Amidase and

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Both cis- and trans-chrysanthemic nitriles and amides were resolved into highly enantiopure amides and acids by Rhodococcus sp. whole cells [85]. The overall enantioselectivity of reactions of nitriles originated from the combined effects of a higher (lJ )-selective amidase and a (IJ )-selective nitrile hydratase (Figure 6.29). Chrysanthemic acids are related to constituents of pyrethrum flowers and insecticides. 

We also found that the occurrence of aldoxime dehydratase is as wide as that for nitrile-degrading enzymes such as nitrile hydratase, amidase and/or nitri-lase. All of the nitrile degraders hitherto isolated contained aldoxime dehydratase activities. The author would like to propose that the pathway in which aldoximes are successively degraded via nitrile could be named as the aldoxime-nitrile pathway (Fig. 1). 

Hydrolases Epoxide hydrolase Amidases and esterases Glycosidases... 

Carboxylesterases and amidases catalyze hydrolysis of carboxy esters and carboxy amides to the corresponding carboxylic acids and alcohols or amines. In general those enzymes capable of catalyzing hydrolysis of carboxy esters are also amidases, and vice versa (110). The role of these enzymes in metabolsim of drugs and insecticides has been reviewed (111, 112). In addition to the interest in mammalian metabolism of drugs and environmental chemicals, microbial esterases have been used for enantioselective hydrolyses (113, 114). 

Metabolic reactions in the liver and the small intestine are well documented [24]. However, only sparse information is available on drug metabolism in the eolon. Drug metabolism in the colon can be brought about by the host enzymes in the epithelial cells or by the microbial enzymes in the gut flora. Metabolie aetivities in the wall of the colon can be attributed to the eytochrome P450, esterases, amidases, and various transferases [25]. Reductive drug metabolism does not appear to be important at this site. 

The chemical stability of the amide bond is high. When the surfactant containing an amide bond was subjected to 1 M sodium hydroxide during five days at room temperature, only 5% of the amide surfactant was cleaved. The corresponding experiment performed in 1 M HCl resulted in no hydrolysis. The amide bond was, however, found to be slowly hydrolyzed when lipase from Candida antarctica or peptidase was used as catalyst. Amidase and lipase from Mucor miehei was found to be ineffective. Despite the very high chemical stability, the amide surfactant biodegrades by a similar path in the... 

Two other immobilized enzymes have reached large scale industrial application penicillin amidase and lipase. 

Hydrolyses Esters and Amides. The plasma, liver, kidney, and intestines contain a wide variety of nonspecific amidases and esterases. These catalyze the metabolism of esters and amides, ultimately leading to the formation of amines, alcohols, and carboxylic acids. Kinetically, amide hydrolysis is much slower than ester hydrolysis. These hydrolyses may exhibit stereoselectivity. 

Core structures of four B-lactam antibiotic families. The ring marked in each structure is the -lactam ring. The penicillins are susceptible to bacterial metabolism and inactivation by amidases and lactamases at the points shown. Note that the carbapenems have a different stereochemical configuration in the lactam ring that apparently imparts resistance to lactamases. Substituents for the penicillin and cephalosporin families are shown in Figures 43-2 and 43-6, respectively. 

Table 12.6 summarizes results obtained in the synthesis of antibiotics in the presence of undissolved substrates catalyzed by penicillin amidase, and... 

The title retains the trivial name for enzymes with the systematic name of urea amidohydrolase and the Enzyme Commission code number of EC 3.5.1.5. Ureases are hydrolases acting on C-N bonds (nonpeptide) in linear amides and thus belong to a group that includes glutaminase, form-amidase, and formyltetrahydrofolate deformylase. The title is plural to emphasize that urease activity may be exhibited by several protein species. Urease, singular, has come to mean by common usage, that particular enzymic protein first crystallized by Sumner from jack bean... 

