Amino acylases from Aspergillus

F. Schneider, Purification and partial charaderization of amino-acylase from Aspergillus oryzae, in Metalloproteins,... 

The substrate specificity of the amino acylase from Aspergillus oryzae is very broad, and a wide range of proteinogenic and non-proteinogenic N-acetyl and N-chloroacetyl amino acids are transformed in the presence of the L-amino acylase. The enzyme membrane reactor (Fig. 5) is operated continuously as a loop reactor, and the enzyme is retained by an ultrafiltration hollow-fiber membrane (molecular weight cut off 10000 Dalton). 

Optical resolution of racemic amino acids (methionine, phenylalanine, tryptophan, valine) by the action of L-specific amino acylase from Aspergillus oryzae (Tanabe Seiyaku Co., Ltd.). The theoretical productivity of 1000 liter immobilized amino acylase columns ranges from 214 kg per day for L-Ala to 715 kg per day for L-Met. 

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... 

In an attempt to find a suitable source of amino acid acylase, Sanzyme, essentially an alpha-amylase complex extracted from a strain of Aspergillus oryzae was explored for the presence of this enzyme. Aspergillus oryzae is known to contain amylase, however, there is no report available for the presence of this enzyme in Sanzyme strain. It was found out that the crude Sanzyme samples contain 7.8 units of amino acylase activity [2], where one unit is defined as 1 micro mole of acetyl methionine hydrolysed/hr/mg of enzyme. It was also found out that the purified form of amino acylase from Sanzyme contain 28.4 units of activity [3] which is high when compared to any other amino acylase reported in literature. 

L-Amino acid acylase from Aspergillus melleus (Amano acylase 30000) was used to hydrolyze racemic 6-acetoxybuspirone to (5)-6-hydroxybuspirone 2 in 96% enantiomeric excess (EE) after 46% conversion. The remaining (l )-6-acetoxy-buspirone with 84% EE was converted to (l )-6-hydroxybuspirone 2 by acid hydrolysis [28]. The EE of both enantiomers could be improved to more than 99% by crystallization as a metastable polymorph. Direct hydroxylation of buspirone to (5 )-6-hydroxbuspirone by Streptomyces antibioticus ATCC 14980 has also been described [28]. 

Enzymatic methods offer in principle the possibility of a direct enantioselective synthesis of amino acids. Enzymes are often used for separation of racemic mixtures, as examplified in the case of methionine. Although racemic methionine is adequate for the animal feed sector, other applications require the enan-tiomerically pure (L)-form. For the resolution, (L)-acylases from Aspergillus sp. are often used, since they can accept a broad spectrum of substrates, are highly active, and very stable under the production conditions. [62]... 

An alternative to extraction crystallization is used to obtain a desired enantiomer after asymmetric hydrolysis by Evonik Industries. In such a way, L-amino acids for infusion solutions or as intermediates for pharmaceuticals are prepared [35,36]. For example, non-proteinogenic amino acids like L-norvaline or L-norleucine are possible products. The racemic A-acteyl-amino acid is converted by acylase 1 from Aspergillus oryzae to yield the enantiopure L-amino acid, acetic acid and the unconverted substrate (Figure 4.7). The product recovery is achieved by crystallization, benefiting from the low solubility of the product. The product mixture is filtrated by an ultrafiltration membrane and the unconverted acetyl-amino acid is reracemized in a subsequent step. The product yield is 80% and the enantiomeric excess 99.5%. 

Acylases have also been applied to the kinetic resolution of amines. Aminoacylase I from Aspergillus melleus was used for the resolution of a range of arylalkylamines and amino alcohols via acylation with methyl 2 methoxyacetate (Figure 14.13) [19]. Excellent chemoselectivity was also observed in all cases, as the amino group was preferentially acylated in the presence of a primary alcohol functionality. However, poor to moderate enantioselectivity was observed, with values <10. The best result (E = 9.3) was obtained with 1 aminoindane 10 during the conversion to ester 38. 

Subsequently, the L-amino acid, l-2, is separated and isolated by a crystallization step, and the remaining N-acetyl D-amino acid is recycled by, e.g., thermal racemization. As a preferred L-aminoacylase, the amino acylase I from Aspergillus oryzae [E.C.3.5.1.14] turned out to be particularly useful. 

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]. 

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. 

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. 

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