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Article No : a02_057

Amino Acids

KARLHEINZ DRAUZ, Degussa AG, Hanau-Wolfgang, Germany

IAN GRAYSON, Degussa, Middlesbrough, United Kingdom

AXEL KLEEMANN, Hanau, Germany

HANS-PETER KRIMMER, Degussa AG, Hanau-Wolfgang, Germany

WOLFGANG LEUCHTENBERGER, Degussa AG, D€usseldorf, Germany

CHRISTOPH WECKBECKER, Degussa AG, Hanau-Wolfgang, Germany

1. Introduction and History . . . . . . . . . . . . . . 1

2. Properties . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Physical Properties and Structure . . . . . . . 2

2.2. Chemical Properties . . . . . . . . . . . . . . . . . 4

2.3. Important Amino Acids . . . . . . . . . . . . . . . 7

2.3.1. Proteinogenic Amino Acids . . . . . . . . . . . . . 7

2.3.2. Other Important Amino Acids . . . . . . . . . . . 9

3. Industrial Production of Amino Acids . . . . 14

3.1. General Methods . . . . . . . . . . . . . . . . . . . . 14

3.2. Production of Specific Amino Acids. . . . . . 15

3.2.1. L-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.2.2. L-Arginine. . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.2.3. L-Aspartic Acid and Asparagine . . . . . . . . . . 16

3.2.4. L-Cystine and L-Cysteine . . . . . . . . . . . . . . . 17

3.2.5. L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . 17

3.2.6. L-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . 19

3.2.7. L-Histidine . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.2.8. L-Hydroxyproline. . . . . . . . . . . . . . . . . . . . . 19

3.2.9. L-Isoleucine. . . . . . . . . . . . . . . . . . . . . . . . . 19

3.2.10. L-Leucine . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.2.11. L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.2.12. D,L-Methionine and L-Methionine . . . . . . . . . 21

3.2.13. L-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . 22

3.2.14. L-Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.2.15. L-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.2.16. L-Threonine. . . . . . . . . . . . . . . . . . . . . . . . . 23

3.2.17. L-Tryptophan. . . . . . . . . . . . . . . . . . . . . . . . 24

3.2.18. L-Tyrosine. . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.2.19. L-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

4. Biochemical and Physiological Significance 25

5. Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

5.1. Human Nutrition . . . . . . . . . . . . . . . . . . . . 27

5.1.1. Supplementation . . . . . . . . . . . . . . . . . . . . . 28

5.1.2. Flavorings, Taste Enhancers, and Sweeteners 30

5.1.3. Other Uses in Foodstuff Technology . . . . . . 31

5.2. Animal Nutrition . . . . . . . . . . . . . . . . . . . . 32

5.3. Pharmaceuticals. . . . . . . . . . . . . . . . . . . . . 35

5.3.1. Nutritive Agents . . . . . . . . . . . . . . . . . . . . . 36

5.3.2. Therapeutic Agents . . . . . . . . . . . . . . . . . . . 36

5.4. Cosmetics. . . . . . . . . . . . . . . . . . . . . . . . . . 41

5.5. Agrochemicals . . . . . . . . . . . . . . . . . . . . . . 41

5.5.1. Herbicides . . . . . . . . . . . . . . . . . . . . . . . . . 42

5.5.2. Fungicides . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.5.3. Insecticides . . . . . . . . . . . . . . . . . . . . . . . . 45

5.5.4. Plant Growth Regulators . . . . . . . . . . . . . . . 46

5.6. Industrial Uses . . . . . . . . . . . . . . . . . . . . . . 46

6. Chemical Analysis . . . . . . . . . . . . . . . . . . . 46

7. Economic Significance . . . . . . . . . . . . . . . . 49

8. Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . 50

References . . . . . . . . . . . . . . . . . . . . . . . . . 51

1. Introduction and History

The proteins, although they occur in an almostinfinite variety, are composedof a relatively smallnumber of basic building blocks, all a-aminoacids. In addition, the amino acids fulfill certainregulatory functions in the metabolism and arerequired for the biosynthesis of other functionalstructures. This review is limited, for the most

part, to the protein-forming amino acids, becausethey are by far the most widely distributed innature and are of considerable economic interest.

The ca. 20 different a-amino acids found inproteins are simple organic compounds, in whichan amino group and a side chain (R) are attachedalpha to the carboxyl function. The R group maybe aliphatic, aromatic, or heterocyclic and maypossess further functionality.

DOI: 10.1002/14356007.a02_057.pub2

Amino acid

Dipeptide

Protein (n > 50)

At present over 200 naturally occurring a-aminoacids are known [1–7]. Table 1 shows the a-amino acids found in proteins, where they occurexclusively as the L-enantiomers. D-Amino acidshave been found only in the cell walls of somebacteria, in peptide antibiotics, and in the cellpools of some plants [10, 14, 15]. The L-aminoacids are commonly abbreviatedwith three-letteror one-letter codes, as shown in Table 1 [4]. Inaddition, the abbreviationGlx or Z is used to referto either glutamic acid or glutamine, and theabbreviation Asx or B is used to refer to eitheraspartic acid or asparagine. More recently theabbreviation Sec or U has been assigned toselenocysteine.

History. The history of amino acid chemistrybegan in 1806, when two French investigators,VAUQUELIN and ROBIQUET, isolated asparaginefrom asparagus juice. It was not until 1925that SCHRYVER and BURTON isolated threoninefrom oat protein, the last of the ca. 20 protein-forming amino acids to be discovered. STRECKERsynthesized alanine in 1850 from acetaldehyde,ammonia, and hydrogen cyanide. ESCHER

established the hypothesis of essential aminoacids. EMIL FISCHER discovered that the aminoacids were building blocks of the proteins.ABDERHALDEN synthesized threonine from acrylicacid derivatives and methanol. ROSE et al. recog-nized threonine as the last of the eight essentialamino acids. D,L-Methionine was producedindustrially in Germany in 1948, and in 1956L-glutamic acid was produced by fermentationin Japan.

Origin of AminoAcids.The first amino acidswere probably produced on the earth more than3�109 years ago via ‘‘prebiotic synthesis’’ in theprimordial atmosphere. The concept of prebioticsynthesis is based on laboratory experiments inwhich glycine, alanine, aspartic acid, glutamicacid, and other compounds were produced by theaction of an electrical discharge on a simulatedprimordial atmosphere consisting of methane,hydrogen, water, and ammonia [16]. Since then,traces of amino acids have been detected inmoonrocks, meteorites, and interstellar space.

2. Properties

2.1. Physical Properties and Structure

a-Amino acids are nonvolatile, white, crystallinecompounds with no defined melting points. Theyare relatively stable on heating, generally decom-posing at 250–300 �C. Both the low volatilityand the thermal stability result from the low-energy dipolar structure (zwitterion, inner salt,betaine), which the amino acids assume in thesolid state.

Evidence for this structure is provided byinfrared and Raman spectra in which the bandstypical of -NH2 and -COOH moieties are absent.Equilibrium in solution also lies almost exclu-sively on the side of the dipolar form; therefore,amino acids are insoluble in nonpolar solventsand usually not very soluble in polar ones. Theonly amino acids that exhibit any appreciablesolubility in alcohol are proline and hydroxypro-line. Solubility in water depends on the pH: theminimum is at the isoelectric point.

This solubility minimum at the isoelectricpoint is quite useful for purifying and recrystal-lizing amino acids. The analytical technique forseparating amino acid mixtures by electrophore-sis is based on the fact that a specific amino aciddoes not migrate in an electric field at its isoelec-tric point, pI, a physical constant for each aminoacid.

The structures and physical properties of themost important a-amino acids are given inSection 2.3.1.

2 Amino Acids Vol. 3

Stereochemistry. With the exception ofglycine, the simplest amino acid (R ¼ H), allnatural a-amino acids are chiral compoundsoccurring in two enantiomeric (mirror-image)forms.

L-Amino acid D-Amino acid

The prefixes L and D express the absoluteconfiguration at the a-carbon atom by means ofthe formal stereochemical relationship to L- or D-glyceraldehyde, the reference substance intro-duced by EMIL FISCHER in 1891. In addition tothe spacial representations shown above, theFischer projections are also universally recog-nized and used:


Polarimetric determination of the specific rota-tion ½a�tD can be used to differentiate between thetwo enantiomers and to check their optical purity.The molecular rotation ½M�tD is less common:

Table 1. Common amino acids of proteins

Trivial name IUPAC

abbreviation

One-

Letter

Code

Protein Content,

g/100 g

[8] [10]

L-Alanine Ala A silk

fibroin

25 29.7

L-Arginine Arg R salmin 87

edostin 17

wool 10

gelatin 8.3

rat liver histone 15.9

L-Asparagine Asn N

L-Aspartic acid Asp D edestin 12

hemoglobin 9–10

barley globulin 10.3

L-Cysteine Cys C wool

keratin

11.9

human hair

keratin

14.4

feather keratin 8.2

L-Cystine Cys-Cys,

(Cys)2, Cyss

C

L-Glutamic acid Glu E gliadin 47

zein 31

wheat gliadin 39.2

maize zein 22.9

L-Glutamine Gln Q

Glycine Gly G gelatin 26

silk fibroin 44

L-Histidine His H hemoglobin 7

L-Hydroxyproline Hyp – gelatin 15

L-Isoleucine Ile I edestin 21

hemoglobin 29

serum proteins 20

oat globulin 4.3

beef serum

albumin

2.6

L-Leucine Leu L edestin 21

hemoglobin 29

serum proteins 20

maize zeins 19

L-Lysine Lys K serum

albumin

13

serum globulin 6

horse myoglobin 15.5

L-Methionine Met M egg

albumin

5

casein 3 4.1

b-lactoglobulin 3.2

L-Phenylalanine Phe F zein 8

egg albumin 5 7.7

serum albumin 7.8

L-Proline Pro P gelatin 17 16.3

gliadin 13

salmin 6.9

casein 10.6

L-Serine Ser S silk

fibroin

13 16.2

trypsinogen 16.7

pepsin 12.2

L-Threonine Thr T casein 4

(Continued)

Table 1 (Continued)

Trivial name IUPAC

abbreviation

One-

Letter

Code

Protein Content,

g/100 g

[8] [10]

human hair

keratin

8.5

avidin 10.5

L-Tryptophan Trp W fibrin 3

egg lysozyme 10.6

L-Tyrosine Tyr Y fibrin 6

silk fibroin 13

papain 14.7

L-Valine Val V casein 8

beef sinew 17.4

beef aorta 17.6

Vol. 3 Amino Acids 3

½M�tD ¼ Mr

100�½a�tD

Mr molecular mass; t temperature; D589.3 nm (wavelength of the sodium D line).Other methods for investigating the structure ofamino acid enantiomers include theCotton effect(change in molecular rotation as a function of thewavelength of plane-polarized light), as reflectedin optical rotational dispersion (reversal of thedirection of the molecular rotation at the wave-length of the absorption maximum), and circulardichroism (differing absorption for left- andright-handed circularly polarized light). L-Ami-no acids exhibit a positive carbonyl Cotton ef-fect, D-amino acids a negative one.

Isoleucine, threonine, and hydroxyprolineeach contain two chiral carbon atoms; therefore,they appear in four stereoisomeric forms. Cys-tine, which likewise contains two chiral carbons,has only three stereoisomers: L-, D-, and meso-cystine, the meso form having a plane ofsymmetry. According to the Cahn–Ingold–Prelog rule (R, S rule) [17], all proteinogenicL-a-amino acids, with the exception of L-cysteineand L-cystine, are S; the D-a-amino acids are R.According to this system, L-threonine, for exam-ple, is termed (2S, 3R)-threonine. However, theL- and D-descriptors are still inwidespread use forthe simple L-a-amino acids.

Absorption Spectra. Aliphatic amino acidsexhibit no absorption in the UV region above220 nm, with the exception of cystine (240 nm).The aromatic amino acids, phenylalanine, tyro-sine, and tryptophan, absorb between 250 and300 nm [1, vol. 2]. The exact position of themaximum and the molar extinction coefficiente are affected by the pH of the aqueous solution.

Two infrared bands are especially character-istic of amino acids: 1560–1600 cm�1 (–COO�)and ca. 3070 cm�1 ð�NHþ

3 Þ. These bands arealso evidence of the bipolar nature of the aminoacids.

2.2. Chemical Properties

Acidity and Basicity. The chemical prop-erties of the a-amino acids are primarily theproperties of the amino and carboxyl groups.Amino acids react with strong acids as proton

acceptors (bases) and with strong bases as protondonors (acids). In acidic medium they arepresent predominantly as cations; in basic medi-um they are present predominantly as anions.Figure 1 shows the titration curves of glycine,glutamic acid, and lysine. The pK1 and pK2

values correspond to the inflection points ofthe titration curve, the pH value where theconcentrations of the zwitterion form andthe cationic or anionic form are equal. The pK1,pK2, and pI values can be calculated from thetitration curves with the Henderson–Hasselbalchequation:

Amino acids with additional basic or acidicgroups (arginine, lysine, glutamic acid, cysteine)exhibit additional pK values. Amino acids act asbuffers in the region of their pK values.

Figure 1. Titration curves of glycine, glutamic acid, andlysine [9]


The pK1 values show the amino acids to beconsiderably stronger acids than acetic acid.However, because of intramolecular protonationof the amine moiety by the carboxyl group,aqueous solutions of amino acids are onlyweaklyacidic. The pH values of aqueous monoamino-monocarboxylic acids lie between 5.5 and 6.0.Solutions of the acidic amino acids aspartic acidand glutamic acid have pH values of ca. 2. Theweakly basic amino acid histidine has a pH of 7.5in aqueous solution; the more strongly basicamino acids lysine and arginine have pH valuesof ca. 11–12. These differences in acidities andbasicities are utilized in the separation of aminoacid mixtures by ion-exchange chromatographyand electrophoresis.

Reactions. Because of their bifunctionaland sometimes trifunctional character, thea-amino acids are capable of taking part in avariety of chemical reactions. Comprehensivetreatments of these may be found in monographsand reviews [1, 9–12, 18].

The introduction of groups protecting theamino, carboxyl, and side-chain functions isespecially important for peptide synthesis [13].Additionally, a-amino acids play a prominentrole as intermediates in the synthesis of hetero-cycles [19]. The chiral a-amino acids and theirderivatives are inexpensive and, for the mostpart, readily available synthons for numerousnatural products and pharmaceuticals. In manycases optically active amino acids can also beused to induce chirality during the course of asynthetic process [20].

a-Amino acids form chelate-like complexeswith heavy metal ions. The best known are thedark blue, easily crystallized copper chelates:

Bis(glycinato) copper(II) hydrate

This complex formation can be used to protectboth the a-amino and the carboxyl groupduring synthesis of e-N-acetyl derivatives orcarbamates of lysine or other side-chain deriva-tives. In these copper or cobalt complexes thea-carbon atom is activated sufficiently to react

with aldehydes. A well-known example is thealkaline condensation of the glycine–coppercomplex with acetaldehyde, resulting in threo-nine. Especially important is the reaction of a-amino acids with ninhydrin to form a blue-violetdye, the basis of a sensitive optical method fordetecting amino acids (see Chap. 6).

Free amino acids react with nitrous acidto yield a-hydroxycarboxylic acids, with reten-tion of configuration. Volumetric measurementof the nitrogen gas set free is the basis ofVan Slyke’s method for the quantitative analysisof amino acids. Reaction of amino acidesters with nitrous acid gives the acid-labilediazocarboxylic acid esters. Treatment of N-al-kyl- orN-arylamino acidswith nitrous acid yieldsthe N-nitroso derivatives, which can be dehy-drated to sydnones in the presence of aceticanhydride:

a-Amino acids have found use as organoca-talysts for asymmetric organic reactions, forexample asymmetric versions of the aldol orthe Mannich reactions [21]. The amino acidsmost often employed are L- or D-proline, theirderivatives, or short-chain peptides based onproline.

Oxidizing agents attack the amino group,converting the amino acid into an iminocar-boxylic acid. These are unstable and eitherhydrolyze to a-oxocarboxylic acids or decom-pose, after decarboxylation, into ammoniaand the aldehyde containing one carbonatom less. Diketones, triketones, N-bromosucci-nimide, or silver oxide may serve as theoxidizing agent. The best known exampleis the reaction with ninhydrin. Oxidative deami-nation also can be carried out enzymaticallywith D- or L-amino acid oxidases. This alsoproceeds via the a-iminocarboxylic acids,which subsequently are hydrolyzed to a-oxoacids:


This enantioselective enzymatic oxidative de-amination is the basis of analytical methods forthe determination of amino acid enantiomers.

The N-acylation of a-amino acids with acylchlorides or anhydrides under Schotten-Baumannconditions produces N-acyl-a-amino acids,which have numerous uses. For example, theN-acetyl derivatives of D,L-amino acids (e.g.,alanine, valine, methionine, phenylalanine, tryp-tophan) are intermediates in the production ofL-amino acids by enzymatic resolution usingaminoacylases (see Section 3.1). Amino acidsacylated with naturally occurring fatty acid resi-dues are used industrially as biodegradablesurfactants.

If free amino acids are heated above 200 �C,especially in the presence of soda lime or metalions, they readily decarboxylate to form amines.The enzymatic decarboxylation of amino acidsgives biogenic amines. Some of these amines arephysiologically active neurotransmitters (hista-mine, tyramine, dopamine, serotonin).

