Aspartic acid production

Enzymatic Process. Chemically synthesized substrates can be converted to the corresponding amino acids by the catalytic action of an enzyme or the microbial cells as an enzyme source, t - Alanine production from L-aspartic acid, L-aspartic acid production from fumaric acid, L-cysteine production from DL-2-aminothiazoline-4-catboxyhc acid, D-phenylglycine (and D-/> -hydtoxyphenylglycine) production from DL-phenyUiydantoin (and DL-/)-hydroxyphenylhydantoin), and L-tryptophan production from indole and DL-serine have been in operation as commercial processes. Some of the other processes shown in Table 10 are at a technical level high enough to be useful for commercial production (24). Representative chemical reactions used ia the enzymatic process are shown ia Figure 6. 

Aspaitase ill immobilized R. calx cells catalyzes, L-aspartic acid production from fimrarate and ammonia. 

Fig. 14 L-Aspartic acid production using an L-aspartic acid ammonia lyase.

The first prejudice that biocatalysts are too expensive is only partly true. If the cost per mol or per unit weight is calculated they certainly are expensive. For example, penicillin amidase costs 10 000/kg on a bulk scale. On the other hand the cost contribution of penicillin amidase in the splitting of penicillin G is only 1/kg of product11171. In the case of L-aspartic acid production the cost contribution of aspartase is even lower, 0.1 /kg. This demonstrates that it is not the absolute catalyst cost but the cost contribution of the catalyst to the final product cost that has to be considered and compared. This is also true for chemical catalysts e. g., the bulk price of BINAP is 40 000/kg11171. Important parameters influencing the cost contribution are the total turnover number (mol product/mol catalyst) and the turnover frequency (mol product/mol catalyst and unit time). 

We are planning industrialization of these continuous L-alanine and D-aspartic acid production systems using immobilized P. dacunhae in the near future. 

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. 

The use of immobilized aspartase or whole cells for L-aspartic acid production has been described by many investigators (Chibata, 1978 Fusee et al 1981 Smith et al., 1982 Wood and Gallon, 1984). The complete development of such a process consists of two major steps, reportedly carried out as follows (Hamilton et al., 1985). 

Land, depreciating, 280-281 Lang Factor method, 191-192 Langmuir-Hinshelwood kinetics, 704 L-aspartic acid production bacteria, filtering, 1014 continuous crystallizers, 1014 continuous filtration, 1014 filtering, 1014 intermediate storage, 1014 ion exchange column, 1014 overview, 1007... 

Fusee, M.C., Swann, W.E., Calton, G.J., 1981. Immobilization of Escherichia coli cells containing aspartase activity with polyurethane and its application for L-aspartic acid production. Applied and Environmental Microbiology 42,672-676. 

Huanga, J., Jinb, N., Katsudab, T., Fukudaa, H., Yamaji, H., 2009. Immobilization of Escherichia coli cells using poly-ethyleneimine-coated porous support particles for L-aspartic acid production. Biochemical Engineering Journal 46,65-68. 

It is evident that the area of water-soluble polymer covets a multitude of appHcations and encompasses a broad spectmm of compositions. Proteins (qv) and other biological materials ate coveted elsewhere in the Eniyclopedia. One of the products of this type, poly(aspartic acid), may be developed into interesting biodegradable commercial appHcations (70,71). 

There are numerous further appHcations for which maleic anhydride serves as a raw material. These appHcations prove the versatiHty of this molecule. The popular artificial sweetener aspartame [22839-47-0] is a dipeptide with one amino acid (l-aspartic acid [56-84-8]) which is produced from maleic anhydride as the starting material. Processes have been reported for production of poly(aspartic acid) [26063-13-8] (184—186) with appHcations for this biodegradable polymer aimed at detergent builders, water treatment, and poly(acryHc acid) [9003-01-4] replacement (184,187,188) (see Detergency). 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

A solution of 88.5 parts of L-phenylalanine methyl ester hydrochloride in 100 parts of water is neutralized by the addition of dilute aqueous potassium bicarbonate, then is extracted with approximately 900 parts of ethyl acetate. The resulting organic solution is washed with water and dried over anhydrous magnesium sulfate. To that solution is then added 200 parts of N-benzyloxycarbonyl-L-aspartic acid-a-p-nitrophenyl, -benzyl diester, and that reaction mixture is kept at room temperature for about 24 hours, then at approximately 65°C for about 24 hours. The reaction mixture is cooled to room temperature, diluted with approximately 390 parts of cyclohexane, then cooled to approximately -18°C in order to complete crystallization. The resulting crystalline product is isolated by filtration and dried to afford -benzyl N-benzyloxycarbonvI-L-aspartyl-L-phenylalanine methyl ester, melting at about 118.5°-119.5°C. 

