Aminoacylase process

In the fine chemicals industry, enantiomerically pure amino acids are mainly produced by the aminoacylase process, the amidase process, and the hydantoinase/ carbamoylase process, all three of which are suitable for I- and D-amino acids. Dehydrogenases and transaminases are now becoming established for reduction processes. 

Enzymatic hydrolysis is also used for the preparation of L-amino acids. Racemic D- and L-amino acids and their acyl-derivatives obtained chemically can be resolved enzymatically to yield their natural L-forms. Aminoacylases such as that from Pispergillus OTj e specifically hydrolyze L-enantiomers of acyl-DL-amino acids. The resulting L-amino acid can be separated readily from the unchanged acyl-D form which is racemized and subjected to further hydrolysis. Several L-amino acids, eg, methionine [63-68-3], phenylalanine [63-91-2], tryptophan [73-22-3], and valine [72-18-4] have been manufactured by this process in Japan and production costs have been reduced by 40% through the appHcation of immobilized cell technology (75). Cyclohexane chloride, which is a by-product in nylon manufacture, is chemically converted to DL-amino-S-caprolactam [105-60-2] (23) which is resolved and/or racemized to (24)... 

To develop a continuous process, the immobilisation of aminoacylase of Aspergillus oryzae by a variety of methods was studied, for example ionic binding to DEAE-Sephadex, covalent binding to iodo-acetyl cellulose and entrapment in polyacrylamide gel. Ionic binding to DEAE-Sephadex was chosen because the method of preparation was easy, activity was high and stable, and regeneration was possible. 

Since there is no commercially available D-aminoacylase, the production process of D-amino acids involves cloning of the D-aminoacylase and the whole cells containing the recombinant d-aminoacylase are used in biotransformation of /V-acetyl-D-amino acid, d-Amino acids can be generated in large quantities at low cost using whole-cell biotransformation [23]. 

This unnatural acid is used as a chiral intermediate for the synthesis of a number of products. Chemical asymmetric synthesis was very difficult and so the stereoselective synthetic properties of enzymes were exploited to carry out a selective reduction reaction. The stereoselective hydrolysis of protein amino acid esters had already been commercialised by Tanabe in Japan using immobilised aminoacylase, and selective reduction reactions using whole yeast cells are already used in a number of processes, such as the selective reduction of the anti-cancer drag Coriolin. 

DL-Amino acid resolution processes for various amino acids had already been successfully pioneered by the Tanabe Seiyabu Co. using immobilised, oryzae aminoacylase acting on DL-N-acetyl acids. This step was first carried out as a continuous immobilized enzyme process in 1969. 

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. 

Traditional commercial requirements for L-phenylalanine have been small (less than 50 ton/a) and had been satisfied by the use of aminoacylase to resolve chemically synthesised DL-N-acetylphenylalanine. However with the advent of aspartame as a high intensity sweetener a very big derived demand for L-phenylalanine was generated. As a result a number of companies began to develop bioconversion and fermentation processes to produce L-phenylalanine. 

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. 

The majority of industrial biocatalytic processes involve the use of hydrolytic enzymes including proteases, transaminases, glycosidases, aminoacylases, and lipases as well as several additional enzyme classes... 

Another large successful commercial application of enzymes is in the amino acid industry. Amino acids for food and feed fortification, nutritional supplements, or as feedstock for downstream products can be made by fermentation processes, from protein hydrolysates or by chemical synthesis. While chemical synthesis is cheaper for a number of amino acids,, it often produces a racemic mixture. The racemic mixture is successfully resolved on a commercial scale by acylating the amino acids, then using an aminoacylase to remove the acyl group from the L-amino acid and separating the free L-amino acid from the still acylated-D-amino acid. Ajinamoto and other companies, especially in Japan, make large amounts of amino acids by this process. 

Aminoacylase. Production of L-amino acids by Tanabe Seiyaku Company using aminoacylase adsorbed on DEAE-Sephadex represents the first industrial use of an immobilized enzyme (3,29). The process uses the enzyme to resolve the racemic mixture of an amino acid, derived by chemical synthesis, by biospecific hydrolysis of the acyl-amino acid followed by separation of the L-amino acid from the acyl-D-amino acid by crystallization. The D-forms are then racemized and passed back through the reactor thus improving the yield. The entire process, including a fixed-bed bioreactor, is a continuous, automated operation. 

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],... 

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. 

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. 

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]. 

Aminoacylase is another hydrolytic enzyme of industrial impact used in the multiton process for the production of L-amino acids for human and animal nutrition from the corresponding racemates. The process is based on the enantiospecificity of the enzyme to selectively hydrolyze the L-enantiomer of the previously acylated racemate so that the L-amino acid is easily separated from the acylated D-amino acid that, after racemization, is recycled back into the enzyme stage (Sato and Tosa 1993a). This process has the historical record of being the first large scale process conducted with immobilized enzyme (Chibata et al. 1987). 

The demand for L-amino acids for food and medical applications is growing fast. Both chemical and microbial processes can be used for their production. However, the chemical routes lack stereoselectivity, thus leading to lower productivity. In Japan the immobilized enzyme aminoacylase has been used for the production of L-amino acids, of which methionine is the most important, since 1996. 

Biocatalytic resolution plays a major role in the industrial scale synthesis of a wide variety of optically pure amino acids. Tanabe uses an L-spe-cific aminoacylase for the manufacture of several L-amino acids, immobilized on DEAE-Sephadex. Degussa on the other hand, uses the free acylase in a membrane bioreactor. The process is highly efficient in enzyme use, and racemisation of the D-isomer is straightforward, thus providing good economics, and virtually no waste (Scheme 7.4). The process can be further refined by the use of racemase enzymes, which makes dynamic kinetic resolution feasible. 

The chemical synthesis of amino acids leads to a racemic mixture. Only the L-form is usable for medicines and foodstuffs. Immobilized enzymes have been used for the industrial separation of enantiomers of various amino acids, e.g., amino acid acylase immobilized onto DEAE-Sephadex. Processing efficiency in the fermentation of amino acids has been greatly facilitated through the use of immobilized amino acid acylases such as L-glutamate aminoacylase. 

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