The esters are usually prepared by directesterification, e.g., reaction of the a-amino acidwith anhydrous alcohol in the presence of anhy-drous hydrogen chloride. The initial product isthe hydrochloride of the ester, which is liberatedby addition of base. On standing or warming,the free esters of a-amino acids can eliminatealcohol to form 2,5-diketopiperazines:

Amino acid esters are useful intermediatesfor peptide synthesis because the carboxylfunction is protected. The esters may be con-verted to amino alcohols by treatment with astrong reducing agent, such as lithium aluminumhydride.

Several cyclic derivatives of the a-aminoacids are of industrial importance. The hydan-toins or imidazolidine-2,4-diones, which havebeen used as intermediates in the synthesis ofa-amino acids, are most conveniently preparedby treatment of aldehyde cyanohydrins withammonium carbonate or urea. They also may beobtained by reacting amino acids with cyanatesor isocyanates, the reaction proceedingvia the ureidocarboxylic acid (hydantoic acidderivative):

The thiohydantoins are obtained by the reac-tion with isothiocyanates.

Dehydration ofN-acylamino acids with aceticanhydride or carbodiimide yields 1,3-oxazolin-5-ones (azlactones):

Racemization occurs readily during the reac-tion. The azlactones are intermediates in thesynthesis of amino acids.

The N-carboxylic acid anhydrides (Leuchsanhydrides, 1,3-oxazolidine-2,5-diones) are of-ten used in peptide synthesis, especially to pre-pare poly-a-amino acids. These reactive aminoacid derivatives are obtained by treating theamino acid with phosgene in the presence oftertiary amines

or by elimination of benzyl chloride fromN-benzyloxycarbonylamino acid chlorides.


2.3. Important Amino Acids

2.3.1. Proteinogenic Amino Acids

L-Alanine [56-41-7], 2-aminopropionic ac-id, C3H7NO2, Mr 89.09, mp 314 �C (decomp.),½a�25D þ14:47� (c ¼ 10.03 in 6 M HCl), solubili-ty 16.51 (25 �C) g/100 g H2O, pI 6.01, dissocia-tion constants: pK1 2.34, pK2 9.69.

L-Arginine [74-79-3], 2-amino-5-guanidi-novaleric acid, 2-amino-5[(aminoiminomethyl)amino]pentanoic acid, C6H14N4O2, Mr 174.20,mp 244 �C (decomp.), ½a�25D þ27:58� (c ¼ 2 in6 MHCl), solubility 14.87 (20 �C) g/100 gH2O,pI 10.76, dissociation constants: pK1 2.01, pK2

9.04 (a-NH2), pK3 12.48 (guanidyl). Hydrochlo-ride: [1119-34-2], C6H15ClN4O2,Mr 210.66, mp220 �C (decomp.), ½a�25D þ11:7� (c ¼ 5 in H2O),solubility 75.1 (20 �C) g/100 g H2O.

L-Asparagine [70-47-3], 2-aminosucci-namic acid, 2,4-diamino-4-oxobutanoic acid,C4H8N2O3, Mr 132.13, mp 236 �C (decomp.),½a�20D þ32:6� (c ¼ 1 in 0.1 M HCl), solubility3.11 (28 �C) g/100 g H2O, pI 5.41, dissociationconstants: pK1 2.02, pK2 8.8.

L-Aspartic Acid [56-84-8], aminosuccinicacid, 2-amino-1,3-butanedioic acid, C4H7NO4,Mr 133.10, mp 270 �C (decomp., sealed tube),½a�25D þ25:4� (c ¼ 2 in 5 M HCl), solubility0.5 (25 �C) g/100 g H2O, pI 2.98, dissociationconstants: pK1 2.1, pK2 3.86 (b-COOH), pK3

9.82.

L-Cysteine [52-90-4], 2-amino-3-mercap-topropionic acid, 3-mercaptoalanine,C3H7NO2S, Mr 121.16, mp 240 �C (decomp.),½a�25D þ9:7� (c ¼ 8 in 1 M HCl), solubility 28(25 �C) g/100 mL solution, 16 (20 �C) g/100 gH2O, pI 5.02, dissociation constants: pK1

1.71, pK2 8.27 (–SH), pK3 10.78. Hydrochloridemonohydrate: [7048-04-6], C3H10ClNO3S,Mr 175.64, mp (anhydr.) 178 �C (decomp.),½a�25D þ6:53� (calculated as cysteine) (c ¼ 2in 5 M HCl), solubility >100 (20 �C) g/100 gH2O.

L-Cystine [56-89-3], 2, 20-diamino-3, 30-dithiobis(propionic acid), 3, 30-dithiobis(2-ami-nopropanoic acid), C6H12N2O4S2, Mr 240.30,mp 260 �C (decomp.), ½a�25D �212� (c ¼ 1 in1 M HCl), solubility 0.011 (25 �C) g/100 gH2O, pI 5.02, dissociation constants: pK1

1.04, pK2 2.05 (-COOH), pK3 8.0 (–NH2), pK4

10.25 (–NH2).

L-Glutamic Acid [56-86-0], 2-aminogluta-ric acid, 2-amino-1,4-pentanedioic acid,C5H9NO4, Mr 147.13, mp 224–225 �C (de-comp.), ½a�25D þ31:5� (c ¼ 2 in 5 MHCl, 20 �C),solubility ¼ 0.843 (25 �C) g/100 gH2O, pI 3.08,dissociation constants: pK1 2.1, pK2 4.07, pK3

9.47.

L-Glutamine [56-85-9], 2-aminoglutaramicacid, 2,5-diamino-5-oxopentanoic acid,C5H10N2O3,Mr 146.15,mp 185-6

�C (decomp.),½a�25D þ31:8� (c ¼ 2 in 1 M HCl), solubility 3.6(19 �C) g/100 g H2O, pI 5.65, dissociation con-stants: pK1 2.17, pK2 9.13.


Glycine [56-40-6], aminoacetic acid,C2H5NO2,Mr 75.07, mp 262, 292

�C (decomp.),solubility 24.99 (25 �C) g/100 g H2O, pI 6.06,dissociation constants: pK1 2.35, pK2 9.13.

L-Histidine [71-00-1], a-amino-1H-imid-azole-4-propionic acid, 1H-imidazole-4-alanine,C6H9N3O2, Mr 155.16, mp 277, 287 �C (de-comp.), ½a�25D þ13:0� (c ¼ 1 in 6 M HCl), solu-bility 4.29 (25 �C) g/100 g H2O, pI 7.64, disso-ciation constants: pK1 1.77, pK2 6.1 (imidazolyl),pK3 9.18. Hydrochloride monohydrate: [5934-29-2], C6H12ClN3O3, Mr 209.63, mp 259 �C(decomp.), solubility 16.99 (20 �C) g/100 gH2O.

L-Hydroxyproline [51-35-4], trans-4-hy-droxy-2-pyrrolidinecarboxylic acid, C5H9NO3,Mr 131.13, mp 274 �C (decomp.), ½a�22D �75:2�

(c ¼ 2 in H2O), solubility 36.11 (25�C) g/100 g

H2O, pI 5.82, dissociation constants: pK1 1.92,pK2 9.73.

L-Isoleucine [73-32-5], 2-amino-3-methyl-valeric acid, 2-amino-3-methylpentanoic acid,C6H13NO2, Mr 131.18, mp 285-6 �C (decomp.),½a�25D þ40:6� (c ¼ 2 in 6 M HCl), solubility4.117 (25 �C) g/100 g H2O, pI 6.02, dissociationconstants: pK1 2.36, pK2 9.68.

L-Leucine [61-90-5], 2-amino-4-methylva-leric acid, 2-amino-4-methylpentanoic acid, 2-

aminoisocaproic acid, C6H13NO2,Mr 131.18,mp293-5, 314-5 �C (decomp.), ½a�25D �10:9� (c ¼0.52 in H2O), solubility 2.19 (25 �C) g/100 gH2O, pI 5.98, dissociation constants: pK1 2.36,pK2 9.6.

L-Lysine [56-87-1], 2,6-diaminohexanoicacid, 2,6-diaminocaproic acid, C6H14N2O2, Mr

146.19, mp 225-5 �C (decomp.), ½a�25D þ25:9�

(c ¼ 2 in 5 M HCl), solubility >100 (25 �C)g/100 g H2O, pI 9.47, dissociation constants:pK1 2.18, pK2 8.95 (a-NH2), pK3 10.53. Hydro-chloride: [657-27-2], C6H15ClN2O2, Mr 182.65,mp 253-6 �C (decomp.), solubility 72.5 (25 �C)g/100 mL solution.

L-Methionine [63-68-3], 2-amino-4-(methylthio)butyric acid, 2-amino-4-(methylthio)butanoic acid, C5H11NO2S, Mr

149.21,mp 283 �C (decomp.), ½a�28D þ23:4� (c ¼5 in 3 M HCl), solubility 5.37 (20 �C) g/100 gH2O, pI 5.74, dissociation constants: pK1 2.28,pK2 9.21.

L-Phenylalanine [63-91-2], 2-amino-3-phenylpropionic acid, a-aminobenzenepropa-noic acid, C9H11NO2, Mr 165.19, mp 283-4 �C(decomp.), ½a�20D �35:1� (c ¼ 2 in H2O), solubil-ity 2.965 (25 �C) g/100 g H2O, pI 5.48, dissoci-ation constants: pK1 1.83, pK2 9.13.

L-Proline [147-85-3], 2-pyrrolidinecar-boxylic acid, 2-carboxypyrrolidine, C5H9NO2,


Mr 115.13, mp 220–222 �C (decomp.),½a�25D �85:0� (c ¼ 1 in H2O), solubility 162.3(25 �C) g/100 g H2O, pI 6.3, dissociation con-stants: pK1 2.0, pK2 10.6.

L-Serine [56-45-1], 2-amino-3-hydroxy-propionic acid, 2-amino-3-hydroxypropanoic ac-id, C3H7NO3, Mr 105.09, mp 228 �C (decomp.),½a�26D �6:8� (c ¼ 10 in H2O), solubility 35.97(20 �C) g/100 g H2O, pI 5.68, dissociation con-stants: pK1 2.21, pK2 9.15.

L-Threonine [72-19-5], 2-amino-3-hydro-xybutyric acid, 2-amino-3-hydroxybutanoic ac-id, C4H9NO3, Mr 119.12, mp 253 �C (decomp.),½a�26D �28:6� (c ¼ 2 in H2O), solubility 9.03(20 �C) g/100 g H2O, pI 6.16, dissociation con-stants: pK1 2.71, pK2 9.62.

L-Tryptophan [73-22-3], 2-amino-3-(30-indolyl)propionic acid, a-amino-1H-indole-3-propanoic acid, C11H12N2O2, Mr 204.23, mp290–292 �C, 281 �C (decomp.), ½a�25D �32:15�

(c ¼ 1 in H2O), solubility 1.14 (25 �C) g/100 g H2O, pI 5.88, dissociation constants: pK1

2.38, pK2 9.39.

L-Tyrosine [60-18-4], 2-amino-3-(4-hydro-xyphenyl)propionic acid, [3-(4-hydroxyphenyl)]alanine, 2-amino-3-(p-hydroxyphenyl)propionicacid, a-amino-4-hydroxybenzenepropanoic ac-id, C9H11NO3, Mr 181.19, mp 342-4 (sealedtube), 297–298 �C (decomp.), ½a�25D �7:27� (c4 in 6 M HCl), solubility 0.045 (25 �C) g/

100 g H2O, pI 5.63, dissociation constants: pK1

2.2, pK2 9.11, pK3 10.07 (–OH).

L-Valine [72-18-4], 2-amino-3-methylbuty-ric acid, 2-aminoisovaleric acid, C5H11NO2, Mr

117.15, mp 315 �C (decomp.), ½a�20D þ26:7� (c3.4 in 6 MHCl), solubility 8.85 (25 �C) g/100 gH2O, pI 5.96, dissociation constants: pK1 2.32,pK2 9.62.

2.3.2. Other Important Amino Acids

b-Alanine [107-95-9], 3-aminopropionicacid, C3H7NO2,Mr 89.09, dissociation constant:pK1 3.6, occurrence: apple, constituent of pan-tothenic acid, carnosine, anserine.

D-Alanine [338-69-2], 2-aminopropionicacid, D-Ala, C3H7NO2, Mr 89.09, application:LHRH-antagonists, e.g., Abarelix (antineoplas-tic) [22].

D,L-Alanine [302-72-7], 2-aminopropionicacid, C3H7NO2,Mr 89.09,mp 295

�C (decomp.),solubility 16.72 (25 �C) g/100 g H2O, pI 6.11,dissociation constants: pK1 2.35, pK2 9.87.

a-Aminoisobutyric Acid [62-57-7], 2-ami-no-2-methylpropionic acid, Aib, C4H9NO2, Mr

103.12, application: growth hormone secretago-gues, e.g., MK-0677 [23].

L-a-Aminobutyric Acid [1492-24-6], 2-aminobutyric acid, L-Abu, C4H9NO2, Mr


103.12, application: Levetiracetam (anticonvul-sant) [24].

g-Aminobutyric Acid (GABA) [56-12-2],4-aminobutyric acid, C4H9NO2, Mr 103.12, oc-currence: citrus fruits, sugar beet, brain.

D,L-Aspartic Acid [617-45-8], aminosucci-nic acid, 2-amino-1,3-butanedioic acid,C4H7NO4, Mr 133.10, mp 275 �C (decomp.,sealed tube), solubility 0.775 (25 �C) g/100 gH2O, pI 2.98, dissociation constants: pK1 2.1,pK2 3.86 (b-COOH), pK3 9.82.

L-Azetidine-2-carboxylic Acid [2133-34-8], C4H7NO2, Mr 101.10, application: Ximela-gatran (thrombin inhibitor) [25].

Betaine [107-43-7], carboxymethyl-tri-methyl ammonium betaine, C5H11NO2, Mr

117.15, occurrence: sugar beet.

L-Carnitine [541-15-1], (3-carboxy-2-hy-droxypropyl)trimethyl ammonium betaine,C7H15NO3, Mr 161.2, occurrence: Lys metabo-lite, muscle.

L-Citrulline [372-75-8], 2-amino-5-ureido-pentanoic acid, C6H13N3O3, Mr 175.19, mp234–237 �C , 222 �C (decomp.), ½a�25D þ24:2�

(c ¼ 2 in 5 M HCl), solubility 10.3 (20 �C) g/100 g H2O, pI 5.92, dissociation constants: pK1

2.43, pK2 9.41; occurrence: urea cycle, water-melon.

D-Citrulline [13594-51-9], 2-amino-5-ur-eidopentanoic acid, D-Cit, C6H13N3O3, Mr

175.19, application: Cetrorelix (antineoplastic)[26].

Creatine [57-00-1], N-(aminoimino-methyl)-N-methylglycine, N-methylguanetic ac-id, C4H9N3O2,Mr 131.14, occurrence: muscle ofvertebrates.

D-Cyclohexylalanine [58717-02-5], 2-ami-no-3-cyclohexylpropionic acid, D-Cha,C9H17NO2, Mr 171.25, application: (thrombininhibitors) [27].

(3S,4aS,8aS)-Decahydroisoquinolinecar-boxylic acid [115238-58-9], decahydroisoqui-noline-3-carboxylic acid, C10H17NO2, Mr

183.26, application: Nelfinavir (antiviral) [28].

L-2,3-Diaminopropionic Acid [4033-39-0], L-Dap, C3H8N2O2, Mr 104.11, application:Imidapril (antihypertensive) [29].

L-3,4-Dihydroxyphenylalanine (DOPA)[59-92-7], 2-amino-3-(3,4-dihydroxyphenyl)


propionic acid, C9H11NO4, Mr 197.17, occur-rence: Tyr metabolite, faba bean.

D-Glutamic Acid [6893-26-1], 2-aminoglu-taric acid, 2-amino-1,4-pentanedioic acid, D-Glu,C5H9NO4,Mr 147.12, application: Spiroglumide(CCK-B-antagonist) [30].

L-Homocysteine [6027-13-0], 2-amino-4-mercaptobutyric acid, C4H9NO2S, Mr 135.18,occurrence: Met metabolite, mushrooms, appli-cation: Omapatrilat (ACE inhibitor) [31].

D-p-Hydroxyphenylglycine [22818-40-2],amino-(4-hydroxyphenyl)acetic acid, D-Phg(OH), C8H9NO3,Mr 167.15, application: Amox-icillin (antibiotic) [32].

L-5-Hydroxytryptophan [4350-09-8], 2-amino-3-(5-hydroxy-1H-indol-3-yl)propionicacid, C11H12N2O3, Mr 220.22, occurrence: sero-tonin precursor.

D,L-Isoleucine [443-79-8], 2-amino-3-methylvaleric acid, 2-amino-3-methylpentanoicacid, C6H13NO2, Mr 131.18, mp 292 �C (de-comp.), solubility 2.011 (25 �C) g/100 g H2O,

pI 6.04, dissociation constants: pK1 2.32, pK2

9.76.

D,L-Leucine [328-39-2], 2-amino-4-methylvaleric acid, 2-amino-4-methylpentanoicacid, 2-aminoisocaproic acid, C6H13NO2, Mr

131.18, mp 293-5, 332 �C (decomp.), solubility1.00 (25 �C) g/100 g H2O, pI 6.04, dissociationconstants: pK1 2.33, pK2 9.74.

L-tert-Leucine [20859-02-3], 2-amino-3,3-dimethylbutyric acid, L-Tle, C6H13NO2, Mr

131.18, application: div. pharmaceuticals [33].