A more general method for preparation ofa-amino acids is the amidotnalmatesynthesis, a straightforward extension of the malonic ester synthesis (Section 22.7). The reaction begins with conversion of diethyl acetamidomalonate into an eno-late ion by treatment with base, followed by S 2 alkylation with a primary alkyl halide. Hydrolysis of both the amide protecting group and the esters occurs when the alkylated product is warmed with aqueous acid, and decarboxylation then takes place to vield an a-amino acid. For example aspartic acid can be prepared from, ethyl bromoacetate, BrCh CCHEt ... 

Merck s thienamycin synthesis commences with mono (V-silylation of dibenzyl aspartate (13, Scheme 2), the bis(benzyl) ester of aspartic acid (12). Thus, treatment of a cooled (0°C) solution of 13 in ether with trimethylsilyl chloride and triethylamine, followed by filtration to remove the triethylamine hydrochloride by-product, provides 11. When 11 is exposed to the action of one equivalent of tm-butylmagnesium chloride, the active hydrogen attached to nitrogen is removed, and the resultant anion spontaneously condenses with the electrophilic ester carbonyl four atoms away. After hydrolysis of the reaction mixture with 2 n HC1 saturated with ammonium chloride, enantiomerically pure azetidinone ester 10 is formed in 65-70% yield from 13. Although it is conceivable that... 

The noteworthy successes of a relevant model study12 provided the foundation for Merck s thienamycin syntheses. In the first approach (see Schemes 2 and 3), the journey to the natural product commences from a readily available derivative of aspartic acid this route furnishes thienamycin in its naturally occurring enantiomeric form, and is noted for its convergency. During the course of this elegant synthesis, an equally impressive path to thienamycin was under parallel development (see Schemes 4 and 5). This operationally simple route is very efficient (>10% overall yield), and is well suited for the production of racemic thienamycin on a commercial scale.. x... 

The elimination of the amino donor, L-aspartic acid, resulted in an almost complete reduction of activity. Neither cell permeabilisation nor cofactor (pyridoxalphosphate) addition were essential for L-phenylalanine production. Maximum conversion yield occurred (100%, 22 g r) when the amino donor concentration was increased. Aspartic add was a superior amino donor to glutamic add 35 g l 1 was used. 

Table 8.8 Estimated costs of production of L-phenylalanlne by enzymatic methods (100 tonnes per year). PPA = phenylpyruvtc acid, ACA = acetamidodnnamic add, L-ASP = L-aspartic acid.

Figure A8.15 Flow diagram for the continuous production of L-aspartic acid and L-alanine.

Since 1978, several papers have examined the potential of using immobilised cells in fuel production. Microbial cells are used advantageously for industrial purposes, such as Escherichia coli for the continuous production of L-aspartic acid from ammonium fur-marate.5,6 Enzymes from microorganisms are classified as extracellular and intracellular. If whole microbial cells can be immobilised directly, procedures for extraction and purification can be omitted and the loss of intracellular enzyme activity can be kept to a minimum. Whole cells are used as a solid catalyst when they are immobilised onto a solid support. 

Grb-2 facilitates the transduction of an extracellular stimulus to an intracellular signaling pathway, (b) The adaptor protein PSD-95 associates through one of its three PDZ domains with the N-methyl-D-aspartic acid (NMDA) receptor. Another PDZ domain associates with a PDZ domain from neuronal nitric oxide synthase (nNOS). Through its interaction with PSD-95, nNOS is localized to the NMDA receptor. Stimulation by glutamate induces an influx of calcium, which activates nNOS, resulting in the production of nitric oxide. 

In the published synthesis the ozonolysis is performed on the protected product (9) and aldehyde (10) isolated before oxidation, hydrolysis and decarboxylation give aspartic acid. 

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