Enzyme aminoacylase

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. 

Enzymes have been immobilized by covalent binding to polymeric supports, by entrapment in cross-linked polymer gels or by physical adsorption on to suitable materials such as ion-exchangers, with retention of activity. The first example of an industrially useful application of this technique was in the separation of L-amino acids from racemic mixtures. This procedure used the enzyme aminoacylase which is immobilized in suitable columns. The racemic amino acid mixture is acylated chemically to form a-N-acetyl-OL-amino acids. When this is passed through the column, only the N-acetyl-L-amino acids are deacylated by the enzyme and the resulting L-amino acids can then be purified. 

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. 

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. 

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

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... 

Tosa, T., Mori, T., Fuse, N., and Chibata, I., Studies on continuous enzyme reactions 6 Enzymatic properties of DEAE-Sepharose Aminoacylase complex, Agr. Biol. Chem., 33 1047-1056 (1969). 

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. 

Aldelyde dehydrogenase I Aminoacylase-I Metabolic enzymes - Balabanov et al, 2001 Balabanov et al, 2001... 

The immobilization of invertase on aluminium hydroxide (2) was one of the earliest reports of adsorption technology. The use of aminoacylase adsorbed on DEAE-Sephadex for producing L-amino acids from a racemic mixture of their corresponding ethyl esters (4) was the first industrial application of an immobilized enzyme system. The basic disadvantage of this convenient technique is that binding is weak and the enzyme slowly leaches out. However, for many purposes, this slow leakage is not an important handicap. Immobilizing enzymes by adsorption has been extensively reviewed (5, 6, 27). Some special approaches are described (1, 28-30). 

However, the range of types of amino acids that can be resolved in this way is much greater than just the natural substrates (i.e. peptides made up of the twenty coded amino acids), because methods to relax the specificity of the enzymes have been found, in some cases by using organic solvents for the reactions. Penicillin acylase from Escherichia coli and an aminoacylase from Streptovercillium olivoreti-... 

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. 

For a detailed review about aminoacylase-catalyzed resolutions, see a) A. S. Bom-marius in Enzyme Catalysis in Organic... 

L-Methionine has been prepared from fV-acetyl-D, L-me-thionine by deacylation with an aminoacylase.37 The lipase was supported on an ion-exchange resin. The reaction was driven to completion by chromatographic ion-exchange removal of the by-product acetic acid and unreacted substrate, the racemic mixture being fed in pulses to the column. The cross-linked enzyme crystals marketed by Altus Biologies are very useful in enzymatic resolutions, because of their enhanced stability to heat and organic solvents and because they can be reused many times. (For more detail on enantiospecific reactions with enzymes, see Chap. 9.)... 

Aminoacylase kinetics may be used as an example for demonstrating the measurement and modeling of enzyme kinetics. This reaction is of industrial importance in... 

In 1993, a thermostable aminoacylase from Bacillus stearothermophilus was characterized by Sakanyan et al.l51. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids preferentially and even has some dipeptidase activity. Its optimal reaction temperature is 70 °C after incubation for 15 min, 90% of the original activity was retained. The authors write that the similarity of the B. stearothermophilus enzyme sequence with that of other enzymes such as aminoacylase I, acetylornithine deacetylase and carboxypeptidase G2 suggests a common origin. The aminoacylase from B. stearothermophilus is well characterized the gene has been completely sequenced1511, cloned into E. coli and overexpressed[51> 671 and studied for catalytic and stability properties16 1 the intrinsic one Zn2+ ion per subunit seems to have a predominantly structural role and activity can be restored to the apo-enzyme by Co2+ and particularly by Cd2+ (3-fold activity ) but not by Zn2+. 

Apart from the L-specific acylases from kidney and Aspergillus strains it has been shown that similar aminoacylases are widely distributed in microorganisms123 27, 8S1. However, from the viewpoint of costs, those acylases which are practically employed for large scale industrial purposes, are restricted to the enzyme from Aspergillus oryzae (see Sect. 12.3.7). 

As with the D-aminoacylases from Streptomyces sp. the enzymes from Alcaligenes strains have a preference for hydrophobic N-acetyl-amino acids. In this respect, they are similar to the L-specific acylase I from kidney preparations and Aspergillus sp. The Alcaligenesfaecalis enzyme prefers the N-acyl-D-amino acid derivatives from Met, Phe and Leu[951. If a high-affinity substrate residue occupies the hydrophobic side-chain pocket the enzyme even deacylates D-Met methyl esters or N-Ac-D-Met-Xaa dipeptide derivatives. 

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. 

Aminoacylase has also been immobilized on a nylon membrane1661. While the half-life as measured by thermal stability, of 161 d is superior to the data for immobilized acylase (65 d)[6] or soluble enzyme in an EMR191, reactor productivity at 0.136 L-valine kg/L-1d-1 is lower than that for DEAE-Sephadex-immobilized acylase (0.5 kg/L-1d-1)161 or that for a membrane reactor (0.35 kg/L-1d j191. 

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