Aspartame enzymatic

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

Another advantage of biocatalysis is that chemo-, regio-, and stereoselectivities are attainable that are difficult or impossible to achieve by chemical means. A pertinent example is the production of the artificial sweetener, aspartame, which has become somewhat of an industrial commodity. The enzymatic process (Fig. 2.31), operated by the Holland Sweetener Company (a joint venture of DSM and Tosoh), is completely regio- and enantiospecific (Oyama, 1992). 

Enzymatic assay techniques have been developed for several additives by Merck. BIOQUANT kits are available for aspartame (intense sweetener) and nitrate (preservative). Gromes et al. (1995) applied the Bioquant kit to determination of aspartame in yoghurt, quark and confectionery. For low concentrations of aspartame a blank correction procedure was necessary. Recoveries of aspartame were in the range 93-102%. 

Erbeldinger, M. Mesiano, A. J. Russell, A. J. Enzymatic catalysis of formation of Z-aspartame in ionic liquid— An alternative to enzymatic catalysis in organic solvents, Biotechrwl. Prog., 2000, 16(6), 1129-1131. 

Oyama, K. (1986) Enzymatic synthesis of Aspartame in organic solvents. In Biocatalysis in organic media, edited by C.Laane, J.Tramper, M.D.Lilly, pp. 209-224. 

Proteases can be used for the synthesis of peptides in a way analogous to the ester synthesis catalysed by lipases. The most successful industrial example of enzymatic peptide synthesis is described in section 4.6 aspartame synthesis. In the industrial process in Europe the equilibrium position is shifted towards synthesis because the... 

There are highly developed methods for chemical peptide synthesis, both solid phase methods and solution methods. This makes it rather difficult for the enzymatic methods to compete. However, the aspartame example shows that for peptides which have a big market it can be worthwhile to develop an enzymatic process. 

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of p-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synfiiesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step. 

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (7 as a bitter peptide, Om-p-Ala-HCl (OBA), Om-Tau-HCl as salty peptides(2j, and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide(5). The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. 

Proteases have been much less studied than lipases in ionic liquid media and generally require the presence of water for activity. We note that the thermolysin-catalyzed amide coupling of benzoxycarbonyl-L-aspartate and L-phenylalanine methyl ester into Z-aspartame in [BMIm][PF6] was an early example of an enzymatic reaction in an ionic liquid medium [8]. 

There are a number of reported solid-to-solid reactions where products precipitated as salts rather than as neutral compounds. The thermolysin-catalyzed production of the potassium salt of Z-aspartame [51, 52], the commercialized process of aspartame synthesis, where a salt of cationic D-Phe-OMe and anionic Z-aspartame precipitates [53], and the enzymatic conversion of solid Ca-maleate to solid Ca-D-malate [44] are examples of such behavior. 

Scheme 12.1 Enzymatic solid-to-solid synthesis ofZ-L-Asp-L-PheOMe potassium salt (Z-aspartame potassium salt) using inorganic salts [51].

Aspartame , Artificial Sweetener through Enzymatic Peptide Synthesis... 

Figure 7.25 Toyo Soda/DSM process for the enzymatic production of aspartame.

M. Erbeldinger, X. Ni, and P. J. Halling, Kinetics of enzymatic solid-to-solid peptide synthesis synthesis of Z-aspartame and control of acid-base conditions by using inorganic salts, Biotechnol. Bioeng. 2001, 72, 69-76. 

Kamat, S. Critchley, G. Beckman, E. J. Russell, A. J. Biocatalytic Synthesis of Acrylates in Organic Solvents and Supercritical Fluids HI. Does Carbon Dioxide Covalently Modify Enzymes Biotechnol. Bioeng. 1995, 46, 610-620. Kamihira, M. Taniguchi, M. Kobayashi, T. Synthesis of Aspartame Precursors by Enzymatic Reaction in Supercritical Carbon Dioxide. Agric. Biol. Chem. 1987, 51, 3427-3428. 

An example of a nonoccupational exposure is methanol, which is formed endogenously, probably as the result of the activities of intestinal flora or enzymatic processes. It is present in a number of consumer products. Methanol may be present in low concentrations in some foods, juices, and alcoholic beverages. Methanol can also be derived from the intestinal enzymatic hydrolysis of the artificial sweetener aspartame, which results in methanol absorption from the intestine (Butchko et al. 2002). It is estimated that a 355-mL serving of aspartame-sweetened beverages and of various fruit and tomato juices may contribute about 20-100 mg of dietary methanol (Butchko et al. 2002). For comparison purposes, exposure at the current Threshold Limit Value time-weighted average of methanol (262 mg/m3) would result in a daily dose of about 1,500 mg, assuming an 8-hour inhaled volume of 10 m3 of air and absorption of 57%. 

The synthesis of aspartame can be achieved by numerous chemical and enzymatic methods of amide bond formation between (Z)-aspartic acid and either (Z,)-phenylalanine or (Z)-phenylalanine methyl ester. Both approaches have been thoroughly reviewed [10]. The chief difficulty with chemical methods is formation of the non-sweet P-isomer as a by-product. 