D,L-Lysine Hydrochloride [70-53-1],C6H15ClN2O2,Mr 182.65,mp 264

�C (decomp.),solubility 35.98 (20 �C) g/100 g H2O.

D,L-Methionine [59-51-8], 2-amino-4-(methylthio)butyric acid, 2-amino-4-(methylthio)butanoic acid, C5H11NO2S, Mr

149.21, mp 281 �C (decomp.), solubility 3.35(25 �C) g/100 g H2O, pI 5.74, dissociation con-stants: pK1 2.28, pK2 9.21.

S-Methyl-L-Cysteine [1187-84-4],2-ami-no-3-methylthiopropionic acid L-Cys(Me),C4H9NO2S, Mr 135.18, application: Kynosta-tin-272 (antiviral) [34].

L-S-Methylmethionine (Vitamin U)[4727-40-6], 3-amino-3-carboxy-propyl)-di-methyl sulfonium, C6H13NO2S, Mr 163.24, oc-currence: cabbage, asparagus.

D-3-(20-Naphthyl)-Alanine [76985-09-6],2-amino-3-naphthalen-2-ylpropionic acid, D-Nal,


C13H13NO2, Mr 215.25, application: LHRH-antagonists, e.g., Abarelix (antineoplastic) [35].

L-Ornithine [70-26-8], 2,5-diaminovalericacid, C5H12N2O2, Mr 132.16, mp 140 �C (de-comp.), ½a�25D þ16:5� (c ¼ 4.6 in H2O), solubili-ty, pI 9.7, dissociation constants: pK1 1.94, pK2

8.65 (a-NH), pK3 10.76; occurrence: urea cycle,shark liver, application: Atosiban (tocolytic)[36]. Hydrochloride: [3184-13-2],C5H13ClN2O2, Mr 168.6, mp 215 �C (decomp.),½a�25D þ28:3� (calculated as ornithine) (c ¼ 2 in5 MHCl), solubility 54.36 (20 �C) g/100 gH2O.

D-Penicillamine [52-67-5], 2-amino-3-mercapto-3-methylbutyric acid, C5H11NO2S,Mr

149.21, occurrence: hydrolysis product of peni-cillin.

D-Phenylalanine [673-06-3], 2-amino-3-phenylpropionic acid, a-aminobenzenepropa-noic acid, D-Phe, C9H11NO2,Mr 165.19, applica-tion: Nateglinide (antidiabetic) [38].

D,L-Phenylalanine [150-30-1], 2-amino-3-phenylpropionic acid, a-aminobenzenepropa-noic acid, C9H11NO2, Mr 165.19, mp 284–288 �C, 320 �C (decomp.), solubility 1.29(25 �C) g/100 g H2O, pI 5.91, dissociation con-stants: pK1 2.58, pK2 9.24

D-p-Cl-Phenylalanine [14091-08-8], 2-amino-3-(4-chlorophenyl)propionic acid, D-Phe(Cl), C9ClH10NO2, Mr 199.63, application:LHRH antagonists, e.g., Abarelix (antineoplas-tic) [39].

L-p-NO2-Phenylalanine [949-99-5], 2-amino-3-(4-nitrophenyl)propionic acid, L-Phe(NO2), C9H10N2O4,Mr 210.17, application: Zol-mitriptan (antimigraine) [40].

S-Phenyl-L-Cysteine [34317-61-8], 2-ami-no-3-phenylthiopropionic acid, L-Cys(Ph),C9H11NO2S, Mr 197.61, application: Nelfinavir(antiviral) [41].

D-Phenylglycine [875-74-1], 2-aminophe-nyl-acetic acid, D-Phg, C8H9NO2, Mr 151.16,application: Ampicillin (antibiotic) [42].

L-Pipecolic Acid [3105-95-1], piperidine-2-carboxylic acid, L-Pec, C6H11NO2, Mr

129.16, occurrence: legumes, metabolite of Lys,application: Ropivacaine (local anesthetic) [43].

L-Piperazinecarboxylic Acid [147650-70-2], piperazine-2-carboxylic acid, C5H10N2O2,Mr

130.15, application: Indinavir (antiviral) [44].


D-Piperidine-3-carboxylic Acid [25137-00-2], nipecotic acid, C6H11NO2, Mr 129.7, ap-plication: Tiagabine (anticonvulsant) [45].

D,L-Proline [609-36-9], 2-pyrrolidinecar-boxylic acid, 2-carboxypyrrolidine, C5H9NO2,Mr 115.13, mp 205 �C (decomp.), pI 6.3, disso-ciation constants: pK1 2.0, pK2 10.6.

D-Proline [344-25-2], 2-pyrrolidinecar-boxylic acid, 2-carboxypyrrolidine, D-Pro,C5H9NO2, Mr 115.13, application: Eletriptan(antimigraine) [46].

D-3-(30-Pyridyl)-alanine [70702-47-5], 2-amino-3-pyridin-3-ylpropionic acid, D-Pal,C8H10N2O2, Mr 166.18, application: LHRH an-tagonist, e.g. Abarelix (antineoplastic) [47].

L-Pyrrolysine [448235-52-7], N6-[(3R)-1,5-didehydro-3-methyl-D-prolyl]- L-lysine,C12H21N3O3,Mr 255.31, the 22nd natural aminoacid to be discovered, occurrence: archaea as partof methane-producing enzymes [37].

L-Saccharopine [997-68-2], 2-(50-amino-50-carboxypentylamino)pentanedioic acid,C11H20N2O6,Mr 276.27, occurrence: baker’s andbrewer’s yeast.

L-Selenocysteine [10236-58-5], 3-selenyl-L-alanine, Sec, U, C3H7NO2Se, Mr 168.05, the21st natural amino acid to be discovered [48].Often found in the form of its dimer, R,R-sele-nocystine [29621-88-3], C6H12N2O4Se2, Mr

334.09.

L-Selenomethionine [3211-76-5], (S)-2-amino-4-(methylseleno)butanoic acid,C5H11NO2Se, Mr 196.11, occurrence: plant tis-sue, application: dietary supplement [49].

D,L-Serine [302-84-1], 2-amino-3-hydroxy-propionic acid, 2-amino-3-hydroxypropanoic ac-id, C3H7NO3, Mr 105.09, mp 246 �C (decomp.,sealed tube), solubility 5.023 (25 �C) g/100 gH2O, pI 5.68, dissociation constants: pK1 2.21,pK2 9.15.

D-Serine [312-84-5], 2-amino-3-hydroxy-propionic acid, 2-amino-3-hydroxypropanoic ac-id, D-Ser, C3H7NO3, Mr 105.09, application: D-cycloserine (cognition enhancer) [50].

L-Thiazolidine-4-carboxylic Acid [34292-47-7], L-Tia, C4H7NO2S,Mr 133.16, application:Kynostatin-272 (antiviral) [34].

D,L-Threonine [80-68-2], 2-amino-3-hy-droxybutyric acid, 2-amino-3-hydroxybutanoicacid, C4H9NO3, Mr 119.12, mp 234-5 �C (de-comp.), solubility 20.5 (25 �C) g/100 g H2O, pI6.16, dissociation constants: pK1 2.71, pK2 9.62.


L-Thyroxine [51-48-9], 2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propionic acid, C15H11NO4, Mr 269.18, occur-rence: thyroid gland.

D,L-Tryptophan [54-12-6], 2-amino-3-(30-indolyl)propionic acid, a-amino-1H-indole-3-propanoic acid, C11H12N2O2, Mr 204.23, mp285 �C (decomp.), solubility 0.25 (30 �C) g/100 g H2O, pI 5.88, dissociation constants: pK1

2.38, pK2 9.39.

D,L-Tyrosine [556-03-6], 2-amino-3-(4-hy-droxyphenyl)propionic acid, [3-(4-hydroxyphe-nyl)]alanine, 2-amino-3-(p-hydroxyphenyl)pro-pionic acid, a-amino-4-hydroxybenzenepropa-noic acid, C9H11NO3, Mr 181.19, mp 340 �C,318 �C (decomp.), solubility 0.351 (25 �C) g/100 g H2O, pI 5.63, dissociation constants: pK1

2.2, pK2 9.11, pK3 10.07 (–OH).

D,L-Valine [516-06-3], 2-amino-3-methyl-butyric acid, 2-aminoisovaleric acid, C5H11NO2,Mr 117.15, mp 298 �C (decomp., sealed tube),solubility 7.09 (25 �C) g/100 g H2O, pI 6.0,dissociation constants: pK1 2.29, pK2 9.72.

D-Valine [640-68-6], 2-amino-3-methylbu-tyric acid, D-Val, C5H10NO2, Mr 116.14, appli-

cation: Fluvalinate (insecticide) [51].

3. Industrial Production of AminoAcids

Amino acids can be classified as natural, includ-ing the proteinogenic (occurring in proteins)amino acids, or as nonnatural. Originally, only20 amino acids were believed to be geneticallycoded, but the recent discoveries of selenocys-teine and pyrrolysine have demonstrated thatadditional amino acids could be coded by sub-verting a stop codon. The following sections willfocus on production methods for the principalproteinogenic amino acids, which are of majorimportance in nutrition and as feed additives.

3.1. General Methods

Fourbasicprocesses (seeFig.2)are suitable for theproduction of amino acids: chemical synthesis(including asymmetric synthesis), extraction, fer-mentation, and enzymatic routes. The classicalchemical synthesis is applied to produce either theachiral aminoacidglycineor racemicaminoacids,for instanceD,L-methionine. If L-aminoacids are tobe manufactured by this route, the chemical syn-thesis has to be followed by a resolution step. In

Figure 2. Routes for production of amino acids


some cases L-amino acids are directly producedfromprochiral precursors bymeans of enzymes aschiral catalysts. For L-cysteine, transformation ofanother amino acid (cystine) is an important me-thod. Catalytic asymmetric syntheses have beendeveloped for many L-amino acids and theirD-antipodes, but few have been applied on an in-dustrial scale. The extraction process has the ad-vantage of offering access to nearly all the protei-nogenic L-amino acids by isolation from proteinhydrolysates. The starting materials are protein-richproducts, suchaskeratin, feathers,bloodmeal,or technicalgelatin(seeFig.3).Thisprocess isnowgenerally being replaced by other methods, be-cause of customer preference for materials not ofanimal origin. Some amino acids, such as L-tyro-sine, can be economically isolated from plantresidues, for example sugar beet molasses.

The use of overproducing microbial strains(! Biotechnology, 1. General; ! Enzymes, 8.Enzymes inGeneticEngineering) in fermentationprocesses with sucrose or glucose as carbon

source is currently themost economic productionmethod forbulkaminoacids suchasmonosodiumL-glutamate, L-lysine hydrochloride, and L-threo-nine. In the last ten years, this has become themethod of choice for production of other aminoacids, such as L-phenylalanine, L-tryptophan, andL-cysteine.Classical breedingandmutagenesis aswell as recombinant DNA techniques are gener-ally employed to modify the biosynthetic path-way and force the bacteria to overproduce thecorrespondingmetabolites. Industrially usedmu-tants are usually characterizedbygeneticmarkersintroduced by methods of deregulation, e.g.,auxotrophic mutants, in order to release key en-zymesfromstrict regulationbymetabolites (feed-back inhibition and repression). A different waytoobtainoverproducingstrainsconsistsofscreen-ing of regulatory mutants, in that important en-zymes are resistant to toxic compounds, i.e.,analogues or derivatives of amino acids. In suchcases high amino acid concentrations do notinhibit the enzymatic key in the pathway ofbiosynthesis and thus result in high productionrates of the desired amino acid. The fourth meth-od, enzymatic catalysis, useswhole cells or activecell components (enzymes) as suchor, if possible,as immobilized biocatalysts in continuously op-erated reactors. The competitiveness of enzymat-ic processes depends on the availability and priceof substrate, the activity and stability of theinvolved enzyme(s) and on the simplicity ofproduct recovery. For some general reviews onamino acid synthesis, emphasizing the chemicaland biocatalytic methods, see [52–56].

In Table 2 the amino acids and their preferredproduction methods are listed, classified by thesize of production volume. Table 3 specifies theamino acids produced by direct fermentation ofcarbohydrates.

3.2. Production of Specific AminoAcids

3.2.1. L-Alanine

L-Alanine is industrially produced from L-aspar-tic acid by means of immobilized Pseudomonasdacunhae cells in a pressurized bioreactor [69].In direct fermentation microorganisms usuallyaccumulate D,L-alanine because of alanine race-mase also present. With a D-cycloserine resistantFigure 3. Extraction of amino acids


mutant selected from Brevibacterium lactofer-mentum, it is possible to obtain 46 g/L D-alaninewith an enantiomeric excess (e.e.) of 95% [58].An alanine racemase-deficient mutant of Arthro-bacter oxydans was reported, that produces75 g/L L-alanine from glucose with a yield of

52% and 95% e.e. [57]. A small amount of L-alanine is still isolated fromprotein hydrolysates.

3.2.2. L-Arginine

L-Arginine is today produced mainly by fermen-tation. Suitable strains for fermentation are de-regulated mutants derived from Corynebacteri-um glutamicum and Bacillus subtilis accumulat-ing 25–35 g/L L-arginine from glucose [70].Very potent strains of Serratia marcescens de-rived from mutants obtained by transduction,having feedback-insensitive and derepressiveenzymes of arginine biosynthesis and 6-azaur-acil resistance, are able to produce 60–100 g/LL-arginine [59]. A small amount of L-arginine isisolated from protein hydrolysates.

3.2.3. L-Aspartic Acid and Asparagine

L-Aspartic Acid is industrially manufac-tured by an enzymatic process inwhich aspartase(L-aspartate ammonia lyase, EC 4.3.1.1) cata-lyzes the addition of ammonia to fumaric acid[71]. Advantages of the enzymatic productionmethod are higher product concentration andproductivity and the formation of fewer bypro-ducts. Thus L-aspartic acid can be easily separat-ed from the reaction mixture by crystallization.

A process involving continuous production ofL-aspartic acid by means of carrier-fixed aspar-tase isolated from Escherichia coli was firstcommercialized in Japan [72]. An economically

Table 2. Production of amino acids, methods, and production volume

Type Amino acid Preferred production method

I L-Alanine enzymatic catalysis,

fermentation

I L-Asparagine extraction, chemical synthesis

I L-Glutamine fermentation, extraction

I L-Histidine fermentation, extraction

I L-Hydroxyproline fermentation, extraction

I L-Isoleucine fermentation, extraction

I L-Leucine fermentation, extraction

I L-Methionine enzymatic resolution

I L-Proline fermentation, extraction

I L-Serine fermentation, extraction

I L-Tyrosine extraction

I L-Valine enzymatic catalysis,

fermentation

II L-Arginine fermentation, extraction

II L-Cysteine fermentation, enzymatic cataly-

sis (via reduction of L-cystine)

II L-Tryptophan fermentation

III Glycine chemical synthesis

III L-Aspartic acid enzymatic catalysis

III L-Phenylalanine fermentation

III L-Threonine fermentation

IV D,L-Methionine chemical synthesis and enzy-

matic resolution

IV L-Glutamic acid fermentation

IV L-Lysine fermentation

Type I 100–1000 t/a

Type II 1000–8000 t/a

Type III 8000–100 000 t/a

Type IV 100 000–800 000 t/a

Table 3. Amino acids produced by direct fermentation from carbohydrates

Amino acid Type of mutant Amino acid

yield, g/L

Reference

L-Alanine (95% e.e.) Athrobacter oxydans 75 HASHIMOTO and KATSUMATA (1994) [57]

D-Alanine (95% e.e.) Brevibacterium lactofermentum 46 YAHATA et al. (1993) [58]

L-Arginine Serratia marcescens AUr-1 100 CHIBATA et al. (1983) [59]

L-Glutamine Brevibacterium flavum AJ 3409 57 YOSHIHARA et al. (1992) [60]

L-Histidine Serratia marcescens 40 SUGIURA et al. (1987) [61]

L-Isoleucine Escherichia coli H-8461 30 KINO et al. (1993) [62]

L-Leucine Brevibacterium flavum AJ 3686 19.5 YOSHIHARA et al. (1992) [63]

L-Lysine Corynebacterium glutamicum >120 OH et al. (1990) [64]

L-Methionine Pseudomonas putida VKPM V-4167 3.5 University Odessa (1992) [65]

L-Phenylalanine Escherichia coli MWPWJ304/pMW16 51 IKEDA et al. (2003) [56]

L-Proline Brevibacterium flavum AP113 97.5 KOCARIAN et al. (1986) [66]

L-Serine Methylobacterium sp. MN43 65 IKEDA et al. (2003) [56]

L-Threonine Escherichia coli BKIIM B-3996 85 DEBABOV et al. (1990) [67]

L-Tryptophan Corynebacterium glutamicum KY9218/pKW9901 50 IKEDA et al. (1994) [68]

L-Valine Corynebacterium glutamicum VR 3 99 IKEDA et al. (2003) [56]


attractive process for L-aspartic acid uses restingor dried cells with a high aspartase content, e.g., a4.5% suspension of aspartase-containing Brevi-bacterium flavum cells could be recycled in sevenrepeated batches and concentrations up to 166g/L L-aspartate could be achieved [73]. In 1973,an immobilized cell system based on E. coli cellsentrapped in polyacrylamide gel lattice was in-troduced for large-scale production [74]. Thisexample represents the first industrial applicationof immobilized microbial cells in a fixed-bedreactor. Further improvements, immobilizationof the cells in k-carrageenan, provided remark-ably increased operational stability, resulting inbiocatalyst half-lives of almost two years [75].A column packed with the k-carrageenan-im-mobilized system allows a theoretical productiv-ity of 140 g L�1 h�1

L-aspartate.As L-aspartic acid gained importance as inter-

mediate for the manufacture of the dipeptidesweetener aspartame (methyl ester of L-aspar-tyl-L-phenylalanine) improved processes weredeveloped. For continuous production of L-as-partic acid Escherichia coli strains immobilizedwith polyurethane [76] or with polyethylenimineand glass fiber support [77] and k-carrageenan-immobilized Pseudomonas putida [78] havebeen reported and protected, respectively. Lesssuccessful was the search for strains that areoverproducing L-aspartate by fermentation ofsugar. A pyruvate kinase-deficient mutant ofBrevibacterium flavum accumulates up to22.6 g/L L-aspartic acid in glucose-containingmedium [79], not enough to be competitive withthe enzymatic processes. On the other hand,genetic recombination techniques have helpedto improve aspartase-containing strains. AnaspA gene bearing plasmid (pBR322:aspA-par)was able to elevate aspartase formation inEscherichia coli K 12 about 30-fold [80].

