L-aspartic add

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. 

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%. 

Under optimal conditions (pH = 8.0,67 g T1 L-aspartic add, 30°C, 1 1 ratio of enzyme activities) after addition of pyridoxal phosphate, 76 g l 1 L-phenylalanine could be produced within 72 hours (92% conversion). This illustrates how simple biochemical manipulation can increase productivity dramatically. 

Of industrial importance at present is die biotransformation of fumarate to L-aspartic add by Escherichia cdi aspartase. Modified versions have been developed, such as the continuous production of L-aspartic add using duolite-ADS-aspartase. A conversion higher than 99% during 3 months on a production scale has been achieved. 

L-alanine can be prepared from aspartic acid (Figure A8.13). L-Aspartate-(5-decarboxylase produced by Xanthomonas oryzae No 531 has been used to prepared L-alanine in 95% yield from 15% L-aspartic add solution. Other strains, ie Pseudomonas dacunhae or Achromobacter pestifer, give comparable yields of L-alanine. The process has been commercialised by Tanabe. 

L-aspartic add has been produced on an industrial scale by the Tanabe Seiyaku Co Ltd, Japan, in a batch wise process using whole cells of Escherichia coli with high aspartase activity. In this process, L-aspartic add is produced from fumaric add and ammonia using aspartase, as described in Figure A8.13. 

To prevent the formation of byproducts like L-malic add and D-alanine, die cells undergo a pH-treatment to inactive fumarase and alanine racemase. Several reactor conformations have been investigated, but a two reactor system was found to be the most effective. The flow sheet of this two reactor system is given in Figure A8.15. In the first reactor L-aspartic add is formed, which reacts in die second reactor to L-alanine. 

Genetic techniques have been used to improve the ability of microorganisms to accumulate amino acids. Several amino acids are manufactured from their direct precursors by the use of microhially produced enzymes. For example, bacterial L-asparlale -carboxylase is used for the production of L-alanine from L-aspartic add. 

S)-Dimethyl N-(9-phenylfluoren-9-yl)aspartate L-Aspartic add, N-(9-phenyl-9H-fluoren-9-yl)-, dimethyl ester (12) (120230-62-8)... 

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],... 

Recently a number of enzymatic systems have been developed at several chemical companies including Upases (synthesis of enantiotrope alcohols, R-amid, S-amin), nitrilases (R-mandehc acid), amidases (non-proteinogenic L-amino acids), aspartic acid ammonia lyase (L-aspartic add), penicilin acylase (6-Aminopenicilanic acid), acylases (semisynthetic penicillins), etc.( Koeller and Wong, 2001 and references therin). 

A commerdal process for the production of L-asparatic add has been developed based on Escherichia cdi strains with high aspartase activity (101-103). Aspartase catalyzes the stereospedfic addition of ammonia to fumaric add (Fig. 29). L-aspartic add can be enzymatically decarboxylated to yield the product L-alanine (104). 

X Tosa, T. Sato, T. Mori, and I. Chibata, Basic studies for continuous production of L-aspartic add by immobilized Escherichia colt cells, Appl. Microbiol, 27 886 (1974). 

At least two enzymes, an acylase and an amino transferase, are necessary for the bioconversion of ACA to L-phenylalanine. The amino source usually is an amiiu) add, L-aspartic add is often used. 

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... 

The elimination of the amino donor, L-aspartic add, 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 F) when the amino donor concentration was increased. Aspartic add was a superior amino donor to glutamic add 35 g l was used. 

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. 

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