Aspartases fumaric acid

Thus, in the action of the enzyme aspartase, fumaric acid, 39, is transformed into L-aspartic acid, 40. This process can be considered as a result of three steps ... 

L-aspartic acid fumaric acid + NH aspartase E. coii ... 

Many enzymes have absolute specificity for a substrate and will not attack the molecules with common structural features. The enzyme aspartase, found in many plants and bacteria, is such an enzyme [57], It catalyzes the formation of L-aspartate by reversible addition of ammonia to the double bond of fumaric acid. Aspartase, however, does not take part in the addition of ammonia to any other unsaturated acid requiring specific optical and geometrical characteristics. At the other end of the spectrum are enzymes which do not have specificity for a given substrate and act on many molecules with similar structural characteristics. A good example is the enzyme chymotrypsin, which catalyzes hydrolysis of many different peptides or polypeptides as well as amides and esters. 

L-aspartic acid by ammonia addition to fumaric acid by aspartase (from E. coli)... 

Owing to the commercialization of Aspartame the demand for i-aspartic acid increased steeply. i-Aspartate can be produced by enantioselective addition of ammonia to fumaric acid catalyzed by aspartase (E.C. 4.3.1.1) (Figure 7.17). 

The industrial preparation of L-aspartic acid by the amination of ( )-2-butenedioic acid (fumaric acid) made use of some form of immobilized aspartase (free cell), which were replaced by microbial whole cells, resulting in reduced costs and simplified operations 38a d. 

L-aspartic acid Fumaric acid Escherichia coli (aspartase) ... 

It is also possible to convert nonchiral readily available industrial organic chemicals into valuable chiral natural-analogue products. This is demonstrated by the conversion of achiral fumaric acid to L(-)-malic acid with fumarase as the active enzyme. The same compound is converted to the amino acid L(-h)-aspartic acid by Escherichia bacteria that contain the enzyme aspartase. If pseudomonas bacteria are added, another amino acid L-alanine is formed (Eq. 9.10). 

The photochemical carboxylation of pyruvic acid by this process is endergonic by about AG° = 11.5 kcal mol and represents a true uphill photosynthetic pathway. The carbon dioxide fixation product can then act as the source substrate for subsequent biocatalyzed transformations. For example, photogenerated malic acid can act as the source substrate for aspartic acid (Figure 35). In this case, malic acid is dehydrated by fumarase (Fum) and the intermediate fumaric acid is aminated in the presence of aspartase (Asp) to give aspartic acid. 

L-Aspartic acid can be produced by direct fermentation of sugars using bacterial strains. However, commercially it has been produced by the amination of fumaric acid by immobilized bioctalysts that have high aspartase (EC 4.3.1.1) activity in a fixed-bed reactor. Suitable microbes for the industrial bioconversion of fumaric acid to L-aspartic acid include strains of Brevibacterium, Cory-nebacterium, E. coli, and Pseudomonas. A weight yield of 110% can be obtained in the conversion of fumaric acid to aspartic acid as shown in the following ... 

The first commercial production of L-aspartic acid was started in 1973 by the Tanaba Seiyaku Company, Japan. The process uses aspartase contained in whole microorganisms and involves the immobilization of E. coli on polyacrylamide gel or carrageenan. The immobilized cells are then subjected to treatment in order to increase cell permeability. The substrate, fumaric acid, is dissolved in a 25 % ammonia solution and the resulting ammonium fumarate is then passed through the reactor containing the immobilized E. coli. The reaction is exothermic and the reactor has to be designed to remove the heat produced. The conversion of fumaric acid to aspartic acid is more economical than the direct fermentation of sugars. The key to economical production of L-aspartic acid for expanded use is a cheaper and more abundant source of fumaric acid. 

Aspartase is ubiquitous in nature and has been studied in many bacterial species with the specific aim of using the enzyme to make L-aspartic acid [5,7-14], It catalyzes the reversible deamination of aspartic acid to fumaric acid (Scheme 2). This in vivo reaction precedes exclusively in the direction of fumarate, but in vitro it is readily reversible and can go to 100% completion. Much of the early characterization of aspartase was conducted on enzyme purified from strains of Escherichia coli B and W [15,16] and, for this reason, many of the early commercial processes were developed using the E. coU aspartase. 

Ammonia lyases in their natural role are involved in the metabolism of amino acids and also play a role in, for instance, the degradation of amino sugars, but only a limited amount of these enzymes have been characterized biochemically. Application of a broad range of different ammonia and lyases in organic chemical synthesis on an industrial scale has thus far not occurred, which is due to both their limited commercial availability and their lack of stability under process conditions. Exceptions are the commercially applied aspartase, which is an ammonia lyase that is utilized for the synthesis of L-aspartic acid from fumaric acid, and phenylalanine lyase. The latter is an example of a commercial application of an ammonia lyase in a process for the production of L-phenylalanine and more importantly L-phenylalanine derivatives. 

L-aspartic acid ammonia lyase, or aspartase (E.C. 4.3.1.1) is used on a commercial scale by Kyowa Hakko, Mitsubishi, Tanabe and DSM to produce L-aspartic acid, which is used as a building block for the sweetener Aspartame, as a general acidulant and as a chiral building block for synthesis of active ingrediants[1]. The reaction is performed with enzyme preparations from E. coli, Brevibacterium jlavum or other coryneform bacteria either as permeabilized whole cells or as isolated, immobilized enzymes. The process is carried out under an excess of ammonia to drive the reaction equilibrium from fumaric acid (1) in the direction of L-aspartic acid (l-2) (see Scheme 12.6-1) and results in a product of excellent quality (over 99.9% e.e.) at a yield of practically 100%. The process is carried out on a multi-thousand ton scale by the diverse producers of L-aspartic acid. Site directed mutagenesis of aspartase from E. coli by introduction of a Cys430Trp mutation has resulted in significant activation and stabilization of the enzyme P1. 

