Kidney amino acid acylases

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

Amino acid acylases have wide distribution in nature. They are found in animal tissues like kidney [1], molds, bacteria, yeast, and its activity is traced in plants including mushrooms. They serve as effective tools in resolution of L-amino acids from DL-amino acid racemic mixtures. L-amino acids are naturally occurring, physiologically active amino acids which are the building blocks of all the proteins. 

Amino acid acylase Porcine kidney, Aspergillus melleus... 

L-Amino acid acylase from kidneys DL-Acylamino acid — L-Amino acid + D-Acyl-amino acid Production of essential amino aids for human and animal nutrition. 

The preparation of protected derivatives of D-allo- and L-fl//o-threonine by enzymatic hydrolysis of 5(47/)-oxazolones using hog kidney acylase has also been described. This methodology has been extended to a wide variety of amino acids and, at present, constitutes a general procedure to prepare non-quaternary... 

Resolution of Racemic Amines and Amino Acids. Acvlases (EC 3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungal Aspergillus acylasc (AA) are commercially available, inexpensive, and stable. Amino alcohols can be resolved by a number of pathways, including hydrolysis, esterification, and transesterification. 

Acylase (acylase I aminoacylase N-acetyl amino acid amidohydrolase E.C. 3.5.1.14), is one of the best-known enzymes as far as substrate specificity (Chenault, 1989) or use in immobilized (Takahashi, 1989) or membrane reactors (Wandrey, 1977, 1979 Leuchtenberger, 1984 Bommarius, 1992a) is concerned however, its exact mechanism or 3D structure is still not known (Gentzen, 1979 1980). Acylase is available in large, process-scale quantities from two sources, porcine kidney and the mold Aspergillus oryzae. 

Enzymes are chiral molecules with specific catalytic activities. For example, when an acylated amino acid is treated with an enzyme like hog kidney acylase or car-boxypeptidase, the enzyme cleaves the acyl group from just the molecules having the natural (l) configuration. The enzyme does not recognize D-amino acids, so they are unaffected. The resulting mixture of acylated D-amino acid and deacylated L-amino acid is easily separated. Figure 24-5 shows how this selective enzymatic deacylation is accomplished. 

Selective enzymatic deacylation. An acylase enzyme (such as hog kidney acylase or carboxypeptidase) deacylates only the natural L-amino acid. 

The use of enzymes to separate enantiomers. For example, the enantiomers of an amino acid can be acylated and then treated with hog kidney acylase. The enzyme hydrolyzes the acyl group from the natural L-amino acid, but it does not react with the D-amino acid. The resulting mixture of the free L-amino acid and the acylated D-amino acid is easily separated, (p. 1169)... 

Enzymatic Kinetic Resolution of N-Acyl Amino Acids Coupled with Racemization by N-Acyl Amino Acid Racemase Acylases are enzymes hydrolysing the N-acetyl derivatives of amino acids. They require the free carboxylate for activity and have long been used for the kinetic resolution of amino acids. The unreacted enantiomer is usually racemized in a separate step by treatment with acetic anhydride. While acylases from hog kidney have an L-specificity, bacterial acylases with L- and D-specificity of various origins have been isolated and used for the kinetic resolution of N-acetyl amino acids. An industrial process for the production of L-Met and other proteinogenic and non-proteinogenic L-amino acids such as L-Val, L-Phe, L-Norval, or L-aminobutyric acid has been established. Currently, several hundred tons per year of L-methionine are produced by this enzymatic conversion using an enzyme membrane reactor [46]. 

Resolution of DL-alanine (1) is accomplished by heating the N-acetyl derivative (2) in weakly alkaline solution with acylase, a proteinoid preparation from porcine kidney containing an enzyme that promotes rapid hydrolysis of N-acyl derivatives of natural L-amino acids but acts only immeasurably slowly on the unnatural o-isomers. N-Acetyl-DL-alanine (2) can thus be converted into a mixture of l-( + )-alanine (3) and N-acetyl-o-alanine (4). The mixture is easily separable into the components, because the free amino acid (3) is insoluble in ethanol and the N-acetyl derivative (4) is readily soluble in this solvent. Note that, in contrast to the weakly levorotatory o-( - )-alanine (a 14.4°), its acetyl derivative is strongly dextrorotatory. 