L-Asparagine can be isolated as a byproductfrom the production of potato starch. A simplesynthesis of L-asparagine starts from L-asparticacid which is esterified to the b-methyl esterfollowed by treatment with ammonia [81].

3.2.4. L-Cystine and L-Cysteine

L-Cysteine used to be produced almost exclu-sively by hydrolysis of hair or other keratins. The

amino acid isolated was L-cystine, which wasreduced electrolytically to L-cysteine. L-Cysteinehas also been prepared from b-chloro-D,L-alanineand sodium sulfide with cysteine desulfhydrase,an enzyme obtained from, e.g., Citrobacteriumfreundii [82].

Today, however, the main processes for cys-teine production are biological. A direct fermen-tation process has been developed for the manu-facture of L-cystine, using a modified E. colibacterium [83]. The technology has been extend-ed to prepare other modified L-cysteine analo-gues [84]. An enzymatic process for L-cysteinehas been successfully developed using microor-ganisms capable to hydrolyze 2-amino-D2-thia-zoline 4-carboxylic acid (ATC) which is readilyavailable frommethyla-chloroacrylate and thio-urea. A mutant of Pseudomonas thiazolinophi-lum converts D,L-ATC to L-cysteine in 95%molar yield at product concentrations higher than30 g/L [85].

3.2.5. L-Glutamic Acid

In 1957, a soil bacterium was discovered [86]which was able to excrete considerable amountsof L-glutamate. Fermentation processes usingstrains of this bacterium, later called Corynebac-terium glutamicum, have been successfully com-mercialized not only for L-glutamic acid but alsofor the production of other economically impor-tant amino acids [87, 88]. Numerous coryneformmicroorganisms have been isolated and found tobe able to overproduce L-glutamic acid and otheramino acids. Examples of these microorganismsare Brevibacterium flavum, Brevibacteriumlactofermentum, and Microbacterium ammonia-philum. Because of minor differences in thecharacter of those bacteria [89] which are allgram-positive, non-spore-forming, nonmotileand all require biotin for growth, the name ofgenus Corynebacterium was suggested for thesecoryneform bacteria [90].

For industrial production of L-glutamic acid,molasses (sucrose), starch hydrolysates (glu-cose) and ammonium sulfate are generally usedas carbon and nitrogen sources, respectively [91].Key factors in controlling the fermentationare the presence of biotin in optimal concentra-tion – to optimize cell growth and the excretion ofL-glutamate – and sufficient supply of oxygen to


reduce the accumulation of byproducts, such aslactic and succinic acid. In biotin-rich fermenta-tion media the addition of penicillin or cephalo-sporin C favors the overproduction of L-glutamicacid due to effects on the cell membrane. Thesupplementation of fatty acids also results in anincreased permeability of the cells thus enhancingglutamate excretion. In the past themechanism ofglutamate excretion was simply explained as a‘‘leakage’’ or ‘‘overflow’’ phenomenon [92]. Inthe beginning of the 1990s it was reported that aspecific carrier system exists [93, 94], which isresponsible for active glutamate transport inCorynebacterium glutamicum. Generally the in-tracellular accumulation of glutamate does notreach levels sufficient for feedback control inglutamate overproducers due to rapid excretionof glutamate. However, the regulatory mechan-isms of L-glutamic acid biosynthesis have beenstudied intensively to obtain mutants with in-creased productivity. Two enzymes have beenshown to play key roles in the biosynthesis ofL-glutamic acid [95].

1. Phosphoenolpyruvate carboxylase (PEPC)catalyzes carboxylation of phosphoenolpyr-uvate to yield oxaloacetate; it is inhibited by L-aspartic acid and repressed by both L-asparticand L-glutamic acids.

2. a-Ketoglutarate dehydrogenase (KDH) con-verts a-ketoglutarate to succinyl-CoA. In L-glutamate overproducing strains KDH limitsfurther oxidation of a-ketoglutarate to carbondioxide and succinate, thus favoring forma-tion of L-glutamic acid.

In L-glutamate overproducing strains the Km val-ue of KDH for a-ketoglutarate was nearly twomagnitudes lower than that of L-glutamic aciddehydrogenase (GDH) which catalyzes the laststep, the reductive amination of a-ketoglutarateto L-glutamate. Consequently, Vmax of GDH wasproven to be about 150 times higher than that ofKDH. The Corynebacterium glutamicum gdhgene has been isolated and characterized [96].A strain ofMicrobacterium ammoniaphilum cul-tured under biotin-deficient conditions produced58% of L-glutamic acid formed from glucose viaphosphoenolpyruvate, citrate, and of a-ketoglu-tarate and theother 42%via the tricarboxylic acid(TCA) or the glyoxylate cycle [97]. In large-scaleproduction the formation of trehalose very often

reduces the product yield. Trehalose consists oftwoa-1,1 bound glucosemolecules and is excret-ed by the bacteria as a material with protects thecell against high osmotic pressure (osmoprotec-tant). A process was recently developed andsuccessfully industrialized in which trehaloseformation is controlled and decreased by cultur-ing theoverproducingmutant inmediacontaininginvert sugar from molasses [98, 99].

Today wild type isolates as well as mutantsdeveloped by classical breeding or even strainsconstructed by modern techniques, using cellfusion or recombinant DNA methods [100], areavailable for industrial L-glutamate production.In addition a new type of fermentation processwas reported which uses a strain that overpro-duces L-glutamic acid and L-lysine simultaneous-ly. The cultivation of an auxotrophic regulatorymutant of Brevibacterium lactofermentum in amedium, supplemented with polyoxyethylene-sorbitan monopalmitate as surface-active agent,resulted in the accumulation of 162 g L-aminoacids per liter (105 g L-lysine � HCl þ 57 gL-glutamic acid). This corresponds to a produc-tion rate of 3.8 g (L-lysine � HCl þ L-glutamicacid) per liter per hour [101].

The success of an economic production basi-cally depends on the experience in fermentationtechnology, in up- and downstream processingand on the skill of employees in research, devel-opment, and production. A multi-step inocula-tion procedure followed by the fed-batch mode(! Biocatalysis, 1. General, Chap. 5.) in themain fermentation up to 500 m3 scale is stillthe preferred technology for the production ofL-glutamic acid.

Critical operations are the steam-forcedsterilization of the fermenter and batchwise orcontinuous sterilization of the culture medium toprevent contamination by foreignmicrobes. Dur-ing fermentation the temperature, pH, dissolvedoxygen, and the sugar consumption have to becontrolled as important process parameters.When the fermentation is completed after 40–60 h, theproductionstrainmayhaveaccumulatedmore than 150 g/L L-glutamic acid. After deacti-vation of the broth, the recovery of the product isstartedbybiomass separationusingcentrifugesorultrafiltration units, concentration of the centri-fugate or filtrate, followed by crystallization,filtration and drying. A scheme of productionsteps is given in Figure 4.


3.2.6. L-Glutamine

Microbial L-glutamine producers were selectedfrom wild type glutamate-producing coryneformbacteria. A sulfaguanidine resistant mutant ofBrevibacterium flavum accumulates 41 g/L L-glutamine in 48 h from 10% glucose [102]. Ayield of 44% was achieved by the mutant Bre-vibacterium flavum AJ3409 [60].

3.2.7. L-Histidine

Efficient L-histidine fermentation can be per-formed with strains of Corynebacterium gluta-micum and Serratia marcescens. A mutant ofCorynebacterium glutamicum having resistance

to 8-azaguanine, 1,2,4-triazole-3-alanine, 6-mer-captoguanine, 6-methylpurine, 5-methyltrypto-phan and 2-thiouracil, produces 15 g/L of L-histidine. Only after introduction of these resis-tance markers by mutagenesis is the mutantcapable of releasing this amount of L-histidineinto the nutrient solution. A strain of Serratiamarcescens having both characters, feedback-insensitive and derepressed histidine enzymescombined with transductional techniques and6-methylpurine resistance, accumulates 23 g/LL-histidine [103]. By amplification of the geneshisG, hisD, hisB, hisC in Serratiamarcescens thefinal concentration of L-histidine could be ele-vated from 28 g/L to 40 g/L [61].

L-Histidine can also be produced by isolationfrom blood-meal hydrolysates [104].

3.2.8. L-Hydroxyproline

L-Hydroxyproline (trans-4-hydroxy-L-proline,2S,4R-hydroxyproline) is today mainly obtainedby enzymatic hydroxylation of L-proline withgenetically modified E. coli [105, 106]. Theproline 4-hydroxylase specifically produces the4-trans isomer. This method has almostcompletely replaced the isolation from hydro-lysates of gelatin or other collagens. An unusualfeature of this amino acid is that it is not incor-porated into collagen during biosynthesis at theribosome, but is formed from L-proline by aposttranslational hydroxylation reaction.

3.2.9. L-Isoleucine

An advantageous fermentation method for pro-duction of L-isoleucine is the use of chemicallysynthesized substrates that only require a fewsteps to be converted to L-isoleucine. Amongthese the natural precursors 2-ketobutyrate[107] or D,L-2-hydroxybutyrate [108] have beenused for the production with Corynebacteriumglutamicum. A leucine requiring mutant ofCorynebacterium glutamicum, with increasedD-lactate utilization and consuming D,L-2-hydro-xybutyrate, accumulates 13.4 g/L L-isoleucine.However, exploitation of this process is hamperedby formation of byproducts [109]. Sugar based L-isoleucine processes have been developed withstrains of Corynebacterium glutamicum [110],

Figure 4. Flow diagram of fermentation and downstreamprocessing of L-glutamic acid


Serratia marcescens [111], and Escherichia coli[112–114]. The mutant Escherichia coli H-8285,being resistant to thiaisoleucine, arginine hydro-xamate, and D,L-ethionine accumulates 26 g/L L-isoleucine in 45 h in a fed-batch process [115].Introduction of resistance to 6-dimethylamino-purine in strain H-8285 resulted in a mutantEscherichia coli H-8461 that increased L-isoleu-cine accumulation to 30.2 g/L [62]. The biosyn-thesis of L-isoleucine has been investigated indetail on the level of involved genes [63], thusrecombinant strains are being constructed withhigh productivity and selectivity. An appropriatebalance of homoserine dehydrogenase and threo-nine dehydratase activities in the construct Cory-nebacterium glutamicum DR17/pECM3::ilvA(V323A) with feedback-resistant aspartate kinasecreate a specific productivity of 0.052 g L-isoleu-cine per gram dry biomass per hour [116]. Therecombinant strain Escherichia coli AJ13100produces L-isoleucine from glucose with highselectivity in 30% yield [117].

3.2.10. L-Leucine

L-Leucine can be manufactured by fermentation,precursor fermentation, or, less commonly today,by isolation from protein hydrolysates. Using afed-batch culture of Corynebacterium glutami-cum ATCC 13032 32 g/L 2-ketoisocaproate canbe converted to 24 g/L L-leucine by the transam-inase B reaction [118]. With the method of directfermentation L-leucine can be produced by eithera-aminobutyric acid-resistant mutants of Serra-tia marcescens or by 2-thiazole-alanine-resistantcoryneform strains [119].Brevibacterium flavumAJ3686 accumulates 19.5 g/L L-leucine (15%yield) [120].

3.2.11. L-Lysine

The most potent microorganisms to overproduceL-lysine are mutants derived from Corynebacte-rium glutamicum, a gram-positive bacterium firstintroduced as an L-glutamate producingmicrobe;thewild strains themselves are not able to excreteL-lysine. Mainly auxotrophic and regulatory mu-tants of this bacterium have been developed byclassical breedingmethods andmutagenesis. Thefollowing techniques have been applied to chan-

nel the metabolic pathway in the biosynthesis ofamino acids:

Screening for auxotrophic mutants, in order torelease key enzymes, for instance aspartatekinase, from strict regulation by metabolites(feedback inhibition).

Screening for regulatory mutants, in which as-partate kinase is resistant to toxic analogues ofL-lysine, such as S-(2-aminoethyl)-L-cysteine(AEC) or O-(2-aminoethyl)-L-serine. In suchstrains high lysine concentrations do not in-hibit the enzymatic key step, the formation of4-aspartylphosphate from L-aspartase cata-lyzed by aspartate kinase.

Screening for mutants having amino acid auxot-rophy combined with deregulation.

Screening for regulatory mutants having addi-tional enzyme defects (reduced pyruvate ki-nase) and low levels of other enzymes (citratesynthase).

As a modern technique, cell fusion with themethod of protoplast fusion has been successive-ly applied for breeding of industrial microorgan-isms [121, 122]. This technique allows the com-bination of positive characteristics of differentstrains such as high selectivity and high produc-tivity. In fermentation with media of inhibitoryosmotic stress (i.e., high substrate concentrationresults in high osmotic pressure on the cell thatinhibits the performance of the mutant) the sugarconsumption rate and L-lysine production rate ofsome mutants can be stimulated by the additionof glycine [123]. Another attractive approach isthe development of thermophilic strains. A mu-tant ofCorynebacterium thermoaminogeneswaspatented which is capable of growing at a tem-perature > 40 �C and accumulating L-lysine inthe culture medium [124].

Meanwhile, regulation of lysine excretion inoverproducing strains is known in detail [125,126]. Thus more attention should be paid toprocess development in the fields of molecularbiology, biochemistry, and physiology and tofinding new approaches for developing improvedoverproducing mutants [127]. The most specificand well-directed methods for strain develop-ment are offered by recombinant DNAtechniques.

In principle all genes encoding the relevantenzymes in L-lysine biosynthesis have been


isolated, characterized, and amplified in coryne-form bacteria to enhance L-lysine formation[128]. As an example, amplification of dapAgene that codes for dihydrodipicolinate synthaseinCorynebacterium glutamicum resulted in 35%higher overproduction of L-lysine compared tothe parent strain [129]. Another option for strainimprovement is the transformation of dapA genetogether with a lysC gene, coding for aspartatekinase with decreased feed back inhibition inCorynebacterium glutamicum [130].

In fed-batch culture and under appropriateconditions the favorable mutants for lysineproduction are able to reach final concentrationof about 120 g/L L-lysine, calculated as hydro-chloride [64]. Fermentation processes areperformed in big tanks up to 500 m3 size. Anoptimized feeding strategy practiced with acomputer aided process control system may en-able high conversion yield and productivity inlarge scale fed-batch cultivation [131]. Apartfrom the specific fermentation know how,inoculation, sterilization, and feeding strategythe recovery process and the quality ofthe product both can be decisive factors toguarantee competitiveness. The conventionalroute of lysine downstream processing is charac-terized by:

Removal of the bacterial cells from fermentationbroth by separation or ultrafiltration

Absorbing and then collecting lysine in an ionexchange step

Crystallizing or spray drying of lysine as L-lysinehydrochloride

An alternative process consists of biomassseparation, concentration of the fermentationsolution, and filtration of precipitated salts.The liquid product contains up to 50% L-lysinebase, that is stable enough to be marketed [132].In the 1990’s, a new concept for lysineproduction was introduced. Here the lysinecontaining fermentation broth is immediatelyevaporated, spray-dried, and granulated toyield a feed-grade product, which contains lysinesulfate. Its lysine content correspond to thatof a material which contains to at least 60%of L-lysine hydrochloride [133, 134]. Theprocess avoids any waste products usually pres-ent in the conventional L-lysine hydrochloridemanufacture.

3.2.12. D,L-Methionine and L-Methionine

L-Methionine and its antipode D-methionine areof equal nutritive value, thus the racemate candirectly be used as feed additive.

The most economic way for production of D,L-methionine is the chemical process based onacrolein, methyl mercaptan, hydrogen cyanide,and ammonium carbonate (see Fig. 5).b-Methylthiopropionaldehyde, formed by addi-tion of methyl mercaptan to acrolein, is theintermediate that reacts with hydrogen cyanideto give a-hydroxy-g-methylthiobutyronitrile.Treatment with ammonium carbonate leadsto 5-(b-methylthioethyl)hydantoin that issaponified by potassium carbonate giving D,L-methionine in up to 95% yield, calculated onacrolein [135].

The production method of choice for L-methi-onine is still the enzymatic resolution of racemicN-acetyl-methionine using acylase from Asper-gillus oryzae. The production is carried out in acontinuously operated fixed-bed or enzymemembrane reactor [136].

