L-Malic acid production

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. 

Thus far, EMRs have been successfully used with macromolecular substrates, as for the saccharification of cellulose11-13 15 26 and protein hydrolysis,19 or with low molecular weight substrates, as for cellobiose hydrolysis9 and L-malic acid production from fumaric acid.20 Bench and large scale plants are already in operation for the preparation of N-acetyl-D,L derivatives from L-amino acids by means of acylase and the production of L-malic acid from fumaric acid by means of fumarase.20 In the case of fumarase, conversions of up to 86% with resolution rates of up to 85% have been attained. 

Fumi M, Sakata N, Otsuki O et al. (1988) A bioreactor-crystaUizer for L-malic acid production. 

Battat, E., Peleg, Y., Bercovitz, A. et al. (1991) Optimization of L-malic acid production by Aspergillus flavus in a stirred fermenter. Biotechnol. Bioengng, 37, 1108-1116. 

Tachibana, S., 1966. Studies on C02-fixing fermentation. 1. L-Malic acid production by. Schizophyllum commtme fries. Journal of Fermentation Technology 44,129-132. 

Wang, X., Gong, C.S., Tsao, G.T., 1996. L-malic acid production from fumaric acid by a laboratory Saccharomyces cere-visiae strain SHY 2. Biotechnology Letters 18,1441-1446. 

Fumaric acid is converted to L-malic acid by hydration in the presence of the enzyme fumamse. From the structure of the substrate and the configuration of the product, it is apparent that the hydroxyl group has been added to the si fiice of one of the carbon atoms of the double bond. Each of the trigonal carbon atoms of an alkene has its fiice specified separately. The molecule of fumaric acid shown below is viewed fixjm the re-re fiice. 

The following chapters will be devoted to the production of j8-poly(L-malic acid) or its salt by fermentation, its Isolation, and physico-chemical characterization. The biosynthesis, degradation, and presumed physiological role will be also considered. 

Danneel, H.J., Busse, M. and Faurie, R. (1995) Pharmaceutical grade L-malic acid from fumaric acid -development of an integrated biotransformation and product purification process. Mededlingen van de Faculteit Landbouwwetenschappen, Rijksuniversiteit te Gent, 60 (4a), 2093—2096. 

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. 

The literature concerning malo—lactic fermentation—bacterial conversion of L-malic acid to L-lactic acid and carbon dioxide in wine—is reviewed, and the current concept of its mechanism is presented. The previously accepted mechanism of this reaction was proposed from work performed a number of years ago subsequently, several workers have presented data which tend to discount it. Currently, it is believed that during malo-lactic fermentation, the major portion of malic acid is directly decarboxylated to lactic acid while a small amount of pyruvic acid (and reduced coenzyme) is formed as an end product, rather than as an intermediate. It is suspected that this small amount of pyruvic acid has extremely important consequences on the intermediary metabolism of the bacteria. 

Tyj"alo—lactic fermentation can be defined as the bacterial conversion of L-malic acid to L-lactic acid and carbon dioxide during storage of new wine. Malic acid is dicarboxylic, but lactic acid is monocarboxylic therefore, the net result of malo-lactic fermentation in wine, aside from the production of carbon dioxide, is a loss in total acidity. In commercial practice, this fermentation is not well understood, and better methods of controlling it are sought. 

In discussing the studies of Brechot et al. (24) and Peynaud et al. (25), Kunkee (I) found it odd that bacteria which ordinarily produce d or DL-lactic acid from glucose produce L-lactic acid in wine as a result of malo-lactic fermentation. Peynaud et al. (26) reported that organisms which produced only D-lactic acid from glucose produced only L-lactic acid from L-malic acid. He postulated further that the malo-lactic fermentation pathway has no free pyruvic acid as an intermediate because the optical nature of L-malic acid would be lost when it was converted to pyruvic acid since pyruvic acid has no asymmetric carbon atom. Therefore, if pyruvic acid were the intermediate, one would expect d, l, or DL-lactic acid as the end product whereas L-lactic acid is always obtained. These results lend considerable support to the hypothesis that free pyruvic... 

London and Meyer (33) have demonstrated the presence of a malic enzyme, E.C. 1.1.1.39, in Streptococcus faecalis. In their system, the organism grows at the expense of L-malic acid, producing carbon dioxide, acetate, and ethyl alcohol and NADH as major end products, and a small amount of lactic acid as a minor end product. The authors speculate that the major function of the malic enzyme is to provide energy, presumably... 

