L-Phenylalanine from phenylpyruvate

As can be seen from Table 8.7 productivity (expressed in g h b is highest for precursor addition. The production of L-phenylalanine from phenylpyruvic add also has the shortest reaction time to obtain hi conversions. The pH commonly used is around 75, quite normal for biological processes. Only the enzyme phenylalanine ammonia lyase shows an optimiim pH of lO.The process temperature varies between 30 and 40°C with an average of 35°C. No extreme temperatures have been reported due to the fact that denaturation occurs at hi temperatures. The optimal concentration for cells frequently used is 10-20 g 1". However, conversion of ACA is done with hi cell mass concentrations in recent studies possibly to compensate for substrate inhibition and thus to maintain hi product concentration. The processes using PPA and ACA need an amino add as amino donor, usually L-aspartic add is used. 

Optically pure opine-tjrpe secondary amine carboxylic acids were also synthesized from amino acids and their analogs, such as L-methionine, L-isoleucine, L-leucine, L-valine, L-phenylalanine, L-alanine, L-threonine, L-serine, and L-phenylalaninol, and a-keto acids, such as glyoxylic, pyruvic, and 2-oxobutyric acids, using the enzyme with regeneration of NADH with FDH from Moraxella sp. C-1 [13]. The absolute configuration of the nascent asymmetric center of the opines was of the D stereochemistry with > 99.9% e.e. One-pot synthesis of N-[l-D-(carboxyl)ethyl]-L-phenylalanine from phenylpyruvic and pyruvic acid by using ODH, FDH, and phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus... 

Similar methods have been used for the syntheses of L-phenylalanine and its analogs. An enzyme membrane reactor system containing Brevibacterium sp. PheDH, yeast FDH, and PEG-NADH was developed for the syntheses of L-phenylalanine from phenylpyruvate and ammonium formate [85]. Asano and Nakazawa synthesized L-phenylalanine, tyrosine, and some other L-amino acids using a dialysis tube containing the Sporosarcina ureae PheDH and yeast FDH [86]. In addition, optically pure three-substituted pyruvates with bulky substituents, such as 5 -2-amino-4-phenylbutyrate and 5-2-amino-5-phenylvalerate, were synthesized from their oxo analogs in a similar way [87]. 

E. Bulot and C. L. Cooney, Selective production of phenylalanine from phenylpyruvic add using growing cells of Corynebacterium glufamicum, Bio-technd. Lett., 7 93 (1985). 

The production of L-phenylalanine from the precursor phenylpyruvic acid by transamination is a process which requires two steps ... 

Some people lack the enzymes necessary to convert L phenylalanine to L tyrosine Any L phenylalanine that they obtain from their diet is diverted along a different meta bolic pathway giving phenylpyruvic acid... 

Two reactions for the production of L-phenylalanine that can be performed particularly well in an enzyme membrane reactor (EMR) are shown in reaction 5 and 6. The recently discovered enzyme phenylalanine dehydrogenase plays an important role. As can be seen, the reactions are coenzyme dependent and the production of L-phenylalanine is by reductive animation of phenylpyruvic add. Electrons can be transported from formic add to phenylpyruvic add so that two substrates have to be used formic add and an a-keto add phenylpyruvic add (reaction 5). Also electrons can be transported from an a-hydroxy add to form phenylpyruvic add which can be aminated so that only one substrate has to be used a-hydroxy acid phenyllactic acid (reaction 6). 

The culture can be used directly for the conversion of phenylpyruvic add to resting cells L-phenylalanine. Therefore, a batch process with resting cells can be carried out, with some glucose added for maintenance (fed-batch fermentation). Another approach is to harvest the cells from the fermentation broth and to use them in a separate bioreactor in higher concentrations than the ones obtained in the cell cultivation. An advantage of the last method can be that the concentration of compounds other than L-phenylalanine is lower, so that downstream processing may be cheaper. 

A possible explanation for the superiority of the amino donor, L-aspartic add, has come from studies carried out on mutants of E. coli, in which only one of the three transaminases that are found in E. coli are present. It is believed that a branched chain transaminase, an aromatic amino add transaminase and an aspartate phenylalanine aspartase can be present in E. coli. The reaction of each of these mutants with different amino donors gave results which indicated that branched chain transminase and aromatic amino add transminase containing mutants were not able to proceed to high levels of conversion of phenylpyruvic add to L-phenylalanine. However, aspartate phenylalanine transaminase containing mutants were able to yield 98% conversion on 100 mmol l 1 phenylpyruvic acid. The explanation for this is probably that both branched chain transaminase and aromatic amino acid transminase are feedback inhibited by L-phenylalanine, whereas aspartate phenylalanine transaminase is not inhibited by L-phenylalanine. In addition, since oxaloacetate, which is produced when aspartic add is used as the amino donor, is readily converted to pyruvic add, no feedback inhibition involving oxaloacetate occurs. The reason for low conversion yield of some E. coli strains might be that these E. cdi strains are defident in the aspartate phenylalanine transaminase. 

This enzyme [EC 1.4.1.20] catalyzes the reaction of l-phenylalanine with NAD and water to produce phenylpyruvate, ammonia, and NADH. The enzymes isolated from Bacillus badius and Sporosarcina ureae are highly specific for L-phenylalanine, whereas that isolated from Bacillus sphaericus also acts on L-tyrosine. 

