Lipases ester formation

Ester formation catalyzed by lipase (Mucor miehei) in conjunction with hydrogenation catalyzed by a rhodium complex Sol-gel immobilization of both catalysts... 

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. 

Lipase has been used in organic solvents to produce useful compounds. For example, Zark and Klibanov (8) reported wide applications of enzymes to esterification in preparing optically active alcohols and acids. Inada et al (9) synthesized polyethylene glycol-modified lipase, which was soluble in organic solvent and active for ester formation. These data reveal that lipases are very useful enzymes for the catalysis different types of reactions with rather wide substrate specificities. In this study, it was found that moditied lipase could also synthesize esters and various lipids in organic solvents. Chemically moditied lipases can help to solve today s problems in esteritication and hopefully make broader use of enzymatic reactions that are attractive to the industry. 

Esters are widespread in fruits and especially those with a relatively low molecular weight usually impart a characteristic fruity note to many foods, e.g. fermented beverages [49]. From the industrial viewpoint, esterases and lipases play an important role in synthetic chemistry, especially for stereoselective ester formations and kinetic resolutions of racemic alcohols [78]. These enzymes are very often easily available as cheap bulk reagents and usually remain active in organic reaction media. Therefore they are the preferred biocatalysts for the production of natural flavour esters, e.g. from short-chain aliphatic and terpenyl alcohols [7, 8], but also to provide enantiopure novel flavour and fragrance compounds for analytical and sensory evaluation purposes [12]. Enantioselectivity is an impor-... 

J. Baratti, and C. Triantaphylides, lipase-catalyzed ester formation in organic solvents. An easy preparative resolution of a-substituted cyclohexanols, Tetrahedron Lett. 1985, 26, 1857-1860. 

It is generally stated that biocatalysis in organic solvents refers to those systems in which the enzymes are suspended (or, sometimes, dissolved) in neat organic solvents in the presence of enough aqueous buffer (less than 5%) to ensure enzymatic activity. However, in the case of hydrolases water is also a substrate and it might be critical to find the water activity (a ) value to which the synthetic reaction (e.g. ester formation) can be optimized. Vahvety et al. [5] found that, in some cases, the activity of Candida rugosa lipase immobihzed on different supports showed the same activity profile versus o but a different absolute rate. With hpase from Burkholderia cepacia (lipase BC), previously known as lipase from Pseudomonas cepacia, and Candida antarctica lipase B (CALB) it was found that the enzyme activity profile versus o and even more the specific activity were dependent on the way the enzyme was freeze dried or immobihzed [6, 7]. A comparison of the transesterification activity of different forms of hpase BC or CALB can be observed in Tables 5.1 and 5.2, respectively. 

Enzymatic reactions in organic media have been a major issue in the field of biocatalysis over the last two decades. Carboxylesterases (mostly lipases) have been used in monophasic organic solution under controlled values of water activity (ajj for catalyzing ester formation the reaction equilibrium can be shifted towards ester formation by interesterification or transesterification [1]. Direct esterification is often hampered by water formation, which may increase o , thus negatively influencing the equihbrium. 

Much of the current interest in making simple derivatives of (+ )-castanospermine (239) can be traced to a seminal publication in 1989, which showed that the alkaloid s anti-HIV activity could be increased by as much as twenty times upon esterification (216). Positionally selective acylation procedures usually involve sequential protection, acylation, and deprotection steps e.g. the preparation of esters at the C-6 and C-7 (217) or the C-8 hydroxy groups (218). Also of interest are procedures that take advantage of enzyme-catalyzed transesterification with activated esters, e.g. the use of subtilisin for ester formation at C-1, pancreatic porcine lipase for preferential reaction at C-6 and C-7 (219-221), and cross-linked enzyme crystals (CLECs) of subtilisin for making the potentially valuable antitumor agent 1-0-butanoylcastanospermine (222). A cautionary note was sounded, however, when it was observed that 6-0-acyl castanospermine esters could equilibrate to a mixture of... 

By far the commonest reaction used in kinetic resolution by enzymes is ester formation or hydrolysis. Normally one enantiomer of the ester is formed or hydrolysed leaving the other untouched so one has the easy job of separating an ester from either an acid or an alcohol. There are broadly two kinds of enzymes that do this job. Lipases hydrolyse esters of chiral alcohols with achiral acids such as 119 while esterases hydrolyse esters of chiral acids and achiral alcohols such as 122. Be warned this definition is by no mans hard and fast If the unreacted component (120 or 123) is wanted, the reaction is run to just over 50% completion, to ensure complete destruction of the unwanted enantiomer, while if the reacted component (121 or 124) is wanted it is best to stop short of 50% completion so that little of the unwanted enantiomer reacts. 

