Aminoacylase

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)... 

The Japanese firm Tanabe Inc Ltd has been operating, since 1969, the optical resolution of DL-amino acids using aminoacylase. The prindple is based on the asymmetrical hydrolysis of N-acyl-DL-amino add by amino acylase which gives the L-amino add and the unhydrolysed acyl-D-amino add. 

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. 

PSL, AMANO) has been found to catalyze the Markovnikov-type addition of thiols to vinyl esters (Scheme 5.23) [114] and acylases aminoacylase [115], penicillin G acylase [116], and acylase from AspergiUus oryzae [117], the same kind of addition of various nitrogen nucleophiles. Unfortunately, most additions described above are nonstereoselective. 

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]. 

Aminoacylases 3.5.1.14 /V-acetyl amino acids amino acids >100... 

AMINO ACID RACEMASE Amino adds, peptides proteins, BIOCHEMICAL NOMENCLATURE AMINO ACID TURNOVER KINETICS AMINOACYLASE AMINOACYL-tRNA HYDROLASE Aminoacyl-tRNA synthetase,... 

Other immobilized enzyme product successes (where annual production of immobilized enzymes has surpassed 1 ton/year) comprise Aminoacylase (Amano), hydantoinase (Smith Kline Beecham), lactase (Valio), lipase (Novo Nordisk), penicillin G acylase (Gist-brocades, Smith Kline Beecham, Rohm) and penicillin V acylase (Novo Nordisk/Gist-brocades) (Poulsen, 1984). 

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. 

A particularly interesting case is the partial hydrolysis of the racemic N-tri-fluoroacetyl derivative 59 with hog kidney aminoacylase (HKA) (EC 3.5.1.14) to prepare chiral isomers of 2-trifluoromethylalanine 60 [53]. The stereochemical preference of hog kidney aminoacylase is to hydrolyze amino acid amides bearing the larger C-2 substituent in the pro-S position [54]. The enzymatic hydrolysis of compound 60 follows this trend. 

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. 

The best-established method for the enzymatic production of L-amino acids is the separation of racemates of N-acetyl-DL-amino acids by acylase I (aminoacylase E.C. 3.5.1.14). N-Acetyl-L-amino acid is cleaved and yields L-amino acids whereas N-acetyl-D-amino acid does not react (Figure 7.10). 

S. Tokuyama, D-Aminoacylase, Eur. Patent Appl. EP 0950706, 1999 (to Daicel Chemical Industries). 

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. 

I. Gentzen, H.-G. Loffler, and F. Schneider, Aminoacylase from Aspergillus oryzae. Comparison with the pig kidney enzyme, Z. Naturforsch. 1980, 35c, 544-550. 

Other important applications in the food industry running at a large scale are the production of L-aspartic add with Escherichia coli entrapped in polyacrilamides [6], the immobilization of thermolysin for the production of aspartame [14], The production of L-alanine by Tanabe Seiyaku [7], the production of frudose concen-centrated syrup [3], the production of L-malic acid by the use of Brevibacterium ammoniagenens immobilized in polyacrilamide by entrapment immobilization methods [11] and L-aminoacids production by immobilized aminoacylase [5],... 

Sato, T. and Tosa, T. (1993) Optical resolution of aminoadds by aminoacylase in Industrial Application of Immobilized Biocatalyst (eds T. Tanaka, T. Tosa and T. Kobayashi) Marcel Dekker, New York, pp. 3-14. 

Scheme 42 Continuous flow optical resolution of acetyl-D,L-phenylalanine using an immobilized aminoacylase enzyme.

E.C. 3.5.1.4) used to prepare amino acids, usually through resolution, and also penicillin G acylase (penicillin G amidohydrolase) (E.C. 3.5.1.11), used in the manufacture of semisynthetic penicillins.152 153 Immobilized penicillin G acylase has most recently been used to catalyze the formation of. V-a-phenylacetyl amino acids, which can then be used in peptide coupling reactions (see Section 19.2.3.2).154 Bacterial aminoacylase I (.V-acyl-i.-amino acid amidohydrolase, E.C. 3.5.1.14) has also been used to acylate chiral amines with poor to moderate enantioselectively.155... 

The use of biocatalysis for the production of chemicals started to receive serious interest in the 1960s with the development of immobilized aminoacylases for the production of chirally pure amino acids by Tanabe Seigaku of Japan, as well as the application of penicillin acylase for the production of 6-aminopenicillanic acid (6-APA), a key... 

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... 

A considerable number of industrial bioconversions utilize covalently immobilized biocatalysts. Examples include Penicillin acy-lases V and G, aminoacylases, and aspartase. In some cases the biocatalyst is immobilized through cross-linking and in others the catalyst is captured by a reactive resin. Covalent immobilization often leads to extremely stable catalysts with high potential for reuse and... 

---