Lipase-catalyzed hydrolytic reactions

The enzymatic reactions are performed in the wells of microtiter plates (96-format) in water (as in lipase-catalyzed hydrolytic reaction of (.S)-13C-4/(i )-4), which is followed by a standard automatic extraction step. Depending on the particular substrate to be assayed and the type of solvent used, it may be necessary to remove the solvent. However, this is often not necessary. For enzymatic reactions in organic medium, solvent extraction is not required. For NMR analysis such solvents as CDC13, Dg-DMSO, or D20 are used. A minimum of about 6 pmol of substrate/product per milliliter of solvent is needed. Although the flow-through cell system does not need too much solvent (about 1L in 24 h), the solvents can be mixed with the undeuterated form in 1 9 ratios to reduce costs. 

The NHase/amidase system in R. erythropolis A4 was also used to transform cyano cyclitols into their carboxyhc acid analogs [21, 22]. As these transformations were coupled to lipase-catalyzed hydrolytic reactions, they are described in detail in the section focused on artificial cascades (see the following paragraphs). 

Two different types of enzymatic time-temperature integrators are described. The first, under the tradename of I-point, is based on a lipase-catalyzed hydrolysis reaction (125). The lipase is stored in a nonaqueous environment containing glycerol. The indicator contains two components that are mixed when the indicator is activated. The operating principle is as follows Upon activation, two volumes of reagents are mixed with each other. Lipase is thereby exposed to its substrate, here a triglyceride. At low temperatures there will be almost no hydrolytic reaction. As the temperature increases, hydrolysis accelerates and protons are liberated. A pH indicator is dissolved in the system. The indicator is selected to shift color after a certain amount of acid has been liberated by the enzyme-catalyzed process. Since the catalytic activity is influenced both by temperature and time, this indicator strip is said to be a time-temperature integrator. 

Hydrolases Catalyze hydrolytic reactions Esterases, lipases, proteases, glycosidases, phosphatases... 

The hydrolytic reaction catalyzed by lipases generally takes place at the oil-water interface. The hydrolytic activity in the presence of triacylglycerols is the basis characteristic of lipases (Table 4). However, hydrolytic activity of different lipases may not always give a good indication of the potential synthesis activity (Table 4). 

Lipases are able to catalyze many hydrolytic and esterification reactions in the presence of different substrates. The type of substrate is a key factor affecting the activity and productivity of lipase-catalyzed reactions. 

In a lipase-catalyzed reaction, the acyl group of the ester is transferred to the hydroxyl group of the serine residue to form the acylated enzyme. The acyl group is then transferred to an external nucleophile with the return of the enzyme to its preacylated state to restart the catalytic cycle. A variety of nucleophiles can participate in this process. For example, reaction in the presence of water results in hydrolysis, reaction in alcohol results in esterification or transesterification, and reaction in amine results in amination. Kirchner et al.3 reported that it was possible to use hydrolytic enzymes under conditions of limited moisture to catalyze the formation of esters, and this is now becoming very popular for the resolution of alcohols.4... 

The main drawback of the processes catalyzed by oxidoreductases in comparison with hydrolytic reactions mediated by lipases resides in the necessity of cofactors for this reason microorganisms are normally used instead of isolated enzymes. However, they present a very important advantage-the possibility to obtain only one enantiomer in the reaction-so that higher yields can be achieved than in normal kinetic resolution processes catalyzed by lipases. 

Lipases generally show low hydrolytic activity when their ester substrates are dissolved in aqueous media and present in imimeric form. A pronounced increase in activity is observed when the substrate concentration reaches the solubiUty limit and a separate phase is formed. In the case of surfactants this impUes that a possible increase in activity can be expected above the CMC. Attempts to investigate how the hydrolysis is affected by micelUzation were made for the linear surfactant 1 of Fig. 4. The CMC of this surfactant is 10 mM, and a marked change in the activity of the MML is indeed observed when this concentration is exceeded, see Fig. 6. The initial reaction is faster (steeper slope) above the CMC. When CALB was used to catalyze the reaction, no increase of the reaction rate was observed above the CMC. It was also found that the rate, expressed in moles of surfactant consumed per minute, was independent of the start concentration (same slope). A tentative explanation to the fact that the MML but not the CALB-catalyzed hydrolysis is accelerated by the presence of micelles may be that MML but not CALB is able... 

