Phenylalanine enzymation method

Some chemicals such as iadigo, tryptophan, and phenylalanine are overproduced ia bacteria by pathway engineering (36—38). In this method, the enzymes iavolved ia the entire pathway are overproduced. In addition, the host bacterium is also altered such that the carbon flow is directed toward the engiaeered pathway (38). E. colih.2LS been modified to overproduce iadigo and tryptophan and phenylalanine. CoTjnebacteriumglutamicum has been engiaeered to overproduce tryptophan from 28 to 43 g/L. Similarly, attempts are underway to overproduce several vitamins by pathway engineering (34,38). 

Consider reaction schemes for the production of L-phenylalanine by enzymatic methods. Now match each of the following substrates with the enzyme(s) responsible for L-phenylalanine formation. 

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

Some enzymes with improved single amino acid specificity are commercially available. An example is phenylalanine dehydrogenase (EC 1.4.1.1), derived from bacterial sources, which acts on phenylalanine with the simultaneous conversion of NAD to NADH. Quantitation of the phenylalanine is based on determining the amount of NADH produced using standard procedures. In the direct methods, the absorbance at 340 nm is measured, whereas in the colorimetric methods, the reaction is coupled to an electron acceptor... 

The liver is also the principal metabolic center for hydrophobic amino acids, and hence changes in plasma concentrations or metabolism of these molecules is a good measure of the functional capacity of the liver. Two of the commonly used aromatic amino acids are phenylalanine and tyrosine, which are primarily metabolized by cytosolic enzymes in the liver [1,114-117]. Hydroxylation of phenylalanine to tyrosine by phenylalanine hydroxylase is very efficient by the liver first pass effect. In normal functioning liver, conversion of tyrosine to 4-hy-droxyphenylpyruvate by tyrosine transaminase and subsequent biotransformation to homogentisic acidby 4-hydroxyphenylpyruvic acid dioxygenase liberates CO2 from the C-1 position of the parent amino acid (Fig. 5) [1,118]. Thus, the C-1 position of phenylalanine or tyrosine is typically labeled with and the expired C02 is proportional to the metabolic activity of liver cytosolic enzymes, which corresponds to functional hepatic reserve. Oral or intravenous administration of the amino acids is possible [115]. This method is amenable to the continuous hepatic function measurement approach by monitoring changes in the spectral properties of tyrosine pre- and post-administration of the marker. 

Aspartame has been assayed by a flow injection analysis biosensor employing an immobilized enzyme (pronase) which cleaves the peptide bond. The resulting phenylalanine methyl ester is then detected by an L-amino acid oxidase electrode. This method was applied to analysis of aspartame in foods [82]. 

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... 

An example of a project of this nature is the screening for microorganisms to produce aspartame (28) from a precursor that is easy synthesized by chemical methods. Microorganisms were screened for their ability to catalyze the trans-addition of ammonia across the double bond of /V-fumaryl-L-phenylalanine methyl ester (FumPM) (74) (Scheme 19.43).403 This is essentially the reaction of a mutated aspartase because the native enzyme has such strict substrate specificity (see Section 19.2.4). Although the literature touts this as a successful screening effort, this process has not been practiced commercially because the yields are extremely low.404405... 

Another immobilization method was described by Maeda and coworkers [344], They developed a facile and inexpensive preparation method for the formation of an enzyme-polymeric membrane on the inner wall of the microchannel (PTFE) through cross-linking polymerization in a laminar flow. With this approach, a-chymotrypsin was immobilized successfully. The activity of the immobilized enzyme was tested using N-glutaryl-L-phenylalanine p-nitroanilide as substrate, and the reaction products were analyzed offline by HPLC. There was no significant difference in the hydrolysis efficiency compared to solution-phase batchwise reactions using the same enzyme/substrate molar ratio (Scheme 4.87). 

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. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

In contrast to the various fermentation and bioconversion approaches that have used enzyme stereoselectivity to syntlresize L-phenylalanine as a single isomer, additional methods have been developed that rely on the same stereoselectivity to resolve racemic mixtures of chemically synthesized amino acids. One such general approach, which has been successfully commercialized for L-phenylalanine production, relies on cleavage of the L-isomer of a D,L-a-amino acid amide mixture by an enantiospecific amidase enzyme. The general procedure operated... 

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