Another example is provided by malic acid, a chiral molecule which also contains a prochiral center (see Eq. 9-74). In this case replacement of the pro-R or pro-S hydrogen atom by another atom or group would yield a pair of diastereoisomers rather than enantiomers. Therefore, these hydrogen atoms are diastereotopic. When L-malic acid is dehydrated by fumarate hydratase (Chapter 13) the hydrogen in the pro-R position is removed but that in the pro-S position is not touched. This can be demonstrated by allowing the dehydration product, fumarate, to be hydrated to malate in 2HzO (Eq. 9-74). The malate formed contains deuterium in the pro-R position. If this malate is now isolated and placed with another portion of enzyme in H20, the deuterium is removed cleanly. The fumarate produced contains no deuterium. 

Figure 10-9 Representation of the course of enzyme-induced hydration of fumaric acid (trans-butenedioic acid) to give L-malic acid (L-2-hydroxy-butanedioic acid). If the enzyme complexes with either—C02H (carboxyl) group of fumaric acid, and then adds OH from its right hand and H from its left, the proper stereoisomer (l) is produced by antarafacial addition to the double bond. At least three particular points of contact must occur between enzyme and substrate to provide the observed stereospecificity of the addition. Thus, if the enzyme functions equally well with the alkenic hydrogen or the carboxyl toward its mouth (as shown in the drawing) the reaction still will give antarafacial addition, but o,L-malic acid will be the product.

The aza-Baylis-Hillman reaction of 4-X-C6H4CH=NTs with CH2=CHCOMe, catalysed by PI13P in the newly designed chiral ionic liquid (121), derived from l-(—)-malic acid, gave products with up to 84% ee. This example represents the first highly enantioselective asymmetric reaction in which a chiral medium is the sole source of chirality.176... 

FIG. 21 Schematic diagram for the production of L-malic acid from fumaric acid using a six-compartment (Zi-Z6) ED stack composed of anionic (a) and cationic (c) membranes, as extracted from Sridhar (1987). As ammonium fumarate is formed, it is enzymatically converted into ammonium malate in an external bioreactor (R). The combined system is also provided with a series of storage tanks for the acidic cathode-(Dl) and anode-(D6) rinsing solutions, raw materials (D2), ammonium fumarate (D3), ammonium malate (D4), and final product (D5). 

Mourgues, J., Robert, L., and Hanine, H. 1997. Extraction and purification of /),/.-malic acid, produced by chemical synthesis, L-malic acid produced by microbiological synthesis or susceptible to be recovered during the manufacturing of food products. Industries Alimentaires et Agricoles 114, 379-384. 

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],... 

Product Glycidylbutyrate Butyl glucosides Styrene oxides Oligosaccharides D-pantothenic acid L-malic acid L-methionine L-valine R-mandelic acid L-Camitine Aspartame L-aspartate Cyclodextrins (S)-CPA 6-APA Cocoa Butter Acrylamide HFCS... 

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. 

The proof just given made no assumptions as to actual configurations. It can also be stated starting from the knowledge that fumarase hydrates fumaric acid to L-malic acid by anti addition on the Si-Si face. Hence, if the reaction is carried out in 2H20, the product is e/yf/iro-L-[3-2H]malic acid with (S) configuration at C-2, and (R) at C-3, 88. Hence, the sequence of reactions just discussed can be represented as follows ... 

While the wine contains several g/L of L-malic acid before MLR, it usually only contains between 200 mg/L and 300 mg/L of citric acid. Although the citric acid is only present in low concentrations, it is of considerable importance. On the one hand, its metabolic pathway leads to production of acetic acid, in other words, it increases the volatile acidity of the wine. However, the most important enological significance associated with fermentation of citrate is the production of diacetyl and other acetonic compounds, which affect the wine aroma. 

Ethanol production is essentially redox neutral however metabolism associated with biomass production generates nett NADH, which is oxidised largely by glycerol production. Other important NADH oxidising reactions with flavour implications are the production of 2,3-butanediol, L-malic acid and succinic add. When glycerol production is stimulated by non-growth associated reactions (i.e. osmotic stress) NAD+ reduction occurs by other reactions including the oxidation of acetaldehyde to acetic acid... 

In the presence of NAD, L-malic acid is oxidised to oxaloacetate in a reaction catalysed by L-malate deshydrogenase (l-MDH). The reaction equilibrium is forced in the direction of the products by the elimination of oxaloacetate, via its reaction with 1-glutamate, resulting in the production of L-aspartate. This reaction is catalysed by glutamate-oxaloacetate-transaminase (GOT) ... 

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). 

P7-32j The production of L-malic acid (used in medicines and food additives) was produced over immobilized cells of Bacillus flavum MA-3 (Proc. 2nd Joint China/USA Chemical Engineering Conference, Beijing, China, Vol. lll,p. 1033, 1997],... 

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