Brevibacterium, malic acid

The major portion of malic acid currently produced at an approximate 10,000 t/y is racemic, because it originates from petrochemically produced fumaric acid. The L-form can also be generated from fumaric acid by its hydration with immobilized cells of Brevibacterium or Corynebacterium. 

This process has been operated successfully by the Tanabe Seiyaku Co. in Japan since 1973. Similar processes have since been commercialised by other companies, such as the Kyowa Hakko Co., often using different immobilisation methods such as polyurethane. The same iimnobilized cell approach has also been used by Tanabe since 1974 in their coimnercial process for the production of L-malic acid from fumarate using the hydratase activity of Brevibacterium ammoniagenes cells. 

L-malic acid by addition of water to fumaric acid by fumarase from Brevibacterium... 

Other important applications in the food industry running at a large scale are the production of L-aspartic add with Escherichia coli entrapped in polyacrilamides [6], the immobilization of thermolysin for the production of aspartame [14], The production of L-alanine by Tanabe Seiyaku [7], the production of frudose concen-centrated syrup [3], the production of L-malic acid by the use of Brevibacterium ammoniagenens immobilized in polyacrilamide by entrapment immobilization methods [11] and L-aminoacids production by immobilized aminoacylase [5],... 

A similar process is also used for the production of L-malic acid from fumarate, in this case using a hydratase enzyme derived from Brevibacterium ammoniagenes. Another variation of the Tanabe technology involves the synthesis of L-alanine from L-aspartic acid through the use of immobilized whole cells (P dacunae) containing aspartate-decarboxylase. 

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. 

Malic acid Acetate, ethanol, fumarate, glucose, propionate Aspergillus jlavus Brevibacterium Jlavum Lactobacillus brevis Paecilomyces varioii Pichia membranaefaciens... 

L-Malic acid by water addition to fumaric acid Fumarase (Brevibacterium)... 

Three immobilized enzyme or microbial cell systems currently used industrially in synthesis of chiral amino acids plus one presently under development are described. L-amino acids are produced by enzymatic hydrolysis of DL-acylamino acid with aminoacylase immobilized by ionic binding to DEAE-Sephadex. Escherichia coli cells immobilized by K-carrageenan crosslinked with glutaraldehyde and hexamethylenediamine are used to convert fumaric acid and cimmonia to L-aspartic acid and Brevibacterium flavum cells similarly immobilized are used to hydrate fumaric acid to L-malic acid. The decarboxylation of L-aspcirtic acid by immobilized Pseudomonas dacunhae to L-alanine is currently under investigation. 

In 1974 we succeeded in the industrial production of L-malic acid from fumaric acid by Brevibacterium ananoniagenes cells immobilized by the polyacrylamide gel method [9, 10]. The asymmetric reaction catalyzed by the fumarase activity of the cells is shown below. 

COMPARISON OF PRODUCTIVITIES OF Brevibacterium ammoniagenes AND Brevibacterium flavum IMMOBILIZED WITH POLYACRYLAMIDE AND WITH CARRAGEENAN FOR PRODUCTION OF L-MALIC ACID... 

Production of L-malic acid from fumaric acid by stereoselective hydration under the action of the intracellular fumarase in Brevibacterium ammoniagenes (Tanabe Seiyaku Co., Ltd). A 1000-liter column bioreactor can typically yield 30 tons of L-malic acid per month. 

Brevibacterium gel Polyacrylamide gel Fumaric acid L-Malic acid... 

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. ... 

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