Resolution of racemic alcohols by acylation (Table 6) is as popular as that by hydrolysis. Because of the simplicity of reactions in nonaqueous media, acylation routes are often preferred. As in hydrolytic reactions, selectivity of esterification may depend on the structure of the acylating agent. Whereas Candida cylindracea lipase-catalyzed acylation of racemic-Ot-methylbenzyl alcohol [98-85-1] (59) with butyric acid has an enantiomeric value E of 20, acylation with dodecanoic acid increases the E value to 46 (16). Not only acids but also anhydrides are used as acylating agents. Pseudomonasjl. lipase (PFL), for example, catalyzed acylation of OC-phenethanol [98-85-1] (59) with acetic anhydride in 42% yield and 92% selectivity (74). 

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)). 

Enzymes can generally catalyze their reactions in both directions. This is of special interest for the preparative use of hydrolytic enzymes such as lipases/esterases, where the reverse reaction can be performed in organic solvents. 

Lipases catalyze the hydrolytic cleavage of triglycerides into fatty acids and glycerol, or into fatty acids and mono or di-glycerides, at oil interfaces in nature. However, this hydrolytic reaction can be reversed and transformed into reactions of esterification (inter or transesterification), alcoholysis or aminolysis by engineering the medium polarity or the water content of the medium. Therefore, substrates for lipases can be esters, like the natural triglyceride substrates in hydrolytic reactions or, if the reaction is reversed, carboxylic acids, alcohols, amines or esters. The reaction medium not only determines the direction of the reaction (hydrolytic or synthetic), but also determines the solubility and stability of lipase substrates. Therefore, lipase activity and selectivity are strongly influenced by reaction medium. 

Enzymes have been used for biocatalysis in organic solvents since the early 1980s, and in ILs since 2000 [17]. As biological catalysts, enzymes accelerate reactions but do not affect the equilibrium distribution. Hence, hydrolytic enzymes that, as an example, under normal circumstances in aqueous solutions hydrolyze esters and amides, will, when placed in water-free conditions, also catalyze the reverse reaction, the condensation of an acid with an alcohol or an amine, to give esters and amides, respectively. Further, under water-free conditions hydrolytic enzymes can accept alternative nucleophiles to catalyze reactions such as transesterifications. An important industrial example of this is the lipase-catalyzed transesterification of triglycerides to obtain fats with a desired melting point [18]. 

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. 

Lipases are used to catalyze hydrolytic, esterification and transesterification reactions. These reactions alter the physical properties of fats and oils and thereby produce a wide range of products (Mukherjee, 1990). Lipases have also been used for the kinetic resolution of Isomers of alcohols or fatty acids (Hills, et 1990). The lipases... 

Since Klibanov [21] nonstrated the first example of enzymatic esterification and transesteri ation in organic solvents, many enzymatic readions in anhydrous media have beat reported. It is now well known that some hydrolytic enzymes are stalde even in organic solvents and can be used for certain types of am nsatioii reactions that are difficult or impossible to achieve in aqueous media [22-28]. Most of these reactions ccmcern lipase-catalyzed esterifications and transesteriftations [29]. 

BMY 14802 88 has also been prepared by lipase-catalyzed resolution of racemic BMY 14802 acetate ester 90 [148]. Lipase from Geotrichum candidum (GC-20 from Amano Enzyme Co.) catalyzed the hydrolysis of acetate 90 to / -(+)-BMY 14802 (Fig. 28) in a biphasic solvent system in 48% reaction yield (theoretical maximum yield is 50%) and 98% e.e. The rate and enantioselectivity of the hydrolytic reaction was dependent on the organic solvent used. The enantioselectivity E values) ranged from 1 in the absence of solvent to more than 100 in dichloromethane and toluene. S-(—)-BMY 14802 was also prepared by the chemical hydrolysis of undesired BMY 14802 acetate obtained during enzymatic resolution process. 

In the case of lipase [64], acid anhydride residues of activated PM with a molecular weight of 13,000 were coupled to the amino groups on the surface of the enz3nme molecule. PM-lipase thus prepared was soluble and active in hydrophobic media, and catalyzed not only an ester hydrolytic reaction in aqueous solution but also ester synthesis or ester exchange reactions in organic solvents. These characteristics of PM-lipase were the same... 

Enzyme-catalyzed regioselective or enantioselective reactions are useful techniques in the organic synthesis of various bioactive substances. Numerous investigators have report on such reactions in aqueous [82] or microaqueous oi anic environments [83], Recently, the authors reported the regioselective deacetylation of peracetylated monosaccharide derivative by PEG-lipase from C. cylindracea [84]. Using a series of peracetylated methyl hexopyranosides as the substrates, it was demonstrated that PEG-lipase catalyzed the ester hydrolytic reaction only at C-4 and C-6 and never hydrolyzed the acetyl groups at C-2... 

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