Aspartases

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

A possible explanation for the superiority of the amino donor, L-aspartic add, has come from studies carried out on mutants of E. coli, in which only one of the three transaminases that are found in E. coli are present. It is believed that a branched chain transaminase, an aromatic amino add transaminase and an aspartate phenylalanine aspartase can be present in E. coli. The reaction of each of these mutants with different amino donors gave results which indicated that branched chain transminase and aromatic amino add transminase containing mutants were not able to proceed to high levels of conversion of phenylpyruvic add to L-phenylalanine. However, aspartate phenylalanine transaminase containing mutants were able to yield 98% conversion on 100 mmol l 1 phenylpyruvic acid. The explanation for this is probably that both branched chain transaminase and aromatic amino acid transminase are feedback inhibited by L-phenylalanine, whereas aspartate phenylalanine transaminase is not inhibited by L-phenylalanine. In addition, since oxaloacetate, which is produced when aspartic add is used as the amino donor, is readily converted to pyruvic add, no feedback inhibition involving oxaloacetate occurs. The reason for low conversion yield of some E. coli strains might be that these E. cdi strains are defident in the aspartate phenylalanine transaminase. 

Of industrial importance at present is die biotransformation of fumarate to L-aspartic add by Escherichia cdi aspartase. Modified versions have been developed, such as the continuous production of L-aspartic add using duolite-ADS-aspartase. A conversion higher than 99% during 3 months on a production scale has been achieved. 

L-aspartic add has been produced on an industrial scale by the Tanabe Seiyaku Co Ltd, Japan, in a batch wise process using whole cells of Escherichia coli with high aspartase activity. In this process, L-aspartic add is produced from fumaric add and ammonia using aspartase, as described in Figure A8.13. 

Das granulierte Gel wird in 1 m Ammoniumfumarat p< 8,5 suspendiert. Man gibt 1 mMol Magnesium-Ionen hinzu und halt 24-38 Stdn. bei 37°. Dabei steigert sich die Aspartase-Aktivitat in den immobilisierten Zellen um den Faktor 9-107,8. Der Katalysator besitzt eine biologische Halbwertszeit von 120 Tagen. 

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. 

Interest in sol-gel processing was awakened by the work of Avnir et al. in 1990 who performed successful experiments with such enzymes as [1-glucosidasc, alkaline phosphatase, chitinase and aspartase [68]. This gave impetus to their own systematic study of the entrapment of biopolymers in a silica matrix as well as those of other teams [69-79]. The results have been summarized and discussed in numerous review articles (see, e.g., Refs. [41—43,45—49,51,80—85]). 

This enzyme [EC 4.3.1.1], also known as aspartase and fumaric aminase, catalyzes the conversion of aspartate to fumarate and ammonia. 

A valuable clue to the manner in which iron pump functions, came in 1957 when an enzyme of molar mass 110 kg/mol was discovered by Jens Skou which hydrolyses ATP only if Na and K+ ions are present in addition to Mgt+ required for all ATPases (enzymes catalysing the hydrolysis of ATP). The activity of this enzyme correlates quantitatively with the extent of ion transport. Another important clue was provided by the observation that this ATPase is phosphorylated at an aspartase site only in the presence of Na+ and Mg2+ ions. The phosphorylated product is hydrolysed if K+ ions are present. It has been also observed that the enzyme undergoes a conformational change when it is phosphorylated. 

Figure 25. Trace enantiomer analysis of L-aspartic acid (obtained by enzymatic amination of fumarate with L-aspartase and determined as the A -trifluoroacetyl-O-methyl ester) on i.-Chirasil-Val31 [20 mx0.25 mm (i.d.) glass capillary column, 90 CC, 0.4 bar hydrogen] with detection by GLC MS selected ion monitoring (mj 198.1) and multiscanning chromatography (MSC, 8 scans)186.

Methyl-aspartase aminiert 3-Alkyl- bzw. 3-Halogen-substituierte Fumarsauren zu (2S,JS)-3-Alkyl- bzw. (2R,3S)-3-Halogen-asparaginsauren. In Deuterium-oxid wer-den die stereospezifiseh deuterierten Aminosauren erhalten2,3. 

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

Other enzymes that catalyze elimination reactions that produce fumarate are aspartate ammonia-lyase (aspartase),63 argininosuccinate lyase (Fig. 24-10, reaction g),64/65 and adenylosuccinate lyase... 

Fig. 25-15). In every case it is NH3 or an amine, rather than an OH group, that is eliminated. However, the mechanisms probably resemble that of fumarate hydratase. Sequence analysis indicated that all of these enzymes belong to a single fumarase-aspartase family.64 65 The three-dimensional structure of aspartate ammonia-lyase resembles that of fumarate hydratase, but the catalytic site lacks the essential HI 88 of fumarate hydratase. However, the pKa values deduced from the pH dependence of Vmax are similar to those for fumarase.64 3-Methylaspartate lyase catalyzes the same kind of reaction to produce ammonia plus czs-mesaconate.63 Its sequence is not related to that of fumarase and it may contain a dehydroalanine residue (Chapter 14).66... 

Ash, amount from tissue 31 Asparagine (Asn, N) 53s Aspartase 526 Aspartate 737s... 

Fumarase. See Fumarate hydratase Fumarase-aspartase family 685 Fumarate 481s, 516s, 683s Fumarate hydratase (fumarase) 526, 683,688 acid-base catalysis 471 concerted reaction 685 Fumarase A 688 Fumarase B 688 Fumarase C 683 mechanism 683 - 685 pH dependence 684 rates of substrate exchange 684 turnover number of 683 Fumarate reductase 785 Fumarylpyruvate 690s Function of state R 476 Fungal infections 20 Fungi 20... 

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). 

Naidja, A., and Huang, P. M. (1996). Deamination of aspartic acid by aspartase-Ca-montmorillonite complex. J. Mol. Catal. A Chem. 106, 255-265. 

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