Aspartases aspartame

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). 

An example of a project of this nature is the screening for microorganisms to produce aspartame (28) from a precursor that is easy synthesized by chemical methods. Microorganisms were screened for their ability to catalyze the trans-addition of ammonia across the double bond of /V-fumaryl-L-phenylalanine methyl ester (FumPM) (74) (Scheme 19.43).403 This is essentially the reaction of a mutated aspartase because the native enzyme has such strict substrate specificity (see Section 19.2.4). Although the literature touts this as a successful screening effort, this process has not been practiced commercially because the yields are extremely low.404405... 

Alanine and aspartic acid are produced commercially utilizing enzymes. In the case of alanine, the process of decarboxylation of aspartic acid by the aspartate decarboxylase from Pseudomonas dacunhae is commercialized. The annual world production of alanine is about 200 tons. Aspartic acid is produced commercially by condensing fumarate and ammonia using aspartase from Escherichia coli. This process has been made more convenient with an enzyme immobilization technique. Aspartic acid is used primarily as a raw material with phenylalanine to produce aspartame, a noncaloric sweetener. Production and sales of aspartame have increased rapidly since its introduction in 1981. Tyrosine, valine, leucine, isoleucine, serine, threonine, arginine, glutamine, proline, histidine, cit-rulline, L-dopa, homoserine, ornithine, cysteine, tryptophan, and phenylalanine also can be produced by enzymatic methods. 

Decarboxylases of phenylalanine, tyrosine, and lysine and ammonia lyases of histidine, glutamine, and asparagine are also highly selective. Guilbault et al. (1988) described a potentiometric enzyme sensor for the determination of the artificial sweetener aspartame (L-aspartyl-L-phen-ylalanine methylester) based on L-aspartase (EC 4.3.1.1). The ammonia liberated in the enzyme reaction created a slope of 30 mV/decade for the enzyme-covered ammonia sensitive electrode. The specificity of the sensor was excellent however, the measuring time of 40 min per sample appears not to be acceptable. The measuring time has been decreased to about 20 min by coimmobilizing carboxypeptidase A with L-aspartase (Fatibello-Filho et al., 1988). 

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. 

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]. 

Aspartame Processed food, dietary food Enzyme aspartase -F NH3 gas electrode... 

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