Amidase enzymes

APA may be either obtained directly from special Penicillium strains or by hydrolysis of penicillin Q with the aid of amidase enzymes. A major problem in the synthesis of different amides from 6-APA is the acid- and base-sensitivity of its -lactam ring which is usually very unstable outside of the pH range from 3 to 6. One synthesis of ampidllin applies the condensation of 6-APA with a mixed anhydride of N-protected phenylglydne. Catalytic hydrogenation removes the N-protecting group. Yields are low (2 30%) (without scheme). 

Rhodococcus erythropolis NCIMB 11540 has been employed as biocatalyst for the conversion of (R)- or (.S )-cyanohydrins to the corresponding (R)- or (S)-a-hydroxycarboxylic acids with an optical purity of up to >99% enatiomeric excess (ee) [27-29] the chiral cyanohydrins can separately be produced using hydroxynitrile lyase from Hevea braziliensis or from Prunus anygdalis [30]. Using the combined NHase-amidase enzyme system of the Rhodococcus erythropolis NCIMB 11 540, the chiral cyanohydrins were first hydrolyzed to the... 

Bacteria, Aberdeen, UK), was found to have a highly active nitrile hydratase/amidase enzyme system, based on whole-cell biotransformation experiments. Subsequently, individual enzymes (nitrile hydratase and amidase) from this strain were cloned and expressed separately in E. coli However, distribution of some strains or other materials from these public collections may be limited, usually as a result of the restrictions on their commercial use imposed by intellectual property rights. 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, enzymes that catalyze the hydrolysis of hydantoins 7-11,147). Because synthetic hydantoins are accessible by a variety of chemical syntheses, including Strecker reactions, enan-tioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, because hydantoins are easily racemized chemically or enzymatically by appropriate racemases, dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases have been reported 147). However, if asymmetric induction is poor or if inversion of enantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of L-methionine as part of a whole cell system E. coll) (Figure 22) 148). 

PAN was for a long time thought to be resistant to microbial attack. However, various bacteria that produced nitrile-converting enzymes were isolated from waste-waters of factories producing PAN fibre. Eor example, a nitrile hydratase/amidase enzyme system was studied from Mesorhizobium sp. E28 [68]. Also, bacteria (namely Ralstonia solanacearum and Acidovorax avenae) were used for the removal of acrylic acid from such waste-waters [69]. Later, on the basis of NMR... 

Generally, enzymatic hydrolysis of nitriles to the corresponding acids can either proceed stepwise, which is the case for catalysis by the nitrile hydratase/amidase enzyme system, or in one step in the case of nitrilases. Both systems have been investigated for surface hydrolysis of PAN [10], Complete hydrolysis with either system was monitored by quantification of ammonia and/or polyacrylic acid formed as a consequence of hydrolysis of nitrile groups [70-72], As a result, considerable increases in colour levels (e.g. 156% for commercial nitrilase) were found upon dyeing [72],... 

During our longstanding interest in the biohydrolysis of nitriles, we found that whole cell preparations of certain Rhodococci, such as R. erythropolis A4 (formerly R. equi A4), R. sp. R312, and R. erythropolis NCIMB 11540, containing the nitrile hydratase/amidase enzyme system, are efficient catalysts for stereoselective microbial hydrolysis of N-protected carbocyclic P-amino nitriles ( )-la-( )-4a, to P-amino acids lc-4c and amides lb-4b, respectively (Scheme 15.1) [33, 34]. 

In contrast to the various fermentation and bioconversion approaches that have used enzyme stereoselectivity to syntlresize L-phenylalanine as a single isomer, additional methods have been developed that rely on the same stereoselectivity to resolve racemic mixtures of chemically synthesized amino acids. One such general approach, which has been successfully commercialized for L-phenylalanine production, relies on cleavage of the L-isomer of a D,L-a-amino acid amide mixture by an enantiospecific amidase enzyme. The general procedure operated... 

Microorganisms which produce a nitrile hydratase also seem to synthesize one or more amidase enzymes [linear amide hydrolase (E.C. 3.5.1)] thus enabling them to hydrolyze nitriles to the corresponding acids in a two-step reaction ... 

E = glutaryl amidase, enzyme from Escherichia coli... 

Thus the hydrolytic reactions of biological interest, as far as drugs are concerned, are primarily those of esters and amides. In the case of esters, esterase enzymes in the plasma and liver inactivate (detoxify) a relatively nonpolar molecule by splitting it into two polar and therefore water-soluble components, an alcohol and an acid. Analogously amidase enzymes split amides into acids and amines (or ammonia). Table 3-3 gives several examples of drug hydrolyses. 

The drug gets metabolized via oxidation of the eyelohexane ring to the eorresponding /(-hydroxy and ffj-hydroxy metabolites. Besides, a minor metabolite whieh oeeurs invariably essentially involves the N-aeetyl structural analogue that eventually results, from the aeetylation of the primary amine caused due to the hydrolysis of the amide system exclusively by amidase enzymes. 

Incubate cells for 2 min at room tanperature in 0.5 mL/well saponin solution (140 mM potassinm glutamate-HCl, pH 6.8, with 1 mg/mL ATP and 0.1 mg/mL saponin) to permeabilize the cell monbranes. Optionally, 50 xM PMSF can be added to the saponin solution to irreversibly inhibit amidase enzymes if endo-cannabinoids are to be added or their endogenous activity is to be studied. 

Aliphatic nitriles are often metabolized in two stages. First they are converted to the corresponding carboxamide by a nitrile hydratase and then to the carboxylic acid by an amidase enzyme (a protease) [626]. 

In 2003, Griengl s group reported the hydrolysis of cyanohydrins by treatment with bacterial cells of Rhodococcus erythropolis NCIMB 11540, which have a highly active nitrile hydratase/amidase enzyme system. In this manner, (R)-2-chloromandelic acid and (R)-2-hydroxy-4-phenylbutyric acid, two important pharmaceutical intermediates, could be prepared in high optical and chemical yield after short reaction times (3 and 1.5 h, respectively) (Scheme 3.43). 

Amidase enzymes (EC 3.5.1.4) belong to the hydrolase family of enzymes and catalyze the hydrolysis of monocarboxylic amides to their corresponding acid products. Conventional amidase classification is done according to substrate profile or is based on their amino acid sequence homology [58]. 

Amidase enzymes catalyze the hydrolysis of amide bonds with considerable divergence noted within the family with respect to substrate spedficity. All amidase enzymes, however, maintain the core a,p,a structure, where the topologies of the C and N terminal halves are similar. 

Aliphatic amidase enzymes demonstrate sequence similarity to the nitrilase superfamily thus indicating some form of evolutionary relationship. These amidases contain a Glu-Lys-Cys catalytic triad and exist as homotetrameric or homohexameric sttuctures that function via a ping-pong (bi-bi) reaction mechanism [60, 61]. 

Longer reaction times led to the diamide product only, with no acid being formed. Further conversion of 2-cyano-3-furan-2-yl-but-2-enamide was attempted using both whole cells of R. rhodochrous ATCC BAA-870 as well as a commercial amidase enzyme preparation (Pseudomonas aeruginosa. Sigma-Aldrich), but neither showed conversion of the substrate (unpublished results). 

---