Acid continued fumaric

Biosynthesis ofS(— )-M llc Acid. Aqueous fumaric acid is converted to levorotatory malic acid by the intracellular enzyme, fumarase, which is produced by various microorganisms. A Japanese process for continuous commercial production of S(—)-mahc acid from fumaric acid is based on the use of immobilized Brevibacteriumflavum cells in carrageenan (32). The yield of pyrogen-free S(—)-mahc acid that is suitable for pharmaceutical use is ca 70% of the theoretical. 

Table 3 summarizes many of the uses mentioned in the literature. The main use of succinic acid in Japan is for bath preparations (314—322). This application in 1994 accounted for nearly 80% of total consumption. After recording a more than 10% yearly increase in the late 1980s, the growth of this apphcation has slowed down, and consumption is decreasing on account of the replacement of succinic acid by fumaric acid for economic reasons. This trend is expected to continue in the coming years. 

B. A deficiency in pyruvate carboxylase results in a diminution of oxaloacetate, the acid that acts as the acceptor for an acetyl group from acetyl-CoA. In order for the TCA cycle to continue efficiently, C4 acids must be replenished. Amino acids whose carbon skeletons feed into the TCA cycle and increase the C4 pool will accomplish this. Glutamine, which is converted to a-ketoglutarate, will lead to an increase in all of the C acids (succinate, fumarate, malate, and oxaloacetate). Alanine and serine are converted to pyruvate, which as a result of the deficiency in pyruvate carboxylase will not increase the C4 pool. Lysine and leucine are ketogenic amino acids and thus also do not increase the C pool. 

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. 

Fumaric acid differs from maleic acid only in that it is in the trans configuration. Upon heating, fumaric acid is isomerized to maleic acid. Continued heating produces maleic anhydride. Fumaric acid is converted to maleic acid by forced rotation around its double bond. The double bond is first weakened by protonation of the carbonyl oxygen. The resultant positive charge is resonance stabilized. The mechanism for conversion of fumaric acid to maleic anhydride is ... 

Brevibacterium ammoniagenes cells immobilized by copolymerization with acrylamide have been used in column form for the continuous production in high yield of pure nicotinamide adenine dinucleotide phosphate, for the continuous production of L-malic acid from fumaric acid, and for study of the NAD-kinase activity of the immobilized cells. The continuous production of L-malic acid has also been achieved with Brevibacterium flavum cells immobilized by gelation in k-carrageenan. ... 

Petruccioli M, Angiani E, Eederici E (1996) Semi continuous fumaric acid production by Rhizopus arrhizus immobilized in polymethane sponge. Process Biochem 31 463 69 Pines O, Shemesh S, Battat E, Goldberg I (1997) Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 48 248-255... 

Petruccioli, M., Angiani, E., Federid, F., 1996. Semi-continuous fumaric acid production by Rhizopus arrhizus immobilized in polyurethane sponge. Process Biochemistry 31,463-469. 

Fumaric acid is used in the plastics industry, in the food industry and as a source of malic add. Although demand has increased rapidly over the last 30 years its production from fermentation has been totally replaced by a chemical method. It is now produced far more cheaply by the catalytic oxidation of hydrocarbons, particularly benzene. With the continuing uncertainties concerning the availability and cost of petroleum, however, fermentation may yet be a viable alternative. 

An important problem in emulsified organic-aqueous systems is that of scale-up, which is concerned with the realization of stable emulsions and the separation of phases after the reaction. The use of biphasic membrane systems that contain the enzyme and keep the two phases separated is likely to solve this problem. In the case of 5-naproxen an ee of 92% has been demonstrated without any decay in activity over a period of two weeks of continuous operation. A number of examples of biocatalytic membrane reactors have been provided by Giorno and Drioli (2000) and include the conversion of fumaric acid to L-aspartic acid, L-aspartic acid to L-alanine, and cortexolone to hydrocortisone and prednisolone. 

