Enzyme catalyzed reaction lipase

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

Kinetic resolutions by means of the selective formation or hydrolysis of an ester group in enzyme-catalyzed reactions proved to be a successful strategy in the enantioseparation of 1,3-oxazine derivatives. Hydrolysis of the racemic laurate ester 275 in the presence of lipase QL resulted in formation of the enantiomerically pure alcohol derivative 276 besides the (23, 3R)-enantiomer of the unreacted ester 275 (Equation 25) <1996TA1241 >. The porcine pancreatic lipase-catalyzed acylation of 3-(tu-hydroxyalkyl)-4-substituted-3,4-dihydro-2/7-l,3-oxazines with vinyl acetate in tetrahydrofuran (THF) took place in an enantioselective fashion, despite the considerable distance of the acylated hydroxy group and the asymmetric center of the molecule <2001PAC167, 2003IJB1958>. 

The monolithic stirrer reactor (MSR, Figure 2), in which monoliths are used as stirrer blades, is a new reactor type for heterogeneously catalyzed liquid and gas-liquid reactions (6). This reactor is thought to be especially useful in the production of fine chemicals and in biochemistry and biotechnology. In this work, we use cordierite monoliths as stirrer blades for enzyme-catalyzed reactions. Conventional enzyme carriers, including chitosan, polyethylenimine and different are used to functionalize the monoliths. Lipase was... 

Knez, Z. Habulin, M. Lipase Catalyzed Esterification in Supercritical Carbon Dioxide. In Biocatalysis in Non-Conventional Media Tramper, J., Vermiie, M. H., Beeftink, H. H., Eds. Elsevier Science Amsterdam, 1992, pp. 401-406. Knez, Z. Habulin, M. Krmelj, V. Enzyme Catalyzed Reactions in Dense Gases. J. Supercrit. Fluids 1998, 14, 17-29. 

Some element reactions for BDF production can be applied widely to oil and fat processing. Since enzyme-catalyzed reactions proceed efficiently under mild conditions, they are suitable for the treatment of materials including unstable compounds. Furthermore, enzymes can convert only a desired compound to its other molecular form because of the strict substrate specificity compared with chemical catalysts. We hope that much attention will be focused on the superiority of enzyme, and that lipase reactions will be applied more and more as the practical process in the oil and fat industry. 

Lipases are a special class of esterases that also catalyze the hydrolytic cleavage of ester bonds, but differ in their substrate spectrum. Lipases have the special capability to catalyze the hydrolysis of water-insoluble substrates such as fats and lipids. Like many other enzyme-catalyzed reactions, the ester hydrolysis is a reversible process, which allows using lipases and other esterases for the synthesis of esters. The use of lipases as catalysts in synthetic chemistry is described elsewhere in this chapter. 

Efficient biochemical processes were developed for the preparation of the two optically active pyrethroid insecticides by a combination of enzyme-catalyzed reactions and chemical transformations. These are based on the findings that a lipase from Arthrobacter species hydrolyzes the acetates of the two important secondary alcohols of synthetic pyrethroids with high enantioselectivity and reaction rate. The two alcohols are 4-hydroxy-3-methy1-2-(2 -propynyl)-2-cyclopentenone (HMPC) and a-cyano-3-phenoxybenzyl alcohol (CPBA). The enzyme gave optically pure (R)-HMPC or (S)-CPBA and the unhydrolyzed esters of their respective antipodes. 

Mensah et al. studied the esterification of propionic acid (P) and isoamyl alcohol (A) to isoamyl propionate and water in the presence of the lipase enzyme [P. Mensah, J. L. Gainer, and G. Carta, Biotechnol. Bioeng., 60 (1998) 434.] The product ester has a pleasant fruity aroma and is used in perfumery and cosmetics. This enzyme-catalyzed reaction is shown below ... 

Water plays different functions in enzyme-catalyzed reactions. A certain level of water in the reaction medium is required to maintain a layer of water around the enzyme molecules to maintain their activity. This water is important for lipases in that it maintains their active three-dimensional conformational state. Water... 

Enzyme-catalyzed reactions are used to produce human mUkfat substitutes for use in infant formulas (46-48). Acidolysis reaction of a mixture of tripalmitin and unsaturated fatty acids using a i -l,3-specific lipase as a biocatalyst afforded TAGs derived entirely from vegetable oils rich in 2-position palmitate with unsaturated fatty acyl groups in the sn- and sn-3 positions (44). These TAGs closely mimic the fatty acid distribution found in human mUkfat, and, when used in infant formula instead of conventional fats, the presence of palmitate in the sn-2 position of the TAGs improved digestibility of the fat and absorption of other important nutrients such as calcium (46, 49). 