The hydrolysis of esters by esterases and of amides by amidases constitutes one of the most common enzymatic reactions of xenobiotics in humans and other animal species. Because both the number of enzymes involved in hydrolytic attack and the number of substrates for them is large, it is not surprising to observe interspecific differences in the disposition of xenobiotics due to variations in these enzymes. In mammals the presence of carboxylesterase that hydrolyzes malathion but is generally absent in insects explains the remarkable selectivity of this insecticide. As with esters, wide differences exist between species in the rates of hydrolysis of various amides in vivo. Fluoracetamide is less toxic to mice than to the American cockroach. This is explained by the faster release of the toxic fluoroacetate in insects as compared with mice. The insecticide dimethoate is susceptible to the attack of both esterases and amidases, yielding nontoxic products. In the rat and mouse, both reactions occur, whereas sheep liver contains only the amidases and that of guinea pig only the esterase. The relative rates of these degradative enzymes in insects are very low as compared with those of mammals, however, and this correlates well with the high selectivity of dimethoate. 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

The serine hydrolase family is one of the largest and most diverse classes of enzymes. They include proteases, peptidases, lipases, esterases, and amidases and play important roles in numerous physiological and pathological process including inflammation [53], angiogenesis [54], cancer [55], and diabetes [56]. This enzyme family catalyzes the hydrolysis of ester, thioester, and amide bonds in a variety of protein and nonprotein substrates. This hydrolysis chemistry is accomplished by the activation of a conserved serine residue, which then attacks the substrate carbonyl. The resulting covalent adduct is then cleaved by a water molecule, restoring the serine to its active state [57] (Scheme 1). 

Immobilized forms of penicillin amidases and acylases have replaced whole-cell biocatalysts for the production of 6-APA and 7-ACA as they can be reused many times, in some cases for over 1000 cycles. Another major advantage is the purity of the enzyme, lacking the /3-lactamase contaminants often present in whole cells. The productivity of these biocatalysts exceeds 2000 kg prod-uct/kg catalyst. A typical process for the production of 6-APA employs immobilized penicillin G acylase covalently attached to a macroporous resin. The process can be run in either batch or continuous modes. The pH of the reaction must be maintained at a value between 7.5 and 8 and requires continuous adjustment to compensate for the drop caused by the phenylacetic acid generated during the course of the reaction. Recycle reactors have been used, as they allow both pH control and the use of packed bed reactors containing the immobilized catalyst. The enzymatic process is cheaper, although not... 

A particularly elegant solution for the assay of proteases and acylases is offered by the fluorogenic detection of free amino acids by decomplexation of copper from calcein, which removes its quenching effect This principle has been used for assays of acylases, amidases, and proteases (Scheme 1.13) [52, 53]. For the case of proteases combinatorial assays are particularly in demand for testing multiple peptides in parallel and determining the cleavage specificity [54]. New solutions... 

The starting material for the acylase process is a racemic mixture of N-acetyl-amino acids 20 which are chemically synthesized by acetylation of D, L-amino acids with acetyl chloride or acetic anhydride in alkaU via the Schotten-Baumann reaction. The kinetic resolution of N-acetyl-D, L-amino acids is achieved by a specific L-acylase from Aspergillus oryzae, which only hydrolyzes the L-enantiomer and produces a mixture of the corresponding L-amino acid, acetate, and N-acetyl-D-amino acid. After separation of the L-amino acid by a crystallization step, the remaining N-acetyl-D-amino acid is recycled by thermal racemization under drastic conditions (Scheme 13.18) [47]. In a similar process racemic amino acid amides are resolved with an L-spedfic amidase and the remaining enantiomer is racemized separately. Although the final yields of the L-form are beyond 50% of the starting material in these multistep processes, the effidency of the whole transformation is much lower than a DKR process with in situ racemization. On the other hand, the structural requirements for the free carboxylate do not allow the identification of derivatives racemizable in situ therefore, the racemization requires... 

Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospeciflcity of over 99%. Despite limited enzyme-substrate compatibility, specific conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using a-chymotrypsin, a peptidase isolated from bovine pancreas. 

Hydrolyses of esters or amides are common reactions catalyzed by ubiquitous esterases, amidases, and... 

Hydrolyses They are of minor significance. Here amidases and glycosidases are important enzymes. Intestinal bacteria with hydrolytic activity split mainly phase II metabolites in the large intestine, with the result that absorbable catabolites then enter the enterohepatic circulation. 

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