An industrial process worked out by the Japanese has enabled the continuous production of L-aspartic acid from ammonium fumarate by using columns packed with immobilized E. ooli. cells... 

Similar experiments have been performed with fumaric and maleic acids (trans and cis l,4-but-2-enedicarboxylic acid). Identical observations were made - at the beginning of the reaction, a broad Bragg reflection centered at 10.0 A is seen to grow in to the patterns. This corresponds to the second stage intercalates of maleate and fumarate. Upon continued addition of the dicarboxylates, a phase at 12.9 A appears. This is the maleate intercalate basal... 

One of these units is converted into a derivative A so that an enantiomeric mixture is generated which can be resolved in the usual manner. The desired enantiomer is used for the continuation of the synthesis, whereas the undesired one is recycled to the starting meso-compound. An illustration of this method is the synthesis of (/-biotin (9) from fumaric acid via the meso-intermediates 5/6 and the enantiomers 7/8 50. 

Aspartase. Tanabe Seiyaku has used aspartase in lysed E. coli cells immobilized by entrapment in polyacrylamide (3) or K-carra-geenan (38) for production of aspartic acid since 1973. Using a substrate stream containing 1 M ammonium fumarate and 1 mM Mg2+ at pH 8.5 and 37°C, a continuous, automated bioreactor with a 120-day half-life will produce L-aspartate at 60% of the cost of a batch fermentation (3). Recently, a process for immobilization of the cells in polyurethane has also been described (37). 

Fumarase. The development and use of this immobilized enzyme by Tanabe Seiyaku for production of L-malic acid is very similar to that of aspartase ( 3). Lysed Brevibacterium ammoniagenes or B. flavin cells are treated with bile acid to destroy enzymatic activity which converts fumarate to succinate. As with aspartase, the cells can be immobilized in polyacrylamide or k-carrageenan gels. Using a substrate stream of 1 M sodium fumarate at pH 7.0 and 37°C, L-malic acid of high purity has been produced since 1974 by a continuous, automated process (3,39) for example, using a 1000-L fixed-bed bioreactor, 42.2 kg L-malic acid per hour was produced continuously for 6 months. 

In this case, the meso isomer is formed. For the sake of completeness, we will continue this exercise by adding an unsymmetrical molecule, AB, to fumaric acid. Suggest the stereochemistry of this product. 

Fumaric acid production using immobilized Rhizopus cells has also been studied. Petruccioli et al. [74] immobilized R. arrhizus NRRL 1526 on polyurethane sponge to carry out repeated batch fumaric acid production from glucose syrup with KOH/KCO3 as the neutralization agent and CO2 source. Although the yield (12.3 g/1) is low, it provides the possibility of using immobilized Rhizopus for the continuous production of fumaric acid. 

Using a column packed with immobilized E. CoZZ cells, conditions for continuous production of L-aspartlc acid from ammonium fumarate were investigated in detail, and an aspartase reactor system was designed. The system is essentially the same as that for the immobilized amlnoacylase system [2]. A solution of 1 M ammonium fumarate (containing 1 mM MgClz, pH 8.5) is passed... 

In this ease, reaction reaches an equilibrium when about 80% of fumaric acid are converted to L-malic acid. We investigated continuous fumarase reactions using immobilized microbial cells. 

Then the conditions for continuous production of L-mallc acid by a column packed with this bile extract treated immobilized cells was studied. When 1 M sodium fumarate (pH 7.0) is passed through the column at 37 C at flow rate of space velocity=0.2 hr the reaction reaches an equiliblium. From the effluent of the column, L-malic acid can be obtained by ordinary methods. Average yield of pyrogen-free pure L-malic acid from consumed fumaric acid is around 70% of the theoretical. Tanabe Seiyaku Co., Ltd. is operating this production system since 1974, and we are satisfied both with the economical efficiency and with the quality of product. 

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