phosphatase from potato was immobilized onto zeolite NaX [118]. Also in this study, adsorption conditions were important in causing even multilayer formation of the enzyme on the zeolite. The influence of the cations in the zeolite support was scrutinized as well, and zeolite NaX turned out to be a better adsorbent than LiX orKX. 

Kanerva et al. resolved ethyl esters of ten alicyclic (i-aminocarboxylic acids by lipase catalysis in organic solvents. The resolutions were based on acylation of the amino group at the / -stereogenic centre with various 2,2,2-trifluoroethyl esters. From the cis and trans racemic esters 42, all four enantiomers of 2-ACPC were prepared. The absolute configurations of 43 and 44 were proved by transformation to the known 2-ACPC enantiomers by hydrolysis and subsequent desalting with an anion-exchange resin [85]. 

Table III. Selective Hydrolysis of Peracetylated Sugars at the C-1 Position by Porcine Pancreatic Lipase Catalysis ...

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. 

Rizzarelli, R, Impallomeni, G., and Montaudo, G. (2004) Evidence for selective hydrolysis of aliphatic copolyesters induced by lipase catalysis. Biomacromolecules, 5, 433-444. 

Due to the similarity between the lipase and protease catalytic triads, the mechanism of lipase catalysis is similar to that of serine protease (Jaeger et al., 1999). a/p Hydrolases have a common catalytic mechanism of ester hydrolysis. First, serine is activated by deprotonation. Subsequently, the nucleophiUty of hydroxyl... 

Aleksandrovic, V., D. Poleti, and J. Djonlagic, Poly(ether-ester)s modified with different amounts of fumaric moieties. Polymer, 43(11) p. 3199. 2002. Rizzarelli, P., G. Impallomeni, and G. Montaudo, Evidence for Selective Hydrolysis of Aliphatic Copolyesters Induced by Lipase Catalysis. Biomacromolecules, 5(2) p. 433. 2003. 

Lipase is an enzyme that catalyzes the hydrolysis of fatty acid esters normally in an aqueous environment in living systems. However, some isolated lipases are stable in organic solvents and can act as catalyst for reverse reactions, esterifications, and transesterifications (Scheme 1) in organic media [1-5]. So far, chiral drugs, liquid crystals, acylated sugar-based surfactants, and functional triglycerides have been synthesized through lipase catalysis [6-10]. 

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. 

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]. 

Furthermore, in the system with coupled lipase and lipoxygenase, the production rate of HP is governed by the first enzymatic reaction and mass transfer. When TL,- is small (0 to 1 mM equiv. 3 mM LA), the kinetic curve has a sigmoid shape due to surface active properties of LA and HP [25]. Hydrolysis of TL and the increase of LA favor the transfer of LA. Such a transfer allows the lipoxygenase reaction to progress. Since lipox-ygenation consumes LA and produces HP, catalysis and transfer demonstrates a reciprocal influence. 

Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product. Many of the enzymes that involve covalent catalysis are hydrolytic enzymes these include proteases, lipases, phosphatases and also acetylcholinesterase. Some of these enzymes possess a serine residue in the active site, which reacts with the substrate to form an acylenzyme intermediate that is attacked by water to complete the hydrolysis (Fignre 3.3). 

The hydrolysis of lipids rarely occurs in a single homogeneous phase, and the behavior of lipases at membrane-solvent and micelle-solvent interfaces has been discussed in detail by Verger and Jain et aP See Micellar Catalysis... 

An interesting example of biocatalysis and chemical catalysis is the synthesis of a derivative of y-aminobutyric acid (GABA) that is an inhibitor for the treatment of neuropathic pain and epilepsy (Scheme 10.4). The key intermediate is a racemic mixture of cis- and trons-diastereoisomer esters obtained by a hydrogenation following a Horner-Emmons reaction. The enzymatic hydrolysis of both diaste-reoisomers, catalyzed by Candida antarctica lipase type B (CALB), yields the corresponding acid intermediate of the GABA derivative. It is of note that both cis- and trans-diastereoisomers of the desired enantiomer of the acid intermediate can be converted into the final product in the downstream chemistry [10]. 

Further findings relevant for the establishment of the chemical nature of enzymatic catalysis and technical apphcation followed within rather short time. Croft-Hill performed the first enzymatic synthesis, that of isomaltose, in 1898, allowing a yeast extract (df-glycosidase) to act on 40% glucose solution (Sumner and Somers, 1953). In 1900 Kastle and Loevenhart found that the hydrolysis of fat and other esters by lipases is a reversible reaction and that enzymatic synthesis can occur in a dilute mixture of alcohol and acid (Sumner and Myiback 1950). This principle was utilized for the synthesis of... 

Classic resolntion has been performed by formation of diastereomeiic salts which could be separated. In a series of synthetic steps and when resolution is one step, it is of utmost importance that the correct chirality is introduced at an early stage. When a racemate is subject to enzyme catalysis, one enantiomer reacts faster than the other and this leads to kinetic resolution (Figure 2.2c). Results of hydrolysis using lipase B from Candida antarctica (CALB) and a range of C-3 secondary butanoates are shown in Table 2.1. 

Moreno, Jose M. Samoza, A. del Campo, Carmen Liama, Emilio F. Sinisterra, Jose V. Organic reactions catalyzed by immobilized lipases. Part I. Hydrolysis of 2-aryl propionic and 2-aryl butyric esters with immobilized Candida cylindracea lipase. J. Mol. Catalysis A Chemical 1995, 95, 179-92. 

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