Immobilized enzyme fumarase

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. 

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. 

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. 

The hydration of fumaric acid [( )-butenedioic acid, 1 R = H] to (S)-2-hydroxybutanedioic acid (2) is catalyzed by the enzyme fumarase. This reaction can be run even on an industrial scale, exploiting the fumarase activity of immobilized microorganisms77. Unfortunately, the substrate spectrum of fumarase is very narrow. Nevertheless, (Z)-2-chlorobutenedioic acid (3, R = Cl) could be diastereo- and enantioselectively hydrated to (2S,3/ )-2-chloro-3-hydroxybu-tanedioic acid (4) on a 50-gram scale, employing commercially available pig heart fumarase [EC 4.2.1.2.]78. 

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. 

The substrate, L-aspartate, is produced from fumarate by an enzyme system involving aspartase, as described in the section on L-aspartate. To produce L-alanine directly from fumarate, the L-alanine-producing column was connected in tandem to an L-aspartate-producing column. In this tandem column system, side reactions caused by fumarase in Escherichia coli and alanine racemase in P. dacunhae reduced the yield. Then, both bacterial cells were separately treated with high temperature and low pH, respectively, and the enzymes responsible for the side reactions were inactivated. Immobilization of these two kinds of bacterial cells with K-carrageenan resulted in the production of L-alanine in a single reactor without the production of the side products, malate and o-alanine (Takamatsu et al. 1982 Chibata et al. 1986b). 

Neufeld, R.J., Peleg, Y, Rokem, J.S., Pines, O., Goldberg, I., 1991. L-maUc acid formation by immobilized Saccharomy-ces cerevisiae amplified for fumarase. Enzyme and Microbial Technology 13,991-996. 

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