The vast majority of amino acid dehydrogenases use ammonium ions as the amine donor. However, recently a novel N-methyl-L-amino acid dehydrogenase (NMAADH), from Pseudomonas putida, was isolated and used to synthesize N-methyl-L-phenylalanine 36 from phenylpyruvic acid 31 and methylamine 35 in 98% yield and greater than 99%e.e. (Scheme 2.15). The enzyme was shown to accept a number of different ketoacids and also use various amine donors. Glucose dehydrogenase from Bacillus suhtilis was used to recycle the NADPH cofactor [17]. 

Although 2-phenylethanol can be synthesised by normal microbial metabolism, the final concentrations in the culture broth of selected microorganisms generally remain very low [110, 111] therefore, de novo synthesis cannot be a strategy for an economically viable bioprocesses. Nevertheless, the microbial production of 2-phenylethanol can be greatly increased by adding the amino acid L-phenylalanine to the medium. The commonly accepted route from l-phenylalanine to 2-phenylethanol in yeasts is by transamination of the amino acid to phenylpyruvate, decarboxylation to phenylacetaldehyde and reduction to the alcohol, first described by Ehrlich [112] and named after him (Scheme 23.8). 

FIGURE 3.1 The biosynthetic pathway from chorismate to L-phenylalanine in Escherichia coli K12. The mnemonic of the genes involved are shown in parentheses below the enzymes responsible for each step. Compound 1 is L-phenylalanine, 2 is chorisimic acid, 3 is prephenic acid, and 4 is phenylpyruvic acid. 

In the mid to late 1980s, many research groups focused on methods and processes to prepare L-phenylalanine (Chapter 3). This was a direct result of the demand for the synthetic, artificial sweetener aspartame. One of the many routes studied was the use of phenylalanine dH (Scheme 19.4, R = C6H5CH2) with phenylpyruvate (PPA) as substrate.57-58 This enzyme from Bacillus sphaericus shows a broad substrate specificity and, thus, has been used to prepare a number of derivatives of L-phenylalanine.59 A phenylalanine dH isolated from a Rhodococcus strain M4 has been used to make L-homophenylalanine (.S )-2-amino-4-pheny I butanoic acid], a key, chiral component in many angiotensin-converting enzyme (ACE) inhibitors.40 More recently, that same phenylalanine dH has been used to synthesize a number of other unnatural amino acids (UAAs) that do not contain an aromatic sidechain.43... 

Murakami et al. also found that the transamination reaction between hydrophobic pyridoxals (36 and 37) and a-amino acids, to produce a-keto acids, was extremely slow for neutral pyridoxals even in the presence of Cu(n) ions [24]. Detailed kinetic analysis of the reactions carried out in the vesicular system indicated that the transformation of the Cu(n) -quinonoid chelate into the Cu(n) -ketimine chelate was kinetically unfavorable compared with the competing formation of the Cu(n)-aldimine chelate from the same quinonoid species. This problem was solved to a certain extent by quaternization of the pyridyl nitrogen in pyridoxal, as Murakami et al. successfully accomplished transamination between catalyst 36 and L-phenylalanine to produce phenylpyruvic acid. 

The use of interm iates as substrates in L-phenylalanine synthesis avoids inhibition by metabolites. Phenylpyruvic add, an intermediate precursor in tfie biosynthesis of L-phenylalanine, can be converted to L-phenylalanine. L-aspartic add is often used as an amino donor. The amino group can only be transfdred from an... 

Although the phenylalanine-derived fragment of cytochalasin D (148) possesses the configuration of the L-amino-acid both D- and L-phenylalanine are equally effective precursors. Incorporation occurs with complete loss of tritium and N from C-2 and extensive loss of tritium from both diastereotopic hydrogens at C-3. These losses could be explained as occurring in part during the course of transamination and phenylpyruvic acid may then be implicated. This acid depressed the incorporation of D-phenylalanine as measured relative to the L-isomer (2S)-[4 - H]- and (2/ 5)-[2- C]-phenylalanine were fed the cytochalasin isolated showed an increased H C ratio, cf. discussion on p. 1. It follows that L-phenylalanine rather than the D-isomer is the primary precursor of cytochalasin D (148). 

D-Amino acids vary in availability with the species. For example d-phenylalanine is used by rat, mouse, and man (15, 35, 727, 730, 962), whereas D-tryptophan is used by the rat (53, 54, 759, 895), is partially used by the mouse and pig (139, 867), and is not used by man (7, 29). The utilization of the D-amino acids is probably determined by the relative rates of absorption of the D-amino acid from the intestine, and of conversion of d- to L-amino acid in the liver (288). The conversion of d- to L-phenylalanine is reduced in vitamin-Be deficiency (52), as is to be expected for a transformation involving transamination to phenylpyruvic acid. Phenylpyruvic and indolepyruvic acids, the a-keto acids corresponding to phenylalanine and tryptophan, may also, to an extent varying with the species, satisfy growTh requirements (e.g., 55, 109, 436, 725, 911). 

Figure. 2. Enzymatic synthesis of L-phenylalanine by reductive amination starting from phenylpyruvate (using EMR technology).

Phenylalanine, synthesized from [ C]cyanide by the Bucherer modification of the Strecker synthesis, was resolved into its l- and D-iso-mers by the action of the d- and L-amino acid oxidases, respectively. The optically active amino acid was separated from phenylpyruvic acid by cation exchange chromatography (3). Similarly, DL-[ F]acyl-p-fluoro-phenylalanine has been subjected to stereospecific deacylation with the fungal enz)nne, L-amino acylase enz)nnatically generated L-[ F]p-fluoro-phenylalanine was separated from the D-acyl amino acid by column chromatography (4). 

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