Amino acylases from Aspergillus oryzae

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... 

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%. 

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. 

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. 

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. 

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