Alternatively, L-methionine may be producedby microbial conversion of the corresponding5-substituted hydantoin. With growing cells ofPseudomonas sp. strain NS671, D,L-5-(2-

Figure 5. Degussa process for production of L-methionine


methylthioethyl)hydantoin was converted to L-methionine; a final concentration of 34 g/L and amolar yield of 93% have been obtained [137].

Biosynthesis of L-methionine and its regula-tion in bacteria is well known. Although somepromising concepts, for example utilization ofsulfate, sulfite, or thiosulfate as sulfur sources formicrobes have been suggested [138], it was notpossible so far to develop strains that are able toexcrete remarkable amounts of L-methionine intothe culture medium.

3.2.13. L-Phenylalanine

Several chemical, biocatalytic, and fermentationmethods for producing L-phenylalanine havebeen developed, some of which are of industrialsignificance (see Fig. 6).

In previous large-scale production processesfor L-phenylalanine two enzymaticmethodswereapplied:

1. Resolution of N-acetyl-D,L-phenylalanine bycarrier-fixed microbial acylase: This processprovided pharmaceutical-grade L-phenylala-nine, but suffered from the disadvantage thatthe D-enantiomer had to be racemized andrecycled.

2. Stereoselective and enantioselective additionof ammonia to trans-cinnamic acid, catalyzedby L-phenylalanine ammonia lyase (PAL,EC 4.3.1.5): PAL-containing Rhodotorula

rubra was used in an industrial process[139] to supply L-phenylalanine for the firstproduction campaign of the sweetener aspar-tame. When continuously operated in an im-mobilized whole cell reactor, the bioconver-sion reached concentration up to 50 g/L L-phenylalanine at a conversion of about 83%[140]. Other processes started from phenyl-pyruvate with L-aspartic acid as amine donorusing immobilized cells of Escherichia coli[141] or from a-acetamidocinnamic acid andimmobilized cells of a Corynebacterium equistrain [142]. In both cases L-phenylalanineconcentrations up to 30 g/L and more (molaryields as high as at least 98%) were reached.

However, fermentation processes based on glu-cose-consuming L-phenylalanine overproducingmutants of E. coli and coryneform strains turnedout to be more economical. The biosyntheticpathway for aromatic amino acids in bacteria isstrictly regulated [143]. L-Phenylalanine isformed in ten enzymatic steps starting fromerythrose-4-phosphate and phosphoenolpyr-uvate. The biosynthesis is governed by the firstkey enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS) which is inhibitedby both L-phenylalanine and L-tyrosine (by actingon the enzyme itself) and repressed by L-tyrosine(by acting on the according gene of the DNA).The other important enzyme is prephenate dehy-dratase (PDT) also inhibited by L-phenylalanine,but stimulated by L-tyrosine. To overcome these

Figure 6. Asymmetric syntheses for production of L-phenylalanine


regulatory mechanisms either auxotrophs ofCorynebacterium glutamicum have beenconstructed or L-phenylalanine analogues, e.g.,4-aminophenylalanine and 4-fluorophenylala-nine, have been applied. The latter variant leadsto resistant mutants of Brevibacterium flavum orlactofermentum [144]. These auxotrophic andregulatory mutants are able to produce more than20 g/L of L-phenylalanine in a medium contain-ing 13% glucose. Similar results can be obtainedby tyrosine auxotrophic regulatory mutants ofE. coli [145].With recombinant DNA techniquesit was possible to improve overproducing strainsof coryneform bacteria as well as of E. coli.Amplification of a deregulated DAHPS gene wasachieved in a phenylalanine producer of Brevi-bacterium lactofermentum [146]. For optimalproduction of L-phenylalanine in fed-batch cul-tivation the critical specific glucose uptake ratehas to be controlled. The specific feed rate duringfermentation has to be adjusted below a criticallimit, since otherwise the E. coli producer willbe forced to excrete acetate [147]. A suitableprofile of the specific glucose feed rate preventsacetate formation and leads to improved L-phe-nylalanine production with a final concentrationup to 46 g/L and a corresponding yield of 18%.L-Phenylalanine is recovered from the fermenta-tion broth either by two-step crystallization or byan ion-exchange resin process. The preferred cellseparation technique is ultrafiltration; and thefiltrates may be treated with activated carbon forfurther purification. Instead of ion-exchange re-sins nonpolar, highly porous synthetic adsorbentsare recommended to remove impurities [148,149]. An alternative process in which a cellseparator is integrated in the fermentation part,thus allowing cell recycling, was suggested forL-phenylalanine production and may lead toprospective developments [150].

3.2.14. L-Proline

L-Proline is still produced to a small extent byisolation from protein hydrolysates, but todaydirect fermentation using analogue-resistant mu-tants of coryneform bacteria or Serratia marces-cens is an economic alternative productionmeth-od [151]. An isoleucine auxotrophic mutant ofBrevibacterium flavum having resistance to sul-faguanidine and D,L-3,4-dehydroproline (DP) is

able to accumulate 40 g/L L-proline. Brevibac-terium flavum AP113 is claimed to produce97.5 g/L L-proline; this mutant is characterizedby isoleucine auxotrophy, resistance to DP, andosmotic pressure and incapable to degradeL-proline [66]. A proline oxidase-less strain ofSerratia marcescens, having resistance to DP,thiazoline-4-carboxylate and azetidine-2-car-boxylate, overproduces 58.5 g/L L-proline intothe culturemedium [152]. By amplification of thegenes proA and proB in this type of regulatorymutant, a construct was obtained which yields75 g/L L-proline [153].

3.2.15. L-Serine

L-Serine is obtained by microbial/ enzymaticconversion of glycine using immobilized restingcells or crude cell extracts. Hyphomicrobiumstrains possess the serine pathway and are ableto produce L-serine from methanol and glycine.Methanol is oxidized by methanol dehydroge-nase to formaldehyde which in turn is convertedin an aldol-like reaction with glycine to L-serine.The reaction is catalyzed by serine hydroxy-methyltransferase (SHMT) [154]. Hyphomicro-bium sp. NCIB10099 was found to produce45 g/L L-serine from 100 g/L glycine and88 g/L methanol in three days [155]. In anenzyme bioreactor with a feedback controlsystem a crude extract fromKlebsiella aerogenescontaining SHMT has been used to synthesizeL-serine from glycine and formaldehyde inthe presence of tetrahydrofolic acid andpyridoxal phosphate. In this bioreactor a serineconcentration of 450 g/L with an 88% molarconversion of glycine at a volumetric productiv-ity of 8.9 g L�1 h�1 could be achieved underoptimized conditions [156]. With whole cells ofEscherichia coliMT-10350 L-serine is producedby treatment of an oxygenated aqueous glycinesolution (485 g/L) with aqueous formaldehydefor 35 h at 50 �C in a molar yield of 89%based on glycine [157]. Extraction of proteinhydrolysates is used to a smaller extent.

3.2.16. L-Threonine

Up to the end of the 1980s, L-threonine wasmainly used for medical purposes, in amino acid


infusion solutions and nutrients. It was manufac-tured by extraction of protein hydrolysates orby fermentation using mutants of coryneformbacteria in amounts of several hundred tons peryear worldwide. The production strains weredeveloped by classical breeding. They wereauxotrophic and resistant to threonine analoguessuch as a-amino-b-hydroxyvalerate (AHV), andreached product concentrations up to 20 g/L.These strains possessed deregulated L-threoninepathways with feedback inhibition-insensitiveaspartate kinase and homoserine dehydrogenase[158, 159]. In the 1990s strain developments,using both conventional methods and recombi-nantDNA techniques, have been very successful.Potent classically selected mutants suggested forindustrial production are the species Brevibac-terium flavum, Providentia rettgeri, Serratiamarcescens, and Escherichia coli. However, inthe competition between the favorable candi-dates, strains of Escherichia coli proved to besuperior to other bacteria. Although the pathwayof L-threonine biosynthesis in Escherichia coli ismuch more regulated than that in Corynebacte-rium glutamicum, new Escherichia coli strainswith excellent yields and productivity in threo-nine formation could be constructed by geneticengineering. L-Threonine is successfully mar-keted as feed additive with a worldwidedemand of more than 10 000 t/a. Productionstrains are based on Escherichia coli K-12constructs harboring plasmids containing thethr operon that consists of the genes thrA,thrB, and thrC [160]. Further improvementsresulted in strains capable to accumulate morethan 80 g/L in about 30 hwith a conversion yieldof more than 40% [161]. The strain stabilitycould be further improved, for example byintegrating the threonine operon into thechromosome [162]. The recovery of feed-gradeL-threonine is rather simple.After fermentation iscompleted, cell mass is removed by centrifuga-tion or ultrafiltration, the filtrate is concentrated,depigmentated and L-threonine isolated by crys-tallization [163].

3.2.17. L-Tryptophan

L-Tryptophan is one of the limiting essentialamino acids required in the diet of pig andpoultry. Amature and growingmarket for L-tryp-

tophan as feed additive is in development,basedon improvedmicrobiological processes, and pro-duction has increased to several thousand tonsper year. Themost attractive production process-es for tryptophan are based on microorganismsused as enzyme sources or as overproducers:

Enzymatic production from various precursorsFermentative production from precursorsDirect fermentative production from carbohy-

drates by auxotrophic and analogue resistantregulatory mutants

L-tryptophan is synthesized from indole, pyru-vate, and ammonia by the enzyme tryptophanase[164] or from indole and L-serine/D,L-serine bytryptophan synthase [165, 166]. Although pro-duction in enzyme bioreactors is quite efficientand concentrations of L-tryptophan up to 200 g/Lcould be achieved by condensation of indole andL-serine [167], these process variants were noteconomic due to the high costs of the startingmaterials. The microbial conversion of biosyn-thetic intermediates such as indole or anthranilicacid to L-tryptophan has also been consideredas alternative for production. Whereas indoleconsuming mutants of Corynebacterium gluta-micum produced about 10 g/L L-tryptophan[168], strains of Bacillus subtilis and Bacillusamyloliquefaciens reached final concentrations> 40 g/L L-tryptophan with anthranilic acid ascarbon source [168, 169]. The process with an-thranilic acid as precursor has been commercial-ized in Japan. However, the manufacturer usinggenetically modified strains derived from Bacil-lus amyloliquefaciens IAM 1521 was forced tostop L-tryptophan production. L-Tryptophan pro-duced by this process was stigmatized because ofside products found in the product causing a newsevere disease termed eosinophilia-myalgia syn-drome (EMS) [170]. One of the problematicimpurities, ‘‘Peak E’’, was identified as 1,10-ethylidene-bis-(L-tryptophan), a product formedby condensation of one molecule acetaldehydewith two molecules of tryptophan [171].

In other processes, i.e., direct fermentationusing overproducing mutants and carbohydratesas carbon sources, formation of such impuritiesdoes not occur. In the 1990s striking progress hasbeen made in the development of auxotrophicand deregulated mutants of Brevibacterium fla-vum,Corynebacterium glutamicum, andBacillus


subtilis. The biosynthesis of L-tryptophan and itsregulation have been reviewed in detail for thedifferent species [172–174]. The precise knowl-edge about the structure of the trp operon inEscherichia coli comprising the trp promoterand the genes trpE, trpD, trpC, trpB, and trpAwhich are coding for the enzymes anthranilatesynthase (AS), phosphoribosyl anthranilatetransferase (PRT), indole glycerol phosphatesynthase (IGP) and tryptophan synthase (TS),respectively, was the benefit for further strainimprovements.

Thus recombinant DNA techniques have beenused to increase the capability of overproductionespecially in strains of Corynebacteriumglutamicum and Escherichia coli. One conceptwas realized successfully by amplification oftrp operon genes together with serAwhich codesfor phosphoglycerate dehydrogenase. This keyenzyme in L-serine biosynthesis shouldprovide enough L-serine in the last step of L-tryptophan formation. Production strains are ableto accumulate 30–50 g/L L-tryptophan withyields higher than 20% based on carbohydrate.Isolation of L-tryptophan is still a major issue,because the amino acid is sensitive to oxygen andheat [167]. However, at a typical productionconcentration of 50 g/L, more than half ofthe L-tryptophan product crystallizes from themedium [68, 175].

3.2.18. L-Tyrosine

L-Tyrosine was, until recently, producedexclusively from protein hydrolysates. Its lowsolubility in water enables a quite simpleisolation of the amino acid. Enzymatic catalysisusing a b-tyrosinase (tyrosine-phenol lyase)from Erwinia herbicola gives tyrosine by athree-component synthesis from phenol,pyruvate, and ammonia. This process has beencommercialized for the enzymatic synthesisof the drug L-DOPA, where phenol is replacedby catechol [54]. L-Tyrosine is formed innature as part of the shikimic acid pathway,as are L-phenylalanine and L-tryptophan.Recombinant DNA technology has been usedto develop strains of Corynebacteriumglutamicum that are specific for L-tyrosine,and production yields of 20–25 g/L have beenobtained [175].

3.2.19. L-Valine

L-Valine is produced industrially in pharmaceu-tical quality by enzymatic resolution ofN-acetyl-D,L-valine. Using direct fermentation thebranched-chained amino L-valine can be pro-duced by either a-aminobutyric acid-resistantmutants of Serratia marcescens or by 2-thiazo-lealanine-resistant coryneform strains [176].Brevibacterium lactofermentum AJ12341 pro-duces 39 g/L L-valine (28% yield) [177].

4. Biochemical and PhysiologicalSignificance

The biosynthesis of amino acids begins withatmospheric nitrogen, which is reduced to am-monia by bacteria and plants. Ammonia is usedby plants, by bacteria, and, to a limited extent, byruminants as a raw material for amino acids.Amino acids, in turn, serve as starting materialsfor the synthesis of proteins and a variety of othernitrogen-containing compounds, such as the pu-rine and pyrimidine bases in nucleic acids. Bac-terial degradation leads, once again, to ammoniaand nitrogen [178].

a-Ketoglutaric acid plays the central role inthe assimilation of ammonia. Its transaminationproduct, glutamic acid, in turn, can provide itsamino group for the synthesis of other aminoacids, e.g., alanine (Fig. 7).

Humans, animals, and some bacteria are in-capable of synthesizing all the necessary aminoacids in their own intermediary metabolism; i.e.,they are heterotrophs and are therefore dependenton the biosynthetic capability of plants. Proteinsthat are consumed as foods by humans and

Figure 7. The transamination of amino acids


animals are hydrolyzed to amino acids by thedigestive enzymes. The amino acids are resorbedin the upper part of the intestine and enter theliver by way of the portal vein. The liver is thecentral organ for metabolism and homoeostasisof the plasma amino-acid level. The body’svarious requirements are met from the pool offree amino acids (Fig. 8), ca. 50 g in adulthumans.

The lion’s share of the amino acids (� 300 g/dfor adults) is required for synthesis of proteins[179]: structural proteins, enzymes, transportproteins, and immune proteins. Additionally,amino acids are required for the synthesis ofoligopeptides and polypeptides that fulfill regu-latory functions in the body, i.e., hormones.Some amino acids or their metabolites are di-rectly active as hormones or facilitate the trans-mission of nerve impulses (neurotransmitters),e.g., serotonin. Furthermore, there are aminoacids that serve special functions, such as methi-onine, which is a methyl group donor. Finally, aseries of amino acids serves as precursors for thebiosynthesis of other structures. For example,glycine is used in the construction of the porphy-rin skeleton. Amino acids are metabolized toproduce energy in the case of a protein-deficientor a protein-excess diet.

The free amino acids in the amino acid poolundergo numerous transformations (Fig. 9), in-volving transamination and oxidative deamina-tion, by which other amino acids can be synthe-sized. The a-keto acids are intermediates and inaddition allow amino acids entrance into thecarbohydrate (through pyruvate) and fatty acid(through acetylcoenzyme A) metabolisms. Adistinction is therefore drawn between gluco-genic and ketogenic amino acids.

D-Amino acids occur in the cell pool of plantsand gram-positive bacteria and as buildingblocks in peptide antibiotics and bacterial cellwalls [10, 14]. They do not occur in human oranimal metabolism; proteins are made exclu-sively of L-amino acids. The traces of D-aminoacids detected in metabolically inert protein(teeth, eye lenses) are believed to originate fromracemization. Orally ingested D-amino acids areresorbed from the intestinal lumen more slowlythan the L-form. The D-enantiomers cannot beutilized or can be utilized only to a slight extent asessential amino acids [180]. The one importantexception is D-methionine. Animals and adulthumans convert D-methionine into L-methionineby transamination. Thea-keto acid ofmethionineis an intermediate. Otherwise, D-amino acids aredegraded with the help of D-amino acid oxidases[181] to be used as an energy source [182].

The major end products of amino acid metab-olism are urea, uric acid, ammonium salts, cre-atinine, and allantoin. The loss of nitrogen via

Figure 8. The amino acid pool and functions of amino acidsin the intermediary metabolism

Figure 9. Intermediary metabolism of amino acids(simplified)


these metabolites stabilizes at about 22 g proteinper day after a few days on a protein-free diet.

Inborn disorders in amino acid metabolism[183] can lead to marked alterations in the excre-tion profile. These disorders usually take the formof an enzyme or transport deficiency [178, 184].Themost common example is phenylketonuria, adisruption of the normal metabolic pathway fromphenylalanine to tyrosine caused by severe limi-tation in the activity of the phenylalanine hy-droxylase [185].

