Phenylpyruvic

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... 

Phenylpyruvic acid can cause mental retardation m infants who are deficient m the enzymes necessary to convert l phenylalanine to l tyrosine This disorder is called phenylketonuria, or PKU disease PKU disease can be detected by a simple test rou tmely administered to newborns It cannot be cured but is controlled by restricting the dietary intake of l phenylalanine In practice this means avoiding foods such as meat that are rich m l phenylalanine... 

Cm.OROCARBONSANDCm.OROHYDROCARBONS - BENZYL Cm ORIDE, BENZAL Cm ORIDE AND BENZOTRICm ORIDE] (Vol 6) Phenylpyruvic acid [156-06-9]... 

Substitution in position 4 displays a more complex influence. Cyclization of the 4-methyl- and 4-ethyl-thiosemicarbazones of phenylpyruvic acid and of the 4-methylthiosemicarbazone of phenyl-glyoxylic acid (103) was readily achieved (104), whereas it was not possible to cyclize the analogous 4-methyl derivatives of pyruvic and glyoxylic acids. It thus appears that cyclization is hindered by substitution in position 4 and that this unfavorable effect can be partly relieved by the known favorable effect of an aryl or aralkyl group in the a-position. 

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. 

Figure 8.7 Fed-batch fermentation of phenylpyruvic acid to L-phenylalanine.

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. 

Application of the desired biotransformation to give a practical and economical process required high molar conversion yields, high amino transferase activities, highly effident product recovery and an inexpensive source of phenylpyruvic add. With genetic and/or biochemical manipulation considerable progress can be made towards meeting some of these requirements. 

Consider the production of L-phenylalanine using P. fluorescerts by precursor (phenylpyruvic add) addition. Explain briefly why ... 

It was proven that the pathway of L-phenylalanine formation involved phenylpyruvic add as intermediate and two steps could be distinguished (see Figure 8.6 section 8.7) ... 

The best results were obtained with L-aspartic add as the amino donor for P. denitrificam and phenylpyruvic add as the amino acceptor. With L-aspartic add, conversion of phenylpyruvic add exceeded 90%. This may be attributed to absence of feedback inhibition of the reaction due to metabolism of file reaction product, oxaloacetic add. When using glutamic acid the conversion of phenylpyruvic add did not exceed 60%. 

Table 8.7 The most important process conditions for the production of L-phenylalanine by direct fermentation precursor addition, phenylpyruvic add (PPA) bloconversion, acetamidocinnamic add (ACA).

A new development is the industrial production of L-phenylalanine by converting phenylpyruvic add with pyridoxalphosphate-dependent phenylalanine transaminase (see Figure A8.16). The biotransformation step is complicated by an unfavourable equilibrium and the need for an amino-donor (aspartic add). For a complete conversion of phenylpyruvic add, oxaloacetic add (deamination product of aspartic add) is decarboxylated enzymatically or chemically to pyruvic add. The use of immobilised . coli (covalent attachment and entrapment of whole cells with polyazetidine) is preferred in this process (Figure A8.17). 

An alkaline pH (- pH 11) is desirable in order to achieve high conversion rates increase solubility of L-phenylalanine inhibit enzymes catalysing degradation of L-phenylalanine and formation of byproducts reduce inhibition of the reaction by the keto form of phenylpyruvic arid. 

Concentrations of 4% phenylpyruvic arid leads to end product inhibition by the L-phenylalanine produced, resulting in lower final yields. 

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