As with the D-aminoacylases from Streptomyces sp. the enzymes from Alcaligenes strains have a preference for hydrophobic N-acetyl-amino acids. In this respect, they are similar to the L-specific acylase I from kidney preparations and Aspergillus sp. The Alcaligenesfaecalis enzyme prefers the N-acyl-D-amino acid derivatives from Met, Phe and Leu[951. If a high-affinity substrate residue occupies the hydrophobic side-chain pocket the enzyme even deacylates D-Met methyl esters or N-Ac-D-Met-Xaa dipeptide derivatives. 

Resolution can also be achieved by the use of an enzyme. Much in the way that kinetic resolution works, an enzyme can react selectively with one enantiomer of the racemate and leave the other enantiomer unreacted and resolved. Partictilarly successful are acylase enzymes which have been applied in a number of synthetically useful transformations [9]. For example, porcine kidney acylase I has been used to prepare unnatural amino acids in almost complete enantiomeric purity (Scheme 4.4) [10]. 

Aminoacylases catalyze the hydrolysis of A-acyl amino acid derivatives, with the acyl groups preferably being acetyl, chloroacetyl, or propionyl. Alternatively, the corresponding A-carbamoyl- and A-formyl derivatives can be used [132], Enzymes of the amino acylase type have been isolated from hog kidney, and from Aspergillus or Penicillium spp. [133-135]. The versatility of this type of enzyme has been demonstrated by the resolution of racemic iV-acetyl tryptophan, -phenylalanine, and -methionine on an industrial scale using colunm reactors (Scheme 2.15) [136, 137]. 

There are two procedures used by chemists that imitate the natural pathway. In one, actual microorganisms are used in fermentation reactions that can directly produce some L-amino acids. In the other, enzymes are used to react with one enantiomer of a racemic pair. As you might guess, it is usually the L isomer that is eaten by the enzyme, because enzymes have evolved in an environment of L isomers. So it is usually the D isomers that are ignored by enzymes. In an extraordinarily clever procedure, this preference for natural, L, enantiomers can be used to achieve a kinetic resolution (Fig. 23.15). The mixture of enantiomers is first acetylated to create amide linkages. An enzyme, hog-kidney acylase, then hydrolyzes the amide linkages of only L-amino acids. So, treatment of the pair of acetylated amino acids leads to formation of the free L-amino acid. The acylat-ed D enantiomer is unaffected by the enzyme, which has evolved to react only with natural L-amino acids. The free L-amino acid can then easily be separated from the residual D acetylated material. 

The catalytic conversion of N-chloroacetylated derivatives of p-amino acids using porcine kidney acylase type I showed significant activity, with the (S)-enantiomer of the acid being formed [35]. A 96% ee together with a 46% conversion was obtained when using a racemic p-methoxyphenyl-substituted substrate, whereas a 95% ee... 

Wada, E., Handa, M., Imamura, K. etal. (2002) Enzymatic synthesis of AZ-acyl-L-amino acids in a glycerol-water system using acylase I from pig kidney. J. Am. Oil Chem. Soc., 79, 41-46. 

Separations may also be accomplished using chiral biological reagents, such as enzymes. Most enzymes, since they are composed of chiral amino acids, will accept only one enantiomer of a molecule as their substrate. Thus, the enzyme hog kidney acylase hydrolyzes only the natural enantiomer of amides. We will develop this idea further in Chapter 15, when we consider the reactions of carboxylic acid derivatives in more detail. 

This observation of the conjugated hydroxy acid is of contiderable interest in the light of the observations tlmt t-amino-substituted lysine is enzymically deaminated and transaminated (160) in the alpha petition and that an c-lysine acylase is present in pig kidney (161). 

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