Fumarase aspartic acid

The addition of water to fiimaric acid catalysed by fumarase is a highly stereospecific reaction and malic acid is formed as the sole product (Figure 2.22, X=H). The ammonia lyase 3-methylaspartase catalyses the similar addition of ammonia to yield L-aspartic acid. When uimatural substrates are used in these reactions (X =/= H), less success is experienced. An increasing X-group gives slow reaction rates. 

Examples of the use of immobilized enzymes in food processing and analysis have been listed by Olson and Richardson (1974) and Hultin (1983). L-aspartic acid and L-malic acid are produced by using enzymes contained in whole microorganisms that are immobilized in a polyacrylamide gel. The enzyme aspartase from Escherichia coli is used for the production of aspartic acid. Fumarase from Brevibacterium ammoni-agenes is used for L-malic acid production. 

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. 

The Mitsubishi Chemical Company has described a process for the commercial production of L-aspartate using an cx-amino-zr-butyric acid resistant mutant of B. flavum [11]. The enzyme is moderately thermal resistant, allowing the process to be run at 45°C. The process is run using immobilized cells in a fed batch system in which the biocatalyst is recycled [4]. An initial problem was the conversion of fumarate to malic acid by an intracellular fumarase activity, which led to low l-aspartic acid yields during the first cycle. This problem was circumvented by preheating the biocatalyst for 1 hour at 45°C, which completely destroyed the fumarase activity [4,11]. Recently, the aspartase gene from B. flavum has been cloned [28] and has presumably been used to improve the efficiency of this process. 

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. ... 

M. Terasawa, S. Nara, H. Yamagata, H. Yu-gawa, Manufacture of d-Aspartic Acid and/or L-Malic Acid with Aspartase and Fumarase, Mitsubishi Petrochemical Co., 1991, JP06014787. 

In succession to the L-aspartic acid production, in 1974 we succeeded in the third industrial application, i.e. the production of L-malic acid from fumarlc acid by immobilized microbial cells. L-Mallc acid is an essential compound in cellular metabolism, and is mainly used in pharmaceutical field. L-Malic acid can be produced by fermentative or enzymatic methods from fumarlc acid by the action of fumarase as follows. 

In general, endogenous metabolism of anaerobic bacteria was found to be more stable, when biocatalysts based on immobilized cells of P. shermanii and E. coli were compared with respect to the reactions shown above (Ikonnikov, 1985). P. shermanii had a higher aspartase activity than P. pentosaceum, P. petersonii and P. technicum (Kalda and Vorobjeva, 1981). After 3 days of incubation with continuous stirring at 37°C and pH 8.5, the extent of substrate conversion (ammonium fumarate) was 95-96% and 75-90% in the case of E. coli K-12 and P. shermanii, respectively. In addition to aspartic acid, the reaction mixtures of the two strains also contained malic acid. Heat treatment of the biomass of P. shermanii (50 C, 1.5 h, pH 5.0) resulted in a complete inactivation of fumarase, while the activity of aspartase was retained (Kalda and Vorobjeva, 1980, 1981). As a result of the elimination of fumarase activity, the yield of L-aspartic acid from ammonium fumarate was increased up to 96-98% the incubation time was also shortened since no substrate was diverted to the side reaction forming malate. 

Reactions 35 and 36 have been shown to be catalyzed by different enzyme fractions and the intermediate has been isolated and partially purified. Earlier reports had indicated that reaction 36 was a hydrolytic step resulting in the formation of arginine plus malic acid. However, recent studies indicate that the formation of malic acid was the result of the presence of fumarase in the enzyme preparation. Of considerable interest is the report that a compound similar to, if not identical with, the end product of reaction 35 is enzymatically formed from arginine plus fumaric acid by extracts of plant and animal tissues, and microorganisms. In this connection it has been reported that one of the components of the condensing enzyme system (reaction 35) is present in yeast extracts as well as in liver preparations. Although ATP is required for synthesis of the intermediate from citrulline plus aspartic acid, it is not needed for the synthesis from arginine plus fumaric acid. 

In this reaction ammonia is either directly removed from aspartic acid or added to the fumaric acid in a manner similar to the removal or addition of water by fumarase or aconitase. Cell-free preparations of the enzyme have been prepared by Virtanen and Tarnanen. ... 

Fig. 1.2 Intermediates of the citric acid cycle showing the relationship between glutamate and aspartate. Pyruvate dehydrogenase complex (1) citrate synthase (2) aconitase (3) isocitrate dehydrogenase (4) a-ketoglutarate dehydrogenase (5) succinyl-CoA synthetase (6) fumarate (7) fumarase dehydratase (8) malate dehydrogenase (9) and aspartate aminotransferase (10)...

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