Lipase in organic media

Valivety, R.H., Hailing, P.J., Peilow, A.D., Macrae, A.R. 1994. Relationship between water activity and catalytic activity of lipases in organic media. Effects of supports, loading and enzyme preparation. Eur. J. Biochem. 222, 461 466. 

Racemic mixtures of carboxylic adds can be resolved by esterification using lipases in organic media. Figure 21 illustrates such a process when racemic a-bromopropionic acid is stereoselectively esterified with n-buta-nol using a lipase from the yeast Candida a/lindraceae (68). This reaction was carried out in virtually anhydrous hexane. 

Liaquat, M. and Apenten, R.K.O., Synthesis of low molecular weight flavor esters using plant seedling lipases in organic media, J. Food Sci., 65, 295, 2000. 

Goto, M., Kamiya, N., Miyata, M., and Nakashio, R, Enzymatic esterification by surfactant-coated lipase in organic media, Biotechnol. Prog., 10, 263-268, 1994. 

Ma F, Hanna MA (1999) Biodiesel production a review. Biores Technol 70 1-15 Magnusson A, Hull K, Holmquists M (2001) Creation of an enantioselective hydrolase by engineered substrate-assisted catalysis. J Am Chem Soc 123 4354 355 Manjon A, Iborra JL, Arocas A (1991) Short-chain flavour ester synthesis by immobilized lipase in organic media. Biotechnol Lett 13 339-345 Margolin A (1996) Novel crystalline catalysts. TIBTECH 14 223-230... 

Goto, M. Noda, S. Kamiya, N. Nakashio, F. Enzymatic resolution of racemic ibuprofen by surfactant-coated lipases in organic media. Biotechnol. Lett, 1996, 18, 839-844. 

Adlercreutz, P. (2013) Immobilisation and application of lipases in organic media. Chem. Soc. Rev. doi 10.1039/c3cs35446f, in press. 

Tang, L., Zhang, H., Shehate, M. M. et al. (2000) Study of the S5mthesis of ascorbate fatty acid esters catalyzed by immobilized lipase in organic media. Biotechnol. Appl. Biochem., 32, 35-39. 

The wide substrate tolerance of lipases is demonstrated by the resolution of organometallic substrates [129-131]. The presence of tin, selenium, or tellurium in the structure of secondary alcohols does not inhibit the lipase activity and enantiopure organometallic alcohols were obtained by acylation in organic media (Figure 6.48). 

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. 

A chemoenzymatic methodology has been developed using indium-mediated allylation (and propargylation) of heterocyclic aldehydes under aqueous conditions followed by Pseudomonas cepacia lipase-catalyzed enantioselective acylation of racemic homoallylic and homo-propargylic alcohols in organic media.192... 

Syntheses of aliphatic polyesters by fermentation and chemical processes have been extensively studied from the viewpoint of biodegradable materials science. Recently, another approach to their production has been made by using an isolated lipase or esterase as catalyst via non-biosynthetic pathways under mild reaction conditions. Lipase and esterase are enzymes which catalyze hydrolysis of esters in an aqueous environment in living systems. Some of them can act as catalyst for the reverse reactions, esterifications and transesterifications, in organic media [1-5]. These catalytic actions have been expanded to... 

The concept of zeolite action was tested in a particular reaction where the enzyme is exposed from the beginning to an acidic environment the esterification of geraniol with acetic acid catalyzed by Candida antarctica lipase B immobilized on zeolite NaA [219]. Lipases have been used for the hydrolysis of triglycerides and due to their ambivalent hydrophobic/hydrophilic properties they are effective biocatalysts for the hydrolysis of hydrophobic substrates [220]. When water-soluble lipases are used in organic media they have to be immobilized on solid supports in order to exhibit significant catalytic activity. 

Catalytic transfer hydrogenations for the reduction of carbon-carbon double bonds are illustrated in Scheme 4.18. Reductions of azide functionalities to amines with lipases suspended in organic media under microwave conditions have also been reported [206]. 

Miyazawa, T., Kurita, S., Ueji, S., Yamada, T. and Shigeru, K., Resolution of mandelic acids by lipase-catalyzed transesterifications in organic media inversion of enantioselectivity mediated by the acyl donor. J. Chem. Soc. Perkin Trans. 1, 1992, 18, 2253-2255. 