The enzymatic approach for the synthesis of aspartame has the advantage of producing only the (Z,)-a-aspartyl compound (the sweet diastereomer). The less expensive racemic (DZ)-phenylalanine can be used as the starting material. The unreacted (D)-phenylalanine can be recovered and racemized for further reaction. By contrast, the chemical methods used industrially also produce the (L)-p-aspartyl compound (non-sweet) in 10-40% yield, for which a purification step is needed. In 1999, the world market for enzymatically synthesized aspartame was reported to be 800 million US [13]. 

Enzymatic methods are now preferred for the commercial production of aspartame from aspartate and phenylalanine. Typically, these use thermolysin (EC 3.4.24.4 derived from Bacillus spp.) to couple benzyloxycarbonyl-protected aspartate (Z-asp) with phenylalanine methyl ester. The resulting Z-APM is then deprotected using standard methods [14-18]. Numerous enzymatic methods have been reported [10]. 

A prominent example of peptide coupling is used for the production of the synthetic dipeptide sweetener aspartame (a-APM) (28) (Chapter 31). The chemical coupling method yields approximately an 80 20 mixture of the a- and P-isomers, whereas the regioselectivity of the enzymatic... 

Oyama, K. Enzymatic Synthesis of Aspartame in Organic Solvents, Biocatalysis in Organic Media, 1986, Wageningen, The Netherlands. Published in Studies in Organic Chemistry, Laane, C., Tramper, J., Lilly, M. D. Eds., Elsevier, Amsterdam, 1987, Vol. 29, p. 209. 

Alanine and aspartic acid are produced commercially utilizing enzymes. In the case of alanine, the process of decarboxylation of aspartic acid by the aspartate decarboxylase from Pseudomonas dacunhae is commercialized. The annual world production of alanine is about 200 tons. Aspartic acid is produced commercially by condensing fumarate and ammonia using aspartase from Escherichia coli. This process has been made more convenient with an enzyme immobilization technique. Aspartic acid is used primarily as a raw material with phenylalanine to produce aspartame, a noncaloric sweetener. Production and sales of aspartame have increased rapidly since its introduction in 1981. Tyrosine, valine, leucine, isoleucine, serine, threonine, arginine, glutamine, proline, histidine, cit-rulline, L-dopa, homoserine, ornithine, cysteine, tryptophan, and phenylalanine also can be produced by enzymatic methods. 

Recently the possibility of total enzymatic synthesis of Aspartame has been suggested (44). It was shown that immobilized penicillinacylase would hydrolyze JJ-phenacetyl L-L-aspartame thus, if thermolysin will accept phenacetyl L-Asp, use of the carbobenzoxyl protecting group and its subsequent removal by hydrogenation would not be required. 

The fermentation of L-Phe has obviously emerged victorious, on purely economic grounds and is now so efficient that even d,L-Phe, which is used by the Holland Sweetener Company in the enzymatic production of aspartame, is obtained by racemization of L-Phe rather than via a chemical procedure. 

A great number of enzymes have already been combined with electrodes. In some cases, several possible enzymatic routes may be selected, depending on the substrate to be analyzed. One such example is that used to determine aspartame (Fig. 3) (18-20). Enzyme electrodes use, ideally, only one enzyme and monitor the main substrate-enzyme reaction, for example, glucose oxidase-glucose. If the main substrate-enzyme reaction is not electrochemically detectable or if signal amplification is required, bi- or trienzymatic sequences may be applied. 

A number of synthetic peptides are significant commercial products, ranging from the sweet dipeptide aspartame (L-aspartyl-L-phenylalanine methyl ester) to clinically used hormones such as insulin and oxytocin. L-Aspartyl-L-phenylalanine methyl ester (3 Scheme 2) is the methyl ester of the C-terminal dipeptide of gastrin. It was found accidently during the synthesis of gastrin that this synthetic sweetener is about 200 times as sweet as sucrose.f This pleasant sweetness without a bitter aftertaste was the reason that L-aspartyl-L-phenylalanine methyl ester was approved in many countries as a food additive, receiving much attention as a low-calorie sweetener. L-Aspartyl-L-phenylalanine methyl ester can be prepared by various chemical routes and the first enzymatic procedure of commercial interest was described by Isowa et al.h l In the industrial process,L-Asp and DL-Phe were chosen as inexpensive raw materials. L-Asp is available very inexpensively, whereas L-Phe is more expensive than DL-Phe. Z-D-Asp acts as a competitive inhibitor, while D-Phe-OMe... 

To illustrate a few aspects of amino add production by enzymatic methods, ffie production of L-phenylalanine will be considered in some detail. L-Phenylalanine is important as an essential amino add for human nutrition and is used as an intermediate for the synthesis of the artifidal sweetener, aspartame. Other examples of the industrial production of amino adds by enzymatic methods are described briefly in Appendix 2. 

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