Lipase catalyzed hydrolysis

Figure 2.6 Mechanism of lipase-catalyzed hydrolysis of esters [34].

Pseudomonas aeruginosa lipase-catalyzed hydrolysis of racemic ester 23 proceeds with very low enantioselectivity E = 1.1). Sequential use of error-prone PCR, saturation mutagenesis at chosen spots and DNA shuffling resulted in the formation of a mutant whose enantioselectivity was over 50. 

FIG. 3 Hydroperoxy-fatty acid S5mthesis from triacylglycerol with successive lipase-catalyzed hydrolysis and lipoxygenation. 

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. 

As the majority of enzyme systems in aqueous-organic biphasic media, lipase-catalyzed hydrolysis uses substrates that are insoluble in aqueous media and yield hydrophobic products (Fig. 3). Therefore, the aqueous phase serves as a biocatalyst reservoir. 

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 ... 

Yang and Russell [7] made comparison of lipase-catalyzed hydrolysis in three different systems organic, biphasic, and reversed micelles. They affirmed that water content is an important factor that distinctly affects every system. Their results demonstrated that activity of lipase in organic-aqueous biphasic media was lower than that obtained in reversed micelles. However, better productivities were obtained in biphasic media, which were the most suitable environment. 

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... 

S. Allenmark, A. Ohlsson, Enantioselectivity of Lipase-Catalyzed Hydrolysis of Some 2-Chloroethyl 2-Arylpropanoates Studied by Chiral Reversed-Phase Liquid Chromatography , Chirality 1992, 4, 98-102. 

H. Uzawa, Y. Nisbida, H. Ohrui, H. Meguro, A New Approach to Determine the Stereospecificity in Lipase Catalyzed Hydrolysis Using Circular Dichroism (CD) Lipases Produce Optically Active Diglycerides from Achiral Triglycerides , Biochem. Biophys. Res. Commun. 1990, 168, 506-511. 

The third group of target molecules comprises chiral carboxylic acid and their derivatives esters, amides and nitriles. Enantiomerically pure esters are prepared in an analogous manner to the enantiomerically pure alcohols discussed earlier [i.e. by esterase- or lipase-catalyzed hydrolysis or (trans)esterification]. However, these reactions are not very interesting in the present context of cascade reactions. Amides can be produced by enantioselective ammoniolysis of esters or even the... 

Scheme 10.4 Synthesis of a GABA inhibitor mediated by a lipase-catalyzed hydrolysis.

Fig. 5. Time course of the lipase-catalyzed hydrolysis of the (R)- and (5)-ester 1 measured with a UV/ Vis plate reader, (a) WT lipase from P. aeruginosa, (b) improved mutant in the first-generation epPCR 16).

Figure 6.7 Lipase catalyzed hydrolysis of dUtiazam intermediate.

The first example of chemoenzymatic DKR of allylic alcohol derivatives was reported by Williams et al. [37]. Cyclic allylic acetates were deracemized by combining a lipase-catalyzed hydrolysis with a racemization via transposition of the acetate group, catalyzed by a Pd(II) complex. Despite a limitation of the process, i.e. long reaction times (19 days), this work was a significant step forward in the combination of enzymes and metals in one pot Some years later, Kim et al. considerably improved the DKR of allylic acetates using a Pd(0) complex for the racemization, which occurs through Tt-allyl(palladium) intermediates. The transesterification is catalyzed by a lipase (Candida antarctica lipase B, CALB) using isopropanol as acyl acceptor (Scheme 5.19) [38]. 

Figure 9.8 Schematic reaction mechanism of a lipase-catalyzed hydrolysis of butyric acid ester.

The Amano researchers found not just drastically different enantiomeric excesses in the Pseudomonas sp. (Amano AH) lipase-catalyzed hydrolysis of methylene-oxypropionyl or -pivaloyl diesters, but a near-total reversal of specificity. Whereas in cyclohexane the different esters yielded half-esters with 88.8-91.4% e.e. -specificity for a triple mutant ( FVL ) of Amano PS lipase, the same transformation with the same enzyme in diisopropyl ether (DIPE) yielded between 68.1 and > 99% e.e. of the S-product (Figure 12.10) (Hirose, 1992,1995). 

W. Ladner and G. M. Whitesides, Lipase-catalyzed hydrolysis as a route to esters of chiral epoxy alcohols, J. Am. Chem. Soc. 1984, 106, 7250-7251. 

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. 

Itoh, T. Takagi, Y. Murakami, T. Hiyama, Y. Tsukube. H, (1996) Crown ethers as regulators of enzymatic reactions enhanced reaction rate and enantioselectivity in lipase-catalyzed hydrolysis of 2-cyano-l-methylethyl acetate J. Org. Chem. 61, 2158-2163. 

The reverse reaction was consisting of lipase-catalyzed hydrolysis of the racemic acetate afforded the (R)-alcohol in high enantiomeric excess (>99% ee). 

Yoshida, N. Aono, M. Tsubuki, T. Awano, K. Kobayashi, T. Enantioselective synthesis of a chiral pyridazinone derivative by lipase-catalyzed hydrolysis. Tetrahedron Asymmetry 2003, 14, 529-535. 

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)). 

HOLLOW-FIBER MEMBRANE REACTOR FOR THE LIPASE CATALYZED HYDROLYSIS SYNTHESIS OF DILTIAZEM... 

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... 

Chen, J.-P., Chang, K.-O. 1993. Lipase-catalyzed hydrolysis of milk fat in lecithin reverse micelles. J. Ferment. Bioeng. 76, 98-104. 

Marangoni, A.G 1994. Candida and Pseudomonas lipase-catalyzed hydrolysis of butteroil in the absence of organic solvents. J. Food Sci. 59, 1096-1099. 

Shirai, K., Jackson, R.L. 1982. Lipoprotein lipase-catalyzed hydrolysis of />-nitrophenyl butyrate. J. Biol. Chem. 257, 1253-1258. 

It should be noted that in the lipase-catalyzed hydrolysis the reaction mixture is biphasic consisting of water and water-insoluble oily substrate, and that the reaction is easily controlled by... 

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