Humans and animals are not capable of pro-ducing all the required L-amino acids in theirintermediary metabolism. Therefore, they aredependent on an external source of these essen-tial amino acids (Table 4). In situations of in-creased requirements (rapid growth, stress, trau-ma), histidine and arginine also become essentialfor humans. Cysteine and tyrosine may be essen-tial for infants during their first few weeks,because their intermediary metabolism does notyet function well enough to produce these frommethionine and phenylalanine in sufficientquantities.

5. Uses

The uses of amino acids have been treated inreview articles [19, 186–189].

5.1. Human Nutrition

In addition to their nutritive value, amino acidsare important flavor precursors and taste enhan-cers. In foods for humans, the flavor uses of

amino acids represent the dominant factor intotal market value. In animal nutrition, aminoacids are used almost exclusively for their nutri-tive value.

Addition of small amounts of amino acids toimprove the nutritive value of proteins is knownas supplementation. Both supplementation andthe combination of proteins with complementaryamino acids are used to increase the biologicvalue of proteins. Usually the supply of at leastone of the essential amino acids lies below therequirement. This, the limiting amino acid, de-termines what percentage of the protein (or, moreprecisely, its amino acids) can be used tomeet thebody’s amino acid requirements. In most cases,methionine is the first limiting amino acid. Some-times it is lysine; now and then it is both together.

The contents of essential amino acids found inseveral animal and vegetable foodstuffs are com-piled in Table 5. Considerable variations may bepresent in the amino acid contents of a givenfoodstuff.

The published requirements for the individualessential amino acids differ. The values (Ta-ble 6) usually contain safety factors and there-fore are higher than the minimum requirement.Requirement values were first determined byROSE [192]; those published by HEGSTED [193]are considered the most reliable at present. Theamino acid requirement pattern suggested by theFAO/WHO [194] is considered optimal for thegreatest part of the population.

The ‘‘average safe level of daily protein intakefor men and women,’’ based on these amino acidrequirement figures, is given as 0.55 g/kg bodyweight. The acute daily protein requirement, how-ever, varies between 0.5 and 2.5 g/kgwith age and

Table 4. Essential (þ) and semiessential (�) amino acids

Baby Adult Rat Chicken Hen Cat Salmon

L-Arginine � � � þ � þ þL-Cysteine þ (?)

Glycine þL-Histidine þ � þ þ � � þL-Isoleucine þ þ þ þ þ þ þL-Leucine þ þ þ þ þ þ þL-Lysine þ þ þ þ þ þ þL-Methionine þ þ þ þ þ þ þL-Phenylalanine þ þ þ þ þ þ þL-Threonine þ þ þ þ þ þ þL-Tryptophan þ þ þ þ þ þ þL-Tyrosine þ (?)

L-Valine þ þ þ þ þ þ þ


constitution [195]. The Deutsche Gesellschaft f€urErn€ahrung (DGE) recommends a daily consump-tion of 0.9 g/kg body weight [196, 197]. TheCommittee on Dietary Allowances, Food andNutrition Board of the National Academy ofSciences (NAS),USA,cites0.8 g/kgasadesirablelevel of daily protein consumption [198, 199].

5.1.1. Supplementation

In general, animal protein contains the essentialamino acids in larger quantities and in a morefavorable ratio than vegetable protein, which isoften deficient in essential amino acids. Lysine isthe first limiting amino acid in wheat, rye, barley,

Table 5. Average amino acid content of some foodstuffs (mg/100 g) a [190]

Food Ile Leu Lys Cyss Met Phe Tyr Thr Trp Val Arg His Protein, Moisture

% content, %

Maize, grain 350 1190 254 147 182 464 363 342 67 M 461 398 258 9.5 12.0

Rice, husked 300 648 299 84 183 406 275 307 98 M 433 650 197 7.5 13.0

Wheat, whole grain 426 871 374 332 196 589 391 382 142 M 577 602 299 12.2 12.0

Wheat, flour, 70–80%

extr. rate

435 840 248 304 174 581 277 321 128 M 493 422 248 10.9 12.0

Potato (Solanum

tuberosum)

76 121 96 12 26 80 55 75 33 M 93 100 30 2.0 78.0

Bean (Phaseolus

vulgaris)

927 1685 1593 188 234 1154 559 878 223 M 1016 1257 627 22.1 11.0

Soybean, milk 171 278 195 57 50 175 133 128 48 M 165 253 84 3.2 92.0

Soy protein, isolate [191] 4147 7119 5777 1008 1092 4644 3458 3211 1080 4210 6767 2378 75.7 4.7

Lettuce, leaves (Lactuca

sativa)

50 83 50 24 – 67 35 54 10 M 71 59 21 1.3 94.8

Tomato (Solanum

lycopersicum)

20 30 32 7 7 20 14 25 9 M 24 24 17 1.1 93.8

Apple (Malus silvestris) 13 23 22 5 3 10 6 14 3 15 10 7 0.4 84.0

Orange (Citrus sinensis) 23 22 43 10 12 30 17 12 6 31 52 12 0.8 87.4

Beef, veal, edible flesh 852 1435 1573 226 478 778 637 812 198 M 886 1118 603 17.7 61.0

Fish, fresh, all types 900 1445 1713 220 539 737 689 861 211 M 1150 1066 665 18.8 74.1

Milk, cows, untreated 162 328 268 28 86 185 163 153 48 M 199 113 92 3.5 87.3

Milk, human 48 104 81 16 19 41 39 53 20 M 54 46 30 1.2 87.6

Cheese, all types 956 1864 1559 76 530 950 973 725 217 M 1393 651 556 18.0 51.0

Egg, whole 778 1091 863 301 416 709 515 634 184 M 847 754 301 12.4 74.0

aChemical determination.MMicrobiological determination.

Table 6. Essential amino acid requirements of humans

Amino acid Suggested patterns of requirements

[194], g/100 g protein

Adult requirement, mg kg�1 d�1

Infant School-age Adult FAO/WHO NAS/NCR ROSEa HEGSTED

b

child 1973 1980 (144)

His 1.4 – – – – – –

Ile 3.5 3.7 1.8 10 12 10 10

Leu 8.0 5.6 2.5 14 16 11 13

Lys 5.2 7.5 2.2 12 12 9 10

Met þ Cysc 2.9 3.4 2.4 13 10 14 13

Phe þ Tyrd 6.3 3.4 2.5 14 16 14 13

Thr 4.4 4.4 1.3 7 8 6 7

Trp 0.85 0.46 0.65 3.5 3 3 3

Val 4.7 4.1 1.8 10 14 14 11

Total 37.3 32.6 15.2 83.5 91 81 80

aFor men.bFor women.cCys can partly cover the total S-amino acid requirement.dTyr can partly cover the total aromatic amino acid requirement.


oats, maize, and millet, whereas methionine isthe first limiting amino acid in meat, milk, soy-beans, and other beans. The second limitingamino acids are usually threonine (wheat, rice)and tryptophan (maize, rice, casein). The limit-ing amino acids for several foodstuffs are listedin Table 7.

Improving the biologic value of vegetableprotein in human nutrition is practiced for eco-nomic and dietary reasons. Combining differentprotein types is not always practical. Often acomplementary protein is unavailable, too ex-pensive, or not of acceptable taste. In these casessupplementation with amino acids is the simplestmethod of increasing the biologic value of pro-teins. There is amonumental volume of literatureon the subject of amino acid supplementation[200–207].

The biologic value is an important criterionfor the evaluation of proteins or amino acidmixtures. It can be determined experimentally[208, 209]. In principle, all methods measure theability of the nutritional protein to replace bodyprotein. Table 8 lists the biologic values of nutri-tional proteins as determined by the minimumrequirement [195]. Whole egg protein is thereference in this scale.

Another scale of evaluation is the proteinefficiency ratio (PER). This is the daily weightgain of young animals, usually rats, under stan-dard feeding conditions (see Table 8). In animproved method, the animals are fed dietsof various protein levels, and the proteinefficiency is then determined using regressionalanalysis.

The net protein retention (NPR) method andthe net protein utilization (NPU) method aremore accurate than the PERmethod because theyconsider the protein (NPR) or total nitrogen(NPU) requirement for maintenance. The‘‘chemical score,’’ in which the availability ofthe amino acids is not considered, is suitablefor a gross estimation of the biologic proteinquality. In this method the amino acid contentis determined analytically and compared withthe amino acid pattern of a reference protein,e.g., the FAO/WHO provisional scoring pattern(Table 9). This method provides an immediatepicture of the size of amino acid gaps and thesequence of limiting amino acids.

Score ¼Content of amino acid in test protein

Content of amino acid in reference protein� 100

Table 7. Limiting amino acids in foodstuffs

Proteins First limiting Second limiting

amino acid amino acid(s)

Peanut Thr Lys and Met

Fish Met Lys

Casein Met Trp

Torula yeast Met

Sesame Lys

Skim milk Met

Beans Met

Sunflower seed Lys Thr

Soy protein Met Lys

Wheat Lys Thr

Rice Lys Thr and Trp

Rye Lys Thr and Trp

Gelatin Trp

Maize Lys Trp and Thr

Table 8. Protein quality of food and minimum requirements (human)

Food Biologic Minimum Protein

value requirement*, efficiency

g kg�1 d�1 ratio (rat)

[195] [195] [190]

Whole egg 100 35 3.92

Beef 92 39 2.30

Cow’s milk 88 40 3.09

Potato 86 [210] 41 [210]** 3.0 [211]

Fish 3.55

Casein 2.50

Soybean 84 42 2.32

Rice 81 44 2.18

Rye flour 76 46

Maize 72 49 1.18

Beans 72 49 1.48

Wheat flour 56 63 0.6

*Of the protein part.**Calculated.

Table 9. FAO/WHO provisional scoring pattern 1973 [194]

Amino acid g/100 g protein

L-Isoleucine 4.0

L-Leucine 7.0

L-Lysine 5.5

L-Methionine þ L-cystine* 3.5

L-Phenylalanine þ L-tyrosine** 6.0

L-Threonine 4.0

L-Tryptophan 1.0

L-Valine 5.0

Total 36.0

*Cys can partly cover the total S-amino acid requirement.**Tyr can partly cover the total aromatic amino acid requirement.


As can be seen in Table 10, addition of ca.0.1–0.5% of the limiting amino acid to suchbasic foodstuffs as wheat, rice, maize, and soy-beans raises the protein efficiency in rat growthtests impressively.

Clinical studies [212–214] and field trialshave solidified the evidence of the benefits tohuman nutrition of amino acid supplementation[215–218]. However, the general supplementa-tion of basic foodstuffs, such as bread and rice, isnot yet practiced extensively. In dietary nutrition,however, supplementation already plays an im-portant role (Table 11).

Some infants exhibit lactose or cow’s milkprotein incompatibility. The formulas marketedfor this condition often are based on isolatedsoybean protein and are supplemented with L-methionine to increase the biologic value. Theadvantageous effects of L-methionine supple-mentation on the physical development of infantshas been demonstrated in a series of clinical

studies [219, 220]. Additionally, the food forpregnant and nursing women, seniors, over-weight persons, and athletes can also be supple-mented. Extruded soy protein, which is used inlarge quantities as a meat extender and vegetari-an meat substitute, can be supplemented withN-acetyl-L-methionine [221].

5.1.2. Flavorings, Taste Enhancers,and Sweeteners

Free amino acids occur in almost all protein-based foods. In some foods their concentration isseveral percent. Foodstuffs having a relativelyhigh concentration of free amino acids includefruit juices [222], cheese [223, 224], beer [225],and seafood [226]. Approximately 85% of thefree amino acids in orange juice is proline, argi-nine, asparagine, g-aminobutyric acid, asparticacid, serine, and alanine [227].

Amino acids are relatively tasteless. Nonethe-less, they contribute to the flavor of foods. Theyhave characteristic synergistic flavor-enhancingand flavor-modifying properties, and they areprecursors of natural aromas [228–230]. Aminoacids and protein hydrolysates are therefore usefuladditives in the food industry. The sodium salt ofL-glutamic acid (MSG) exhibits a particularlypronounced flavor-enhancing effect, leading tothe introduction of a fifth human taste conceptof umami [231], and has been recognized as aflavoring factor for seaweed, sake, miso, andsoy sauce since 1908. The substance is used inconcentrations of 0.1–0.4% as an additive forspices, soups, sauces, meat, and fish, usually incombination with nucleotides [230]. In 2000, theexistence of a specific receptor for umami wasconfirmed [232].

L-Cysteine especially enhances the aroma ofonion [233] and is therefore used to rearomatizedried onions. Glycine, which has a refreshing,

Table 10. Increase of the protein efficiency ratio (PER) by supple-

mentation with amino acids [190, 203]

Food

(protein

content)

L-Lys�HCl,%

L-Thr,

%

D,L-Trp,

%

D,L-Met,

%

PER*

%

Wheat flour

(10%)

0.65

0.2 1.56

0.4 1.63

0.4 0.15 2.67

Rice (7.8%) 1.50

0.2 0.1 2.61

Maize (8.75%) 1.41

0.4 0.07 2.33

Soybean milk

(10%)

2.12

0.3 3.01

Extruded soy

protein (10%)

1.99

0.23 2.62

*Reference protein: casein, PER¼2.50.

Table 11. Amino acids in dietetic products

Protein/protein hydrolysate Supplemented amino acid Use Indication

Cow’s milk, casein, whey protein L-Cyss and/or L-Lys � HCL infant nutrition adapted nutrition

Soy protein L-Met infant nutrition lactose incompatibility and milk

protein allergy

Casein/yeast L-Lys � HCl meal supplement protein malnutrition, in place of

conventional nutrition


sweetish flavor, occurs abundantly in musselsand prawns. It is considered to be an importantflavor component of these products. When usedas an additive for vinegar, pickles, and mayon-naise, it attenuates the sour taste and lends a noteof sweetness to their aroma. D,L-Alanine is usedfor the same purpose in the Far East [234].Glycine is used to mask the aftertaste of thesweetener saccharin [235, 236].

The L- and D-amino acids usually exhibitpronounced flavor differences. Many L-enantio-mers taste weakly bitter, whereas their opticalantipodes, the D-amino acids, taste sweet[237–239] (Table 12). For the most part, dipep-tides and oligopeptides have bitter flavors. Oneof the few exceptions is the methyl ester of thedipeptide L-aspartyl-L-phenylalanine (Aspar-tame) [240, 241], which is 150–200 times sweet-er than sucrose ( ! Sweeteners).

The free amino acids are used widely infoodstuff technology as precursors for aromasand brown food colors [242]. The flavors areformed during foodstuff production, e.g., duringthe ripening of cheese [243, 244], the fermenta-tion of alcoholic beverages [245, 246], or theleavening of dough [247, 248], or foodstuffcooking, e.g., frying, roasting, boiling, by theMaillard reaction between amino acids and re-ducing sugars (nonenzymatic browning) [228,

249, 250]. The Strecker degradation of aminoacids plays a central role in this process. A broadspectrum of aroma-intensive, volatile com-pounds forms [251, 252]. The most importantclasses are aliphatic carbonyl compounds andheterocycles, such as furans, pyrones, pyrroles,pyrrolidines, pyridines, imidazoles, pyrazines,quinoxalines, thiophenes, thiolanes, trithianes,thiazoles, and oxazoles.

It is often possible to assign certain aromas tospecific amino acids [252]. For example, thesulfur-containing amino acid cysteine is primar-ily responsible for the formation of meat flavor.Proline seems to be important for the aroma ofbread crust. Phenylalanine, as well as thebranched-chain amino acids leucine and valine,is important for the characteristic flavor of choc-olate. Valine and leucine are also involved in thearoma of roasted nuts. Methionine plays a keyrole in the aroma of French fries. The flavors ofsuch products as precooked foods, snack articles,and spices may be improved by addition of theproper Maillard aromas. One variation is addingthe precursors of theMaillard aromas, i.e., aminoacid plus sugar, to the foodstuff and allowing thefragrance to form in situ. Some aroma profilesthat can be prepared from amino acids are com-piled in Table 13.

5.1.3. Other Uses in Foodstuff Technology

Amino acids are used in the foodstuff industry forpurposes other than supplementation and flavor-ing. L-Cysteine, for example, is used by the bakedgoods and pasta industry as a flour additive [263,264]. As a reducing agent, it relaxes wheat gluten

Table 12. Tastes of L- and D-amino acids* [239]


Alanine sweet (12–18) sweet (12–18)

Arginine bitter neutral

Asparagine neutral sweet (3–6)

Aspartic acid acidic/neutral acidic/neutral

Cysteine sulfurous sulfurous

Glutamine neutral sweet (8–12)

Glutamic acid acidic/‘‘glutamate-like’’ acidic/neutral

Glycine sweet (25–35) sweet (25–35)

Histidine bitter sweet (2–4)

Isoleucine bitter sweet (8–12)

Leucine bitter sweet (2–5)

Lysine sweet/bitter sweet

Methionine sulfurous sweet/sulfurous

(4–7)

Phenylalanine bitter sweet (1–3)

Proline sweet/bitter (25–40) neutral

Serine sweet (25–35) sweet (30–40)

Threonine sweet (35–45) sweet (40–50)

Tryptophan bitter sweet (0.2–0.4)

Tyrosine bitter sweet (1–3)

Valine bitter sweet (10–14)

*Threshold values for sweet taste in parenthesis, mmol/mL; the

threshold value for sucrose is 10–12 mmol/mL.