Since maleic acid is a cheaper starting material than fumaric acid, the process that is probably the most economical makes use of both a maleate isomerase (E. C. 5.2.1.1) and aspartase (E.C. 4.3.1.1), Scheme 12.6-1. Mitsubishi has succeeded in... 

Mitsubishi has also developed a process for production of D-aspartic acid (d-2) and L-malic acid (4) by incubation of racemic aspartic acid with the exclusively L-selective aspartase in combination with fumarase, thereby preventing the reaction going backwards by conversion of the generated fumaric acid into L-malic acid 4. ... 

The combined utilization in a single reactor of both aspartase from Brevibacterium flavum and aspartate-P-decarboxylase from Pseudomonas dacunhae, thereby catalyzing the reaction from fumaric acid via L-aspartic acid to L-alanine (5), has also been developed by Mitsubishi 5. ... 

Another combination reaction is the biocatalytic production of the herbicide phosphinotricin [L-2-amino-4-(hydroxymethylphosphinyl)butyric acid, (7) in Scheme 12.6-2] by the company Meiji Seika, whereby an amino-transferase that acts on 4-(hydroxymethylphosphinyl)-2-oxo-butyric acid and that utilizes aspartic acid as the amino donor was used in combination with aspartase to generate the amino donor from fumaric acid and ammonia 6. ... 

We attempted continuous production of L-aspartic acid from fumaric acid and ammonia by immobilized Escherichia coli having high aspartase activity [3, 4, 5]. Various methods were tested for the immobilization of microbial cells, and a stable and active enzyme system was obtained by entrapping whole microbial cells in a polyacrylamide gel lattice. 

Enzymes can be either totally specific for a given substrate, to such an extent that they will not tolerate any structural or configurational changes in the substrate, or they can be broadly specific for a given type of functional group, and they will still operate on substrates with structural variations around that functional group. An example of the former is the enzyme aspartase, Asp, which catalyzes both the addition of ammonia to fumaric acid and the elimination of ammonia from L-aspartic acid by the following reactions ... 

Production of L-aspartic acid from fumaric acid by stereoselective addition of ammonia under the action of the intracelluar aspartase in E. coli (Tanabe Seiyaku Co., Ltd.). When a 1000-liter column is used, theoretical yield of L-aspartic acid is 3.4 tons per day (and even considerably higher for mutant strains and plasmid pNKl01-harboring strains). A similar industrial process using the immobilized E. coli aspartase (instead of the whole cells) was established earlier by Kyowa Hakko Kogyo, Co., Ltd.. L-Aspartate is mainly used as a building block for the manufacture of the sweetener aspartame [170]. 

The enzyme aspartase (EC 4.3.1.1.) has long been known to catalyze stereospecific addition of ammonia across the double bond of fumaric acid (216-218), and after the original assignment of stereochemistry to this reaction was corrected, it was realized that addition of ammonia was anti (82). Since the enzyme is commercially available, reaction of fumaric acid 72 in H20/N H3 readily affords (2S, 3R)-[3- Hi]aspartic acid 21a, Hg = H, whereas addition of ammonia to [ Hj] fumaric acid 72, Ha = H, yields (2S, 3S)-[2,3- H2] aspartic acid 21a, H = H (218-221). Recently, aspartase in immobilized... 

E. coli has been used in an improved synthesis of these compounds (118). The enzyme j8-methylaspartase (EC 4.3.1.2) also adds ammonia across the double bond of fumaric acid in the same way as aspartase (222), and so an alternative synthesis is available. When [ H2]fumaric acid 72, Ha = H, was incubated with aspartase in H20, (2S,3R)-[2,3- H2, 3- Hj]aspartic acid 21a, Ha = H, Hb = H, was obtained (47) (Scheme 63). ... 

I)-Aspartases from Escherichia coli and Brevibacterium flavum catalyse the stereospecific addition of ammonia to fumaric acid. Nanning Only-Time in China, Kyowa Hakko Kogyo and Tanabe Seiyaku in Japan produce aspartic acid accordingly. Using an (L)-aspartate-jS-decarboxylase, alanine can be prepared in a subsequent step as well. [62]... 

Addition of hydroxylamine to the double bond of fumaric acid was accelerated by aspartase but unstable N-hydroxyaspartic acid was not isolated (57,154). 

Enzymic amination of fumaric acid using aspartase leads to the formation of L-aspartic acid, which is performed at a capacity of 1,200 t/year (Scheme 2.214)... 

L-Aspartic acid is formed from oxaloacetic acid which originates in the tricarboxylic acid cycle (D 5). Hoifk ever, it may also be formed from fumaric acid in a reversible reaction catalyzed by aspartase ... 

These molecules are derived from aspartic acid, itself formed from members of the Krebs cycle. In plants and micro-organisms fumaric acid is combined with ammonia in the presence of aspartase, whilst in mammals which do not possess aspartase, aspartic acid is formed by reductive deamination of oxaloacetate in a reaction of imknown mechanism. 

Aspartic acid is obtained in 90% yield from fumaric acid by using the aspartase enzyme ... 

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