Processes Using L-Amino Acylases

The enantioselective hydrolysis of racemic N-acetylated a-amino acids d,l-1 at De-gussa represents a long established large-scale process for the production of L-ami-no acids, l-2 [4]. This enzymatic resolution requires an L-aminoacylase as the biocatalyst. The starting materials for this process are readily available, since racemic N-acetyl amino acids d,l-1 can be economically synthesized by acetylation of racemic a-amino acids with acetyl chloride or acetic anhydride under alkaline conditions via the so-called Schotten-Baumann reaction [5]. The enzymatic resolution reaction of N-acetyl d,L-amino acids, d,l-1, is achieved by a stereospecific L-aminoacylase which hydrolyzes only the L-enantiomer and produces a mixture of the corresponding L-amino acid, l-2, acetate, and N-acetyl D-amino acid, d-1 (Fig. 4) [6], 

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. 

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). 

Since the early 1980s this process has been scaled-up to a production level of hundreds of tons per year. Fig. 6 shows the Degussa process for manufacturing l-methionine, l-6 [9 a]. The biocatalyst is produced in bulk quantities and its operational stability is high hence this continuous EMR-acylase process demonstrates high efficiency, especially on a large-scale [9]. 

In addition, the amino acylase process can be also applied in the production of other proteinogenic and non-proteinogenic L-amino acids such as L-valine and l-phenylalanine. It is worth noting that racemases have recently been developed by several companies which allow (in combination with the L-aminoacylases) an extension of the existing process towards a dynamic kinetic resolution reaction [10]. It should be mentioned that the same concept can be also applied for the synthesis of D-amino acids when using a D-aminoacylase as an enzyme. 

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