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

Lipase, which is highly useful for kinetic resolution, however, has a limitation for use in DKR in that it carmot be used for (S)-configuration products. For this purpose, subtiHsin, a protease from Bacillus licheniformis, can replace lipase since it provides complementary enantioselectivity (Scheme 1.4). Subtilisin, however, has been much less frequently employed in resolution compared to lipase because it displays poor catalytic performance in organic media. Subtilisin is inferior to lipase in several properties such as activity, enantioselectivity and stability. Accordingly, the use of the enzyme usually requires some special treatments for activation and stabilization before use. For example, the treatment of subtilisin with surfactants has enhanced substantially its activity and stability up to a synthetically useful level. 

Kvittingen, L., Sjursnes, B., Anthonsen, T. and Hailing, P. (1992) Use of salt hydrates to buffer optimal water level during lipase catalysed synthesis in organic media A practical procedure for organic chemists. Tetrahedron, 48,2793-2802. 

Surfactants have been used to solubilise lipases in organic solvents (Okahata and Mori, 1997). One method starts with mixing aqueous solutions of the surfactant and the enzyme. The enzyme-surfactant complex precipitates and can subsequently be dissolved in organic media. Several surfactants have been tested and especially good results have been obtained with dialkyl glucosyl glutamates. In one case it was shown that the complex consisted of one enzyme molecule surrounded by approximately 150 surfactant molecules. 

Lipases may also be most advantageously applied for kinetic enantiomer separation of racemic alcohols either through esterification in organic media or by hydrolysis of the corresponding acetates in water90 1161. 

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 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]. 

The lyophilization of enzymes from solutions containing salts or amphiphilic compounds is known to increase the activity in organic media by up to several orders of magnitude. Thus, the transesterification activity of a-chymotrypsin was increased 82-fold by co-lyophilization with pentaglyme [75]. The colyophilization of lipases and (poly ethylene)glycol (PEG) led to an enhanced transesterification activity in various ionic liquids [76, 77]. 

Lipase AH (Amano, Najoya, Japan) from Pseudomonas sp. and Lipase PS (Amano, Najoya, Japan) from Pseudomonas cepacia in particular have been demonstrated to be useful for production, especially for enantiomerically pure secondary alcohols in organic media (Hirose, 1995) (Chapter 12, Section 12.6). Despite almost identical amino acid sequences in the two enzymes (their proteins differ only in 16 amino acid residues), these two lipases exhibit opposite enantioselectivity. As no crystal structure of these enzymes was available, all experiments were based on knowledge of the primary sequence. 

Not only do enzymes work in anhydrous organic media, but in this unnatural milieu they acquire remarkable properties such as enhanced stability, altered substrate and enantiomeric specificities, molecular memory, and the ability to catalyze unusual reactions (Klibanov, 1989). Regarding the latter point, hydrolases, such as lipases, catalyze not only transesterifications in organic media but also other types of reactions, including esterification, aminolysis, thiotransesterification, and oximolysis. As all of these reactions compete with hydrolyses, which tend to dominate in aqueous media, some of them proceed to an appreciable extent only in non-aqueous solvents. 

R. Bovara, G. Carrea, G. Ottolina, and S. Riva, Effects of water activity on Vm3X and Km of lipase catalyzed transesterification in organic media, Biotechnol. Lett. 

Hi) Enzymatic Esterifications. A major alternative to the classical basic catalysis is the use of enzymes for esterification, in particular with proteases and lipases.110 112 To make these enzymes, which normally hydrolyze amide or ester linkages, work in the reverse direction of esterification, the reactions have to be performed in organic media, with only the small amount of water necessary to preserve their active conformation. In such reactions, the difficulty is to find those conditions of solvent and temperature compatible with both the solubility of the substrates and the stability and the activity of the enzyme.113,114 In the case of sucrose (Scheme 9), most proteases lead selectively to monoesters at position p nl-ii5,ii6 Ypggg reactions are often performed in DMF, but examples in Me2SO, which is much less toxic, have also been reported, despite the ability... 

The lipase-catalyzed asymmetric transesterification was performed using isopropenyl acetate in organic media affording the ( -alcohol in high enantiomeric excess (>99% ee). 

Shang, C-S. Hsu, C-S. Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media. Biotechnology Letters 2003, 25 413-416. 

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