L-aminoacylase

Although this enzymatic process fills only a niche in the L-lysine market, it is a successful example of a general method for amino acid resolution. It has some superior features compared to the Tanabe L-aminoacylase approach. The L-lysine can be extended to non-protein amino acids such as the use of P. putida aminopeptidase to resolve DL-homophenylalanine to produce precursors for the anti-hypertensive dmg Enalapril. A similar approach has also been used for the production of L-cysteine from DL-2-amino-A2-thiazohne-4-caiboxylate using Sarcina lucea, which is remarkable in that both isomers form L-cysteine. 

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],... 

Fig. 4 Enzymatic resolution of racemic N-acetyl amino acids using an L-aminoacylase.

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 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. 

In summary, a broad range of large-scale applicable biocatalytic methodologies have been developed for the production of L-amino acids in technical quantities. Among these industrially feasible routes, enzymatic resolutions play an important role. In particular, L-aminoacylases, L-amidases, L-hydantoinases in combination with L-carbamoylases, and /l-lactam hydrolases are efficient and technically suitable biocatalysts. In addition, attractive manufacturing processes for L-amino acids by means of asymmetric (bio-)catalytic routes has been realized. Successful examples are reductive amination, transamination, and addition of ammonia to rx,/fun-saturated carbonyl compounds, respectively. 

Table 12.3-11. Comparison of the substrate specificity of D-, and L-aminoacylase in Streptomyces tuirus and Streptomyces olivaceusl2S].

All microorganisms producing D-aminoacylases commonly produce L-aminoacy-lases as well. Therefore, to reach high optical purity of the D-amino acids produced from the respective N-acetyl-D,L-amino acids, the D-aminoacylases have to be separated from the L-aminoacylases (Table 12.3-13). However, this is a disadvantage in view of an industrial application since additional purification steps lead to more expensive enzymes and thus add costs to the whole production process. This is one of several reasons why it is widely accepted today that the production of D-amino acids by enzyme-catalyzed hydrolysis of D,L-hydantoins seems to be more promising than the D-aminoacylase route via N-acetyl-D,L-amino acids. The enzyme-catalyzed synthesis of D-amino acids from the respective D,L-hydantoins is described in Chapter 12.4. 

Tokuyama et al.11721 found that most of acylamino acid racemase-producing strains produce not only acylamino acid racemase but also aminoacylases one of either d- or L-aminoacylase or both of them. Moreover, acylamino acid racemase shows the optimum pH at around 8.0, which is close to that of aminoacylases. Therefore, N-acylamino acid can be converted as a whole into l- or D-amino acids in one step by means of microbial cells of appropriate strains producing either l- or D-aminoacylase in addition to acylamino acid racemase. 

More recently, the discovery and commercialization of L-aminoacylase from Thermococcus litorali was a product of the LINK project between Chirotech Technology and the University of Exeter. The L-aminoacylase of T. litoralis had broad substrate specificity for the hydrolysis of N-acylated a-amino acids, with respect to both the side chain and the N-acyl group. It is especially useful for the enantiospecific hydrolysis of acyl groups, particularly N-benzoyl groups of a-amino acids. This can be used to advantage in synthetic processes that require the enantiospecific deprotection of racemates [29]. 

The enzyme activity of the L-aminoacylase of T. litoralis was maximal at 85°C but with a half life (ti/2) limited to 1.7 h. Reduction of the operating temperature to 70°C yields a significant increase in... 

L-Aminoacylase Alcohols Pseudomonas sp. BA2 Entrapment in calcium alginate beads [73]... 

Immobilized L-aminoacylase (Scheme 6.19) was used to deacylate a range of compounds using this approach. 

Ngamsom B., Hickey A.M., Greenway G.M., Littlechild J.A., Watts R, Wiles C. Development of a high throughput screening tool for biotransformations utilising a thermophilic L-aminoacylase enzyme. J. Mol. Catal. B.Enzymat. 2010 ... 

The hydrogenation technology is now well established and eminently scalable, and indeed has been used by us to provide a very diverse set of amino acids. However, there are some cases where the use of L-aminoacylase is still the method of choice for inserting the chirality by resolution. These are generally where the side chain is susceptible to further transformation by the chemocatalyst, or where it might poison the catalyst, or where the enamide synthesis is difficult. Enamide hydrogenation substrates are generally synthesized... 

We recently used L-aminoacylase to provide the amino acids shown in Scheme 8. In these instances either the aldehydes were not available or they performed very poorly in the Erlenmeyer chemistry for enamide synthesis. These resolutions can be integrated very readily into an overall process, where the unwanted isomer can be racemized and recycled by way of treatment with acetic anhydride and racemization via the azalactone. 

Scheme 8 Amino acids synthesized by L-aminoacylase resolution.

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