There is an inherent problem with either type of enzyme as the reactions are reversible. One way to make the reaction run in the direction of ester formation is to use a non-aqueous solvent (you may be surprised that enzymes function in, say, heptane, in which they are insoluble, but lipases do). One way to make the reaction run in the other direction is to make the alcohol component an enol so that, on hydrolysis, it gives the aldehyde or ketone and does not reverse. 

Good and bad features of enzymes as catalysts Organisms Reduction of Ketones by Baker s Yeast Ester Formation and Hydrolysis by Lipases and Esterases... 

At this moment, fractionating reactors are mostly studied and applied outside the fine-chemical field. Examples are the large-scale production of the fuel ethers MTBE and TAME via reactive distillation. Also, biocatalytic studies have been performed. Malcata and co-workers investigated the integration of ester formation by Upases and distillative separation of the final products ester and water [44]. A number of synthesis reactions have been studied such as the esterification of ethanol and acetic acid to form ethyl acetate and water [45] in an SMB reactor with chemocatalysts (acidic ion exchange resins). Another, fairly similar appUcation was presented by Kawase et al. [46] to manufacture an ester from 2-phenylethanol. Mensah and Carta [47] used a chromatography column with lipases immobilised on resin to produce esters as well. 

Lipase Ester hydrolysis, -formation, Many stable enzymes. Low predictability of State of the art... 

In a typical example, the fatty acid and the PEG were mixed at a molar ratio of 1.9 1. Novozym 435 lipase (0.5-1 gram per kg substrate) was added. At 60°C for 20 hours under vacuum (10-20mm Hg), the acid number was determined to be 8.5-10. C-NMR indicated the conversion of the acid carbon (178 ppm) to the ester carbon (174 ppm), and H-NMR spectra were used to quantify the ester formation by integrating the proton signals of -O-C-CH2OH (3.3-3.6 ppm), -O-C-CH2OCO-R (4.0-4.2 ppm) and CH3- of fatty acids and fatty esters (0.70-0.90 ppm). 

Lipase Polyol-Fatty Acid Esters Formation of ester bonds between fatty acyl group and hydroxyl(s) of polyol, such as AAAGs and saccharide-fatty acid esters Hayes, 2004 Watanabe and Shimoda, 2009 Pyo and Hayes, 2009... 

Lipase-catalyzed intermolecular condensation of diacids with diols results in a mixture of macrocycUc lactones and liuear oligomers. Interestingly, the reaction temperature has a strong effect on the product distribution. The condensation of a,(D-diacids with a,(D-dialcohols catalyzed by Candida glindracea or Pseudomonas sp. Upases leads to macrocycUc lactones at temperatures between 55 and 75°C (91), but at lower temperatures (<45°C) the formation of oligomeric esters predorninates. Optically active trimers and pentamers can be produced at room temperature by PPL or Chromobacterium viscosum Upase-catalyzed condensation of bis (2,2,2-trichloroethyl) (+)-3-meth5ladipate and 1,6-hexanediol (92). 

The 2-ethoxyethanol was a by-product, as shown in Figure 5.13. The formation rate of 2-ethoxyethanol was the same as the conversion rate of the (S)- or (R)-ibuprofen ester one mole of 2-ethoxyethanol was formed when one mole of ester was catalysed. A known concentration of 2-ethoxyethanol was added in the organic phase before the start of the reaction for product inhibition. The plots of the kinetics for the free lipase system are presented in Figure 5.17 and immobilised enzyme (EMR) in Figure 5.18, respectively. The Kw value was 337.94 mmoFl 1 for the free lipase batch system and 354.20 mmoll 1 for immobilised... 

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. 

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]. 

Reaction with lipoprotein lipase results in the loss of approximately 90% of the triacylglycerol of chylomicrons and in the loss of apo C (which remrns to HDL) but not apo E, which is retained. The resulting chy-lotnicron remnant is about half the diameter of the parent chylomicron and is relatively enriched in cholesterol and cholesteryl esters because of the loss of triacylglycerol (Figure 25-3). Similar changes occur to VLDL, with the formation of VLDL remnants or IDL (intermediate-density lipoprotein) (Figure 25-4). 

Both intact carotenoids and their apolar metabolites (retinyl esters) are secreted into the lymphatic system associated with CMs. In the blood circulation, CM particles undergo lipolysis, catalyzed by a lipoprotein lipase, resulting in the formation of CM remnants that are quickly taken up by the liver. In the liver, the remnant-associated carotenoid can be either (1) metabolized into vitamin A and other metabolites, (2) stored, (3) secreted with the bile, or (4) repackaged and released with VLDL particles. In the bloodstream, VLDLs are transformed to LDLs, and then HDLs by delipidation and the carotenoids associated with the lipoprotein particles are finally distributed to extrahepatic tissues (Figure 3.2.2). Time-course studies focusing on carotenoid appearances in different lipoprotein fractions after ingestion showed that CM carotenoid levels peak early (4 to 8 hr) whereas LDL and HDL carotenoid levels reach peaks later (16 to 24 hr). 

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