Table 13. Amino acids for Maillard flavors*

Meat, poultry [253] Cys, Cyss, Gly, Glu, Ala,Met, His, Ser,

Asp, Pro

Bread, cracker, biscuit

[247, 254]

Pro, Lys, Arg, Val, His, Leu, Glu, Phe,

Asp, Gly, Gln

Chocolate, cocoa [255] Leu, Phe, Val, Glu, Ala

Honey [256–258] Phe

Cream, butter [259] Pro, Lys, Ala, Gly, His

Nut, peanut [260] Leu, Val, Ile, Pro, Glu, Gln, His, Phe,

Asp, Asn

Potato [261] Met

Tobacco [257, 262] Asn, Arg, GABA, Gln, Ala, Gly, Orn,

Glu, Asp, Leu, Val, Thr, Pro, Tyr, Phe

*Key amino acids underlined.


proteins (by cleavage of the disulfide linkages),homogenizes the dough, accelerates dough de-velopment, and improves the structure of thebaked product, while allowing shorter kneadingtimes.

Because they are capable of forming com-plexes with metals, amino acids act as antiox-idants for fats and fat-containing foodstuffs[265]. This effect is strengthened by primaryantioxidants, such asa-tocopherol. Melanoi-dines, which are formed during the Maillardreaction, are stronger antioxidants than the aminoacids themselves [266]. Maillard products arealso reported to be preservatives [267]. Glycineapparently exhibits a special preservativeeffect [268].

There has been a recent upsurge in the sale ofsports drinks and ‘‘energy bars’’, foodstuffs con-taining supplements with selected amino acid.These drinks were originally designed for per-formance athletes, but are now in widespreaduse, and the market is growing rapidly [269].Amino acids used in functional beverages andfoods include creatine, which is intended to helpbuild muscle; the branched-chain amino acids L-isoleucine, L-leucine, and L-valine, which areimportant components of cell membraneproteins and assist synthesis of body proteinand reduce central fatigue; and L-arginine,which is a regulatory agent for the circulation(production of NO). These amino acids havea higher rate of uptake in the body than protein,and improve muscle strength and recovery afterexercise [270].

5.2. Animal Nutrition

The use of amino acids for the nutrition ofmonogastric animals is based on the same foun-dation as the supplementation of human food-stuffs and the clinical experiencewith humans. Inpractice, the enrichment of animal feeds andformulated feeds with amino acids, especiallymethionine and lysine, represents far greaterquantities than does human nutrition. By supple-menting feeds or formulated feeds with the firstlimiting amino acid an obvious cost reductioncan be achieved, while maintaining the quality ofthe ration [271].

Of the ca. 20 amino acids found in feedprotein, about one half are essential for mono-

gastric animals (see Table 4). Most natural feedsare relatively poor in methionine and lysine(Table 14). The requirement of our livestock,however, is comparatively high [272].

When formulating a feed mix for a givenanimal type, the manufacturer has twochoices for meeting the requirement of a partic-ular amino acid. He may use either an excessof feed protein that contains large amounts ofthis amino acid or a minimum of natural proteinand supplement it with synthetic amino acid.Because methionine, lysine, and threonine arecommercially available and inexpensive, theyare often used in formulated feed. L-Tryptophan,which inmany cases is the third or fourth limitingamino acid, is becoming more popular as aningredient for feed supplements, particularlyfor pigs.

Amino Acids Content of Feedstuffs. Ef-fective supplementation requires an exact knowl-edge of the natural amino acid content of both theindividual feedstuffs and the formulated feedmix: the desired rates of supplementation mustalways be capable for being measured analyti-cally. Ion-exchange and high-pressure liquidchromatography are reliable and provenmethodsfor this.

The amino acid contents of individual feed-stuffs are published internationally in a largeseries of tabulations (see, e.g., Table 14). How-ever, such data must be current and reliable.

Amino Acid Requirements of Livestock.Determining amino acid requirement of animalsrequires difficult, time-consuming experiments.The values derived from these experiments arenot constants valid for all times but vary depend-ing on environmental (heat stress, disease), ge-netic (sex, breed), and dietary factors (proteinlevel, energy level, feed intake). There are es-sentially three methods for determining theserequirements: carcass or milk analysis, syntheticrations, and semisynthetic rations.

For the first method, the amino acid content ofa carcass is taken as a first-order approximation tothe amino acid requirement of an animal. Thesame method can be applied for young sucklingmammals by analyzing the amino acid content ofthe milk.

In the second method, the test animal is fed asynthetic mixture of all amino acids, along with


an otherwise balanced ration (control group).The requirement for a single amino acid is deter-mined by reducing its content in the diet to zeroand from there on supplementing it stepwise up toan amount where the animal performs as well asthe control group.

The third and most commonly used methodis that a basal diet consisting of typically usedfeeds is formulated to be deficient in one aminoacid but adequate in all other nutrients. To thisbasal diet graded levels of the amino acid inquestion are added until the performance of theanimal approaches a maximum. The response ingrowth to stepwise increasing of amino acidsupplementation follows the law of diminishingreturns and can be described best by an exponen-tial function.

Amino acid recommendations and require-ments vary between species and for each specieswith age. There are several physiological reasonsfor that:

The various proteins deposited in the animal’sbody differ considerably in their amino acidcomposition. In poultry, for example, the pro-portion of lysine ismuch higher inmuscle proteinthan in feather protein. In contrast, methionine

and cystine are required in a higher percentagefor the formation of feather protein and formaintaining metabolic functions than for muscleprotein.

Not only the amino acid composition of thedifferent proteins varies but also the quantity ofeach protein deposited in the animal changeswith age. The daily accretion of muscle proteinincreases up to a certain age depending on thespecies and declines thereafter. The daily proteinrequirement for metabolic functions rises con-tinually as the animal grows. Another reason foramino acid recommendations changing with ageis the fact that young animals have a high poten-tial for growth but at the same time a relativelysmall feed intake capacity which requires a highnutrient density in the diet.

The amino acid recommendations fordomestic monogastric animals are listed inTable 15.

Economics of Amino Acid Supplementa-tion. The purpose of modern, formulated feedmixes is to meet all nutritional requirements ofthe animal at a minimum cost, and aminoacids and proteins are usually among the most

Table 14. Amino acid composition of feedstuffs, wt% [272, 273]

Feedstuff Dry

matter

Crude

protein

Met MetþCys Lys Thr Trp Arg Leu Ile Val His

Alfalfa 88 17.7 0.27 0.50 0.86 0.76 0.25 0.81 0.72 1.27 0.91 0.36

Barley 88 10.6 0.18 0.42 0.38 0.36 0.12 0.53 0.37 0.73 0.52 0.24

Beans, field 88 25.4 0.20 0.52 1.63 0.90 0.22 2.29 1.03 1.89 1.15 0.67

Blood meal 91 88.8 1.03 2.17 7.96 3.85 1.42 3.94 1.17 11.39 7.69 5.65

Corn 88 8.5 0.18 0.37 0.25 0.31 0.06 0.40 0.29 1.05 0.41 0.26

Corn gluten feed 88 19.0 0.32 0.71 0.58 0.68 0.11 0.85 0.59 1.69 0.89 0.59

Corn gluten meal 88 60.6 1.43 2.52 1.02 2.08 0.31 1.93 2.48 10.19 2.79 1.28

Feather meal 91 81.1 0.61 4.74 2.08 3.82 0.54 5.62 3.86 6.79 5.88 0.93

Fish meal 91 62.9 1.77 2.34 4.81 2.64 0.66 3.66 2.57 4.54 3.03 1.78

Meat and bone meal 91 49.1 0.68 1.18 2.51 1.59 0.28 3.45 1.34 2.98 2.04 0.91

Meat meal 91 48.8 0.68 1.24 2.44 1.63 0.30 3.42 1.40 2.99 2.13 0.93

Oat 88 12.6 0.22 0.57 0.53 0.44 0.14 0.87 0.48 0.92 0.66 0.31

Rapeseed meal, 88 34.8 0.70 1.59 1.95 1.53 0.45 2.15 1.37 2.47 1.77 0.97

Rice 88 7.3 0.20 0.37 0.26 0.26 0.09 0.60 0.29 0.59 0.40 0.18

Sesame meal 88 41.1 1.15 1.97 1.01 1.44 0.54 4.86 1.47 2.74 1.85 0.98

Sorghum 88 9.3 0.17 0.34 0.22 0.31 0.10 0.38 0.37 1.21 0.46 0.23

Soybean meal, 44% CP* 88 44.0 0.64 1.31 2.75 1.76 0.57 3.28 2.01 3.44 2.09 1.21

Soybean meal, 48% CP 88 47.6 0.69 1.41 2.98 1.89 0.61 3.52 2.16 3.71 2.23 1.31

Sunflower meal 88 33.5 0.77 1.36 1.19 1.25 0.40 2.75 1.37 2.15 1.66 0.88

Tapioca 88 3.3 0.04 0.09 0.12 0.11 0.04 0.18 0.11 0.19 0.14 0.08

Triticale 88 11.6 0.21 0.49 0.42 0.39 0.12 0.61 0.42 0.79 0.55 0.29

Wheat 88 13.3 0.21 0.50 0.38 0.38 0.15 0.64 0.44 0.87 0.56 0.32

Wheat bran 88 15.7 0.25 0.58 0.64 0.52 0.22 1.07 0.49 0.98 0.72 0.44

Wheat gluten feed 88 14.4 0.22 0.52 0.46 0.46 0.19 0.86 0.45 0.89 0.67 0.39

Wheat gluten meal 88 74.3 1.17 2.79 1.24 1.89 0.68 2.59 2.65 5.20 2.88 1.54

*Crude Protein


expensive components of a feed mix. The per-formance of a feed mix is measured primarily byfeed utilization and weight gain. Both feed utili-zation and weight gain, as well as other factorssuch as laying performance or feather or hairgrowth, are directly dependent on a sufficientsupply of amino acids. Methionine and lysineplay themajor roles. Methionine in the form of D,L-methionine and lysine in the form of L-lysineHCl are used in place of or as a supplement tonatural methionine and lysine sources [276].

Linear programming is the method of choicefor simultaneously optimizing a ration and mini-mizing cost [277]. This method allows simulta-neous consideration of all demands that are made

on the ration. A prerequisite is exact data on thenutrient content of all available feedstuffs andadditives as well as their prices and availabilities.Additionally, the restrictions, i.e., the dietaryrequirements that the ration has to fulfill, mustbe known. Table 16 shows two examples of feedmixes formulated by the linear programmingmethod commonly used.

Protein and Amino Acid Digestibilities ofFeed Ingredients. The nutritive value of pro-tein for monogastric animals is determined notonly by the amino acid composition of the dietbut also by digestibility of the individual aminoacids, in particular the amino acids likely to be

Table 15. Amino acid recommendations, wt%, for poultry [273] and pigs [274, 275]

Species Metabolizable Crude Met Metþ Lys Thr Trp

energy, MJ/kg protein Cys

Broiler

Starter 12.7 21 0.57 0.92 1.27 0.80 0.20

Grower 13.2 18 0.44 0.76 1.00 0.65 0.19

Finisher 13.2 17 0.41 0.74 0.95 0.63 0.11

Laying hen (105 g feed intake per day) 12.0 16 0.38 0.71 0.80 0.52 0.15

Turkey (weeks of age)

0–4 11.7 28 0.66 1.15 1.80 1.06 0.31

5–8 12.1 25 0.59 1.04 1.60 0.95 0.27

9–12 12.6 22 0.53 0.92 1.40 0.84 0.24

13–16 13.0 19 0.46 0.80 1.20 0.73 0.20

> 16 13.4 16 0.43 0.75 1.10 0.67 0.19

Pig (kg live weight)

10–19 10.4 18 0.48 0.87 1.40 0.94 0.25

20–30 10.2 17 0.40 0.74 1.15 0.79 0.23

40–70 9.8 14.5 0.35 0.64 0.95 0.68 0.19

70–105 9.6 13.5 0.30 0.55 0.82 0.59 0.16

Sow

Lactating 9.8 16.5 0.36 0.65 1.00 0.70 0.20

Pregnant 8.9 12.5 0.23 0.42 0.70 0.46 0.14

Table 16. Two examples of formulated feeds

Broiler feed composition, wt% Pig fattening feed

composition, wt%

Yellow corn 28.00 Feed grain (barley, wheat, corn) 35.00

Wheat 20.00 Soybean meal (44% crude protein) 19.00

Soybean meal (48% crude protein) 30.00 Tapioca meal 20.00

Tapioca meal 10.00 Corn gluten feed 15.00

Fat 7.00 Meat and bone meal (45% crude protein) 3.00

Meat and bone meal

(45% crude protein)

3.00 Fat 3.00

Mineral premix 1.25 Beet molasses 2.00

Vitamin-trace element premix 0.50 Mineral premix 2.43

D,L-Methionine 0.25 Vitamin-trace element premix 0.50

100.00D,L-Methionine 0.07

100.00


limiting. Over the last decades, considerableresearch has been carried out that demonstrateslarge differences in amino acid digestibilitiesbetween feeds.

Amino acid digestibilities can be determinedaccording to the ileal or fecal analysis method.The fecal analysis method measures the amountof each amino acid consumed and excreted infeces. The amino acid digestibilities determinedaccording to the ileal analysis method are calcu-lated based on the intake and amount of eachamino acid passing at the end of the distal ileum.The ileal analysis method should be consideredas an improvement on the fecal analysis method,since the protein or amino acid absorbed in thelarge intestine make little or no contribution tothe protein status of the animal. The ileal analysismethod is also very sensitive to differences inamino acid digestibilities, as these result fromprocessing conditions or from inherent differ-ences between samples of the same feedstuff.Digestibilities measured over the entire digestivetract may be altered by resident bacteria. Thebacteria may break down some of the aminoacids, convert them to other amino acids, or evenproduce new amino acids. Measuring aminoacids excreted in the feces will not reflect unab-sorbed amino acid, but rather unabsorbed aminoacids after possible alteration by the bacteria.

Amino acid digestibility values from the lit-erature, determined with the ileal analysis meth-od (Table 17) show large differences in aminoacid digestibilities between feeds and amongdifferent samples of the same feed.

The differences in amino acid digestibility offeeds can be attributed to various factors. Theseinclude, e.g., fiber levels, heat damage duringprocessing and the presence of antinutritional

factors that interfere with nutrient digestion andutilization.

Studies have clearly shown improvements indiet formulation practices, when diets are formu-lated on the basis of ileal digestible rather than onsupply of limiting amino acids. This holds espe-cially true when a good quality protein supple-ment is replaced by protein supplement(s) oflower quality combined with supplementaryamino acids. In consequence, amino acid require-ments should be expressed as ileal digestible,rather than as total amino acid requirements.

Amino Acids as a Measure to Reduce theN-Output from Livestock Production. Ani-mal production accounts for a significant portionof nitrogen containing compounds released intothe environment. In areas with intensive livestockproduction this might result in environmentalproblems, especially if N-requirements for cropfertilization and N-output from livestock produc-tion get out of balance. In this case nitrogencontaining compounds are released into the sur-face and ground water where they accumulate.

The total amount of nitrogen produced isdependent on both the average number of ani-mals per unit available on and the efficiency ofconversion of feed protein into body protein. Thisefficiency is often impaired due to diets that arenot balanced for the specific amino acid require-ments of the animal fed. The portion of proteinthat is fed without supplying an adequate mix ofamino acids is thus excreted without being uti-lized by the animals. Meanwhile the animal’srequirements for amino acids are well knownwhich allows a reduction in the total proteincontent of diets as long as the diet is supplemen-ted with amino acids in accordance with theanimal’s specific requirements. As a result, feedprotein utilization is maximized and also waterintake as a means to excrete excess nitrogen viathe kidney is reduced.

In consequence the N-output from livestockproduction can be reduced down to 65% whensupplemental amino acids are used together witha reduction of the protein content of the diet.

5.3. Pharmaceuticals

The pharmaceutical industry requires aminoacids at a rate between 2000 and 3000 t/a

Table 17. Range of ileal digestibilities (%) of lysine, methionine, and

threonine in different feeds for pigs modified from [274]

Amino acids Lysine Methionine Threonine

Cereal grains

Barley 65–79 72–88 64–76

Wheat 62–81 79–92 51–78

Corn 71–82 88–92 74–79

Protein supplements

Soybean meal (44% CP*) 85–89 77–90 73–81

Canola meal 74–76 81–87 66–67

Meat and bone meal 58–67 72–79 53–62

Cottonseed meal 53–70 65–82 55–69

*Crude Protein


worldwide. More than half of this is used forinfusion solutions. During the last few years thepotential of amino acids and their derivatives asactive ingredients for pharmaceuticals has beenrecognized clearly, and considerable growth canbe predicted.

5.3.1. Nutritive Agents

Infusion Solutions. Parenteral nutritionwith L-amino acid infusion solutions is a well-established component of clinical nutrition ther-apy. A standard infusion solution contains theeight classical essential amino acids, the semi-essential amino acids L-arginine and L-histidine,and several nonessential amino acids, generallyglycine, L-alanine, L-proline, L-serine, and L-glutamic acid.

Also available are special infusion solutionstailored to the requirements of particular groups,such as newborn infants, seniors, or patients withan extreme negative nitrogen balance. Solutionsrich in the branched-chained amino acids leu-cine, isoleucine, and valine and poor in methio-nine and aromatic amino acids are available forliver-disease patients. Solutions containing onlyessential amino acids are available for kidneypatients. Enzymatic protein hydrolysates, whichwere used as infusion solutions until the 1980shave disappeared almost completely from themarket. They were not available in the optimalcomposition, and there were often compatibilityproblems. Only pure, crystalline L-amino acidsare used in modern infusion solutions. The solu-tions (up to 10%), which also contain electro-lytes in addition to amino acids, are sterile andpyrogen-free.