An interesting case of competitive adsorption is provided by the pH effect for an acidic enzyme catalyzed reaction (e.g. lipase). If the pH is too low protonation of an essential amino acid residue of the enzyme may occur with loss in activity. At very high pH again loss in the number of active enzyme molecules may occur, but now because of possible deprotonation of essential residues. Since we are dealing with competitive adsorption effects, at low substrate concentrations, where the rate is controlled by a maximum in the rate will occur at the pH where competitive adsorption is the least. As can be deduced from (3.39) at high substrate concentration the reaction rate will not be affected. 

Although, supercritical carbon dioxide has the advantage of being nontoxic and abundant, it is practically immiscible with water. Therefore, supercritical fluids used as the reaction medium in enzyme-catalyzed reactions include fluoroform, sulfur hexafluoride, and ethane, while lipases are the enzymes utilized in such reactions. ... 

Holmes et al. reported the first enzyme catalyzed reactions in water-in-CO2 microemulsions (67). Two reactions, a lipase-catalyzed hydrolysis and a lipoxygenase-catalyzed peroxidation, were demonstrated in water-in-C02 microemulsions using the surfactant di(l/7,l/7,5/7-octafluoro- -pentyl) sodium sulfosuccinate (di-HCF4). A major concern of enzymatic reactions in CO2 is the pH of the aqueous phase, which is approximately 3 when there is contact with CO2 at elevated pressures. Holmes et al. examined the ability of various buffers to maintain the pH of the aqueous solution in contact with CO2. The biological buffer 2-(A-morpholino)ethanesulfonic acid sodium salt (MES) was the most effective, able to maintain a pH of 5, depending on the pressure, temperature, and buffer concentration. The activity of the enzymes in the water-in-C02 microemulsions was comparable to that in a water-in-heptane microemulsion stabilized by the surfactant AOT, which contains the same head group as di-HCF4. 

Many chemical reactions carried out in supercritical fluid media were discussed in the first edition, and those developments are included in total here after some recent work is described. In the epilogue (chapter 13) of the first edition we made reference to one of the author s work in enzyme catalyzed reactions in supercritical fluids that was (then) soon to appear in the literature. The paper (Hammond et al., 1985) was published while the first edition was in print, and as it turned out, there was a flurry of other activity in SCF-enzyme catalysis many articles describing work with a variety of enzymes, e.g., alkaline phosphatase, polyphenol oxidase, cholesterolase, lipase, etc., were published starting in mid 1985. Practical motivations were a potentially easier workup and purification of a product if the solvent is a gas (i.e., no liquid solvent residues to contend with), faster reaction rates of compounds because of gas-like transport properties, environmental advantages of carbon dioxide, and the like. 

An additional strategy employed by Sih and co-workers involved sequential enzyme-catalyzed reactions. Pseudomonas lipases were found to tolerate a wide range of substrates although the enantioselectivity was generally only moderate. However, by first performing a methanolysis of the oxazolinone followed by a separate enzyme-catalyzed hydrolysis under kinetic resolution conditions, a highly enantio-merically enriched product could be obtained, as shown in Fig. 9-211491. 

In reaction (25) racemization was realized by madelate racemase. However, this transformation is still a process carried out in two batches and therefore, not a dynamic kinetic resolution but certainly the starting point for further investigations by combining a lipase- and a second enzyme-catalyzed reaction in order to perform real dynamic kinetic resolution. 

Enzymes. The use of enzymes is an integral part of many important processes in food production. Hydrolytic enzymes especially are employed on an industrial scale, mainly because no costly regeneration of cofactors is required, in contrast to oxidoreductases. The release of specific fatty acid profiles by lipases in the course of cheese manufacture, or the cleavage by proteases of peptide fragments in protein hydrolyzates that otherwise will cause bitterness are examples for the impact of enzyme-catalyzed reactions on the final flavor of foods (5). 

In this work we have shown that it is possible to use an ezyme to catalyze the polycondensatioh reaction to form polyamides. The large number of polyamides that have been made indicate diat lipases from Candida antarctica and Mucor miehei are rather nonspecific and can be used generally for polyamide synthesis. In a particular example, a water-soluble polyamide has been produced from dimethyl adipate and ethylene triamine via this enzyme-catalyzed reaction at 50-110°C. The enzymatic polymerization is easy to do and... 

From a practical point of view the improved solubility of nonpolar reactants is very important. Low reactant solubility in water is a frequently encountered problem in enzyme-catalyzed reactions, leading to low production capacity per vessel volume. Also, the possibility of using hydrolytic enzymes such as lipases, esterases, peptidases, and amylases to catalyze condensation reactions instead of bond cleavage is of considerable practical importance because it opens new ground for enzyme-catalyzed processes. 

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