The simultaneous administration of carbohy-drates is necessary for optimal utilization of theamino acids. Glucose is normally a separateinfusion. Some commercially available aminoacid infusion solutions contain an energy sourcein the form of sugar alcohols (sorbitol, xylitol),which do not enter into a Maillard reaction withthe amino acids.

Normally, parenteral nutrition is only prac-ticed over a limited time. In principle, however,total parenteral nutrition over many years ispossible. In such a case, all essential nutrients(unsaturated fatty acids, vitamins, and trace ele-ments) must be provided.

Elemental Diets. Enteral nutrition is also ameans of providing the essential nutrients [278].Elemental diets, which were developedoriginally for the astronauts [389], containchemically defined nutritive components. In ad-dition to free amino acids the mixtures generallycontain carbohydrates, fats, minerals, andvitamins in a combination adapted to therequirements. In many cases, elemental dietsare used as an alternative and supplement toparenteral nutrition. They have high nutritionalvalue and are totally resorbable. They are largelyindependent of the digestive function of thepancreas and reduce the intestinal bacteria flora.Amino acid elemental diets generally are usedin cases of anatomic, functional, or enzymaticdefects [279].

Formula diets based on peptides are gainingground as an alternative to elemental diets basedon L-amino acids. According to [280], short-chained peptides are resorbed rapidly via a pep-tide transport system in the gut, therefore in aprocess that is independent of amino acid trans-port. Compositions of nitrogen-free amino acidanalogues (keto acids and hydroxy acids) havecome into use for the special case of kidneyinsufficiency (chronic renal failure).

Elemental diets or formula diets are adminis-tered orally or via a nasogastric tube directly intothe gastrointestinal tract.

5.3.2. Therapeutic Agents

Manytherapeuticagentsarederivativesofnaturalor nonnatural amino acids. Examples are benser-azide, captopril, and dextrothyroxine. They aredescribed under keywords such as! Spasmoly-tics,! Antihypertensives, or! Thyrotherapeu-tic Agents. Only therapeutically useful aminoacids and simple derivatives are treated here.

Amino Acids and Salts. The amino acidsand their simple salts that are important therapeu-tic agents are compiled in Table 18. The proprie-tary names listed represent only a selection.

N-Acetylcysteine [616-91-1], C5H9NO3S,Mr 163.2, mp 109–110 �C, ½a�20D þ5� (c ¼ 3,H2O), is a mucolytic and secretolytic agent. Itis also indicated for the treatment of paracetamolpoisoning.


Table

18.Aminoacidsandtheirsaltsas

drugs

Compound

Form

ula

Mr

Medical

use

Tradenam

es

L-A

lanine

[56-41-7]

C3H7NO2

89.09

parenteralnutrition,stim

ulantofglucagonsecretion

L-A

rginine

[74-79-3]

C6H14N4O2

174.20

parenteralnutrition,stim

ulationofpituitaryreleaseof

growth

horm

oneandprolactin,stim

ulationofpancreatic

releaseofglucagonandinsulin

L-aspartate

[7675-83-4]

C10H21N5O6

307.31

treatm

entofhyperam

monem

iaArgihepar,Laevil,

Potentiator,Sargenor,

Sorbenor

L-glutamate

[4320-30-3]

C11H23N5O6

321.34

treatm

entofhyperam

monem

iaModumate

hydrochloride

[1119-34-2]

C6H15ClN

4O2

210.66

treatm

entofhyperam

monem

ia,electrolyte

concentratefor

Argivene,

R-G

ene,

i.v.solutions

Polilevo

L-pyroglutamate

C11H21N5O5

303.27

2-oxoglutarate

[16856-18-1]

C11H20N4O7

320.3

treatm

entofhepatic

disorders

Leberam

,Anetil,

Argiceto,Eucol

L-A

sparagine

[70-47-3]

C4H8N2O3

132.13

parenteralnutrition

monohydrate

[5794-13-8]

C4H10N2O4

150.13

D,L-A

sparticacid

[617-45-8]

C4H7NO4

133.10

magnesium,tetrahydrate

[52101-01-6]

C8H20N2O12Mg

360.54

cardiacagent,managem

entoffatigue,

mineral

supplement

Magnesium

Verla,

potassium,semihydrate

[923-09-1](anhydrous)

C4H6NO4K

�1/2

H2O

180.20

cardiacagent,managem

entoffatigue,

mineral

supplement

Trommcardin,Trophicard-K

€ ohler

sodium,monohydrate

C4H8NO5Na

173.10

L-A

sparticAcid

[56-84-8]

C4H7NO4

133.10

parenteralnutrition

ferrous,tetrahydrate

C8H2ON2O12Fe

392.1

treatm

entofirondeficiency

Sideryl,Spartocine

magnesium,dihydrate

[2068-80-6]

C8H16N2O10Mg

324.52

managem

entoffatigueandheartconditions

potassium,semihydrate

[1115-63-5]

C4H6NO4K

�1/2

H2O

180.20

managem

entoffatigueandheartconditions

Magnesiocard,

(anhydrous)

Corroverlan

sodium,monohydrate

[3792-50-5]

C4H8NO5Na

173.10

parenteralnutrition

(anhydrous)

Betaine

[107-43-7]

C5H11NO2

117.15

citrate

[17671-50-0]

C11H19NO9

309.27

lipotropic

Flacar,Panstabil

hydrochloride

[590-46-5]

C5H12ClNO2

153.61

lipotropic,gastric

acidifier

Aciventral,Acidol,

Pesim

uriat

monohydrate

[17146-86-0]

C5H13NO3

135.15

lipotropic

Hepaderichol

L-Citrulline

[372-75-8]

C6H13N3O3

175.19

treatm

entofhepatic

disorders

Polilevo

L-Cysteine

[52-90-4]

C3H7NO2S

121.16

treatm

entofdam

aged

skin,topically

inophthalmology,

Reducdyn,Hepa-Loges,

detoxicant

Irradian,Cicatrex,

Felacomp

hydrochloride

[52-89-1]

C3H8ClNO2S

157.62

Cheihepar,Choldestal

hydrochloridemonohydrate

[7048-04-6]

C3H10ClNO3S

175.64

L-Cystine

[56-89-3]

C6H12N2O4S2

240.30

parenteralnutrition,lipotropic

agent,treatm

entofhairand

Cystin

‘‘Brunner’’,

naildam

age

Gerontamin,Pantovipar,

(Continued)


Table

18(Continued)

Compound

Form

ula

Mr

Medical

use

Tradenam

es

Priorin

L-G

lutamic

acid

[56-86-0]

C5H9NO4

147.13

parenteralnutrition,dietary

supplement,treatm

entof

Aciglut,Glutamin-V

erla

hyperam

monem

ia

calcium,dihydrate

[5996-22-5]

C10H20N2O10Ca

368.2

Vivacalcium

hydrochloride

[138-15-8]

C5H10ClNO4

183.54

gastric

acidifier

Bioprotein-H

olzinger,

Pansan,Pepsalara

monosodium,monohydrate

[142-47-2]

C5H10NO5Na

187.13

flavoringandseasoningoffood

(anhydrous)

magnesium,tetrahydrate

[19238-50-7]

C10H24N2O12Mg

388.62

tonics,mineral

supplements

Magnesium

Verla,

Glutergen

potassium,monohydrate

[19473-49-5]

C5H10NO5K

203.24

tonics,mineral

supplements

(anhydrous)

magnesium,hydrobromide

C10H17BrN

2O8Mg

397.5

tranquilizer

Psicosoma,

Psychoverlan

L-G

lutamine

[56-85-9]

C5H10N2O3

146.15

parenteralnutrition,treatm

entofmentaldisordersand

alcoholism

Glycine

[56-40-6]

C2H5NO2

75.07

parenteralnutrition,antacidin

conjunctionwithcalcium

Cicatrex,Felacomp,

carbonate

Gastripan-K

,

Mirogastrin

aluminum,hydrate

[13682-92-3]

C2H6NO4Al(þ

xH2O)

135.1

antacid

Acidrine,

Al-Glycin

L-H

istidine

[71-00-1]

C6H9N3O2

155.16

parenteralnutrition,essential

aminoacid

forinfants

acetate

C8H13N3O4

215.21

hydrochloridemonohydrate

[5934-29-2]

C6H12ClN

3O3

209.63

Anti-rheuma,

Rollkur-Ankermann,

Laristine,

Plexam

ine

L-Isoleucine

[73-32-5]

C6H13NO2

131.18


entofhepatic

coma,

dietary

supplement

L-Leucine

[61-90-5]

C6H13NO2

131.18


entofhepatic

coma,

dietary

numerous

supplement

combinations

D,L-Lysine

[70-54-2]

C6H14N2O2

146.19

form

ationofsaltswithacidic

drugs(toenhance

solubility)

monohydrochloride

[70-53-1]

C6H15ClN

2O2

182.65

geriatric

Jestrosemin

acetylsalicylate

[34220-70-7]

C15H22N2O6

326.34

soluble

form

ofacetylsalicylicacid

(aspirin)forinjection

Aspisol,Delgesic

L-Lysine

[56-87-1]

C6H14N2O2

146.19


supplement,prophylaxisof

numerouscombinations

acetate

[57282-49-2]

C8H18N2O4

206.24

herpes

simplexinfection(?)

L-aspartate

[27348-32-9]

C10H11N3O6

279.30

dietary

supplement

L-glutamate

[5408-52-6]

C11H23N3O6

293.32

dietary

supplement

L-m

alate

[71555-10-7]

C10H20N2O7

280.28

dietary

supplement

monohydrate

[39665-12-8]

C6H16N2O3

164.21

dietary

supplement

monohydrochloride

[657-27-2]

C6H15ClN

2O2

182.65

treatm

entofhypochloremia,alkaloses

Aktivanad,Athensa,

Omnival,Vivioptal

D,L-M

ethionine

[59-51-8]

C5H11NO2S

149.21

lipotropic

andcholereticagent

numerouscombinations

L-M

ethionine

[63-68-3]

C5H11NO2S

149.21


supplement,lipotropic

agent,

treatm

entofparacetam

olpoisoning


N-Acetylcysteine is prepared by reaction ofcysteine hydrochloride monohydrate with aceticanhydride in the presence of sodium acetate [281,282].

Trade names: Exomouc (Bouchara), Flui-mucil (Zambon) and numerous others [283].

Carbocisteine (carbocysteine) [638-23-3],S-carboxymethyl-L-cysteine, C5H9NO4S, Mr

179.2, mp 204–207 �C (decomp.), ½a�20D �34:0to �36.0 � (c ¼ 10, H2O), is used to treat dis-orders of the respiratory tract associated withexcessive mucus.

Synthesis involves S-alkylation of L-cysteinewith chloroacetic acid in the presence of sodiumhydroxide [284, 285].

Trade names: Mucodyne (Sanofi-Aventis)and numerous others [283].

Fudosteine [13189-99-5], S-(3-hydroxy-propyl)- L-cysteine, C6H13NO3S, Mr 179.24, mp198–202 �C, [a]20D �22 � (c ¼ 1, H2O), waslaunched by Mitsubishi Pharma in 2001 for thetreatment of bronchial hypersecretion.

Fudosteine is synthesized by condensation ofL-cysteine with 3-bromopropyl alcohol in aque-ous NaOH, or by condensation of L-cysteine withallyl alcohol by means of aqueous potassiumpersulfate [286].

Trade names: Cleanal, Spelear (MitsubishiPharma).

Levodopa [59-92-7], (S)�3-(3,4-dihydrox-yphenyl)alanine, C9H11NO4 , Mr 197, mpL

-Ornithine

acetate

[60259-81-6]

C7H16N2O4

192.22

parenteralnutrition

L-aspartate

[3230-94-2]

C9H19N3O6

265.27

treatm

entofhepatic

disorders

Hepa-Merz

monohydrochloride

[3184-13-2]

C5H13ClN

2O2

168.62

parenteralnutrition

Ornitaine,Polilevo

2-oxoglutarate

[5191-97-9]

C10H18N2O7

278.14

treatm

entofhepatic

disorders(hyperam

monem

ia)

Ornicetil

D-Phenylalanine

[673-06-3]

C9H11NO2

165.19

antidepressant

L-Phenylalanine

[63-91-2]

C9H11NO2

165.19

parenteralnutrition

L-Proline

[147-85-3]

C5H9NO2

115.13


supplement,startingmaterial

forcaptoprilandenalapril

L-Pyroglutamic

acid

[98-79-3]

C5H7NO3

129.07

form

ationofsaltswithbasic

drugs

D,L-Serine

[302-84-1]

C3H7NO3

105.09

startingmaterialforbenserazide

Aktiferrin

L-Serine

[56-45-1]

C3H7NO3

105.09


supplement

Sulfolitruw

L-Threonine

[72-19-5]

C4H9NO3

119.12


supplement

L-Tryptophan

[73-22-3]

C11H12N2O2

204.23

parenteralnutrition,antidepressant,sleepinducer,dietary

Optimax,Kalma,

supplement

Pacitron

L-Tyrosine

[60-18-4]

C9H11NO3

181.19


supplement

L-V

aline

[72-18-4]

C5H11NO2

117.15


supplement,treatm

entof

hepatic

coma


285.5 �C (decomp.), ½a�20D �12:15� (c ¼ 4 in1 M HCl).

Levodopa is widely used for treatment ofParkinson’s disease, most often in combinationwith peripheral decarboxylase inhibitors such asbenserazide and carbidopa. For manufacture andtrade names ! Parkinsonism Treatment.

Trade names: Larodopa (Roche) and numer-ous others [283].

Mecysteine Hydrochloride [18598-63-5],cysteine methyl ester hydrochloride, methyl L-2-amino-3-mercaptopropionate hydrochloride,C4H10ClNO2S, Mr 171.66, mp 140–141 �C, isused in the treatment of disorders of the respira-tory tract associated with excessive mucus. It isprepared by esterification of L-cysteine hydro-chloride monohydrate with methanol in the pres-ence of hydrogen chloride [287].

Trade names: Acthiol (Joulli�e, France), Ac-tiol (Lirca, Italy), Visclair (Sinclair, UK), Aslos-C (Nissin, J), Epectan (Seiko, Japan), Moltanine(Tohok.-Tokyo Tanabe, Japan), Radcol (NipponUniversal, Japan), Sekinin (TokyoHosei, Japan).

Methiosulfonium Chloride (Iodide)[1115-84-0], L-methylmethionine sulfoniumchloride, vitamin U, C6H14ClNO2S, Mr 199.7;mp 134 �C (decomp.). Iodide [3493-11-6],C6H14INO2S, Mr 291.1.

Methiosulfonium chloride is used for its pro-tective effect on the liver and gastrointestinalmucosa, whereas the iodide finds use for rheu-matic disorders. The compounds are made by

heating L-methionine with methyl chloride ormethyl iodide [288].

Trade names: Chloride: Ardesyl (Beytout,France), withdrawn from the market; Cabagin(Kowa, Japan). Iodide: Lobarthrose (Opodex,France), withdrawn from the market.

Oxitriptan [4350-09-8], (S)-5-hydroxy-tryptophan, C11H12N2O3 , Mr 220, mp 273 �C(decomp.), ½a�22D �32:5� (c ¼ 1 in H2O),½a�22D þ16:0� (c ¼ 1 in 4 M HCl).

This intermediate in mammalian biosynthesisof serotonin is used as an antidepressant. It isproduced either by total synthesis (analogous toL-tryptophan via the 5-benzyloxy derivative)[289–291] or by fermentation with Chromobac-terium violaceum [292].

Trade names: Levothym (Promonta Lund-beck, Germany), L�evotonine (Panpharma,France), Oxyfan (Coli, Italy), Tript-OH (Sig-ma-Tai, Italy).

D-Penicillamine [52-67-5], D-3-mercapto-valine, D-b,b-dimethylcysteine, C5H11NO2S, Mr

149.21, mp 198.5 �C, ½a�25D �63� (c ¼ 0.1, pyri-dine). Hydrochloride [2219-30-9], C5H12CI-NO2S, Mr 185.7, mp 177.5 �C (decomp.)½a�25D �63� (c ¼ 1 in 1 M NaOH).

D-Penicillamine is a chelating agent that aidsthe elimination of toxicmetal ions, e.g., copper inWilson’s disease. It is used, as an alternative togold preparations, in the treatment of severerheumatoid arthritis. It is useful in treating cys-tinuria because it reacts with cystine to formcysteine-penicillamine disulfide, which is muchmore soluble than cystine.

D-Penicillamine is produced by hydrolysisof benzylpenicillin via its Hg(II) complex[293] or by total synthesis. In the synthesis,


isobutyraldehyde, sulfur, and ammonia are con-densed to 5,5-dimethyl-2-isopropyl-D3-thiazo-line, which, on reaction with hydrogen cyanide,gives 5,5-dimethyl-2-isopropyl-thiazolidine-4-carbonitrile. Hydrolysis with boiling hydrochlo-ric acid yields D,L-penicillamine hydrochloride.Cyclization with acetone and formylation leadsto D,L-3-formyl-2,2,5,5-tetramethylthiazolidine-4-carboxylic acid, which can be resolved with(�)-phenylpropanolamine via the diastereomer-ic salts. Hydrolysis with hydrochloric acid leadsto D-penicillamine hydrochloride [294].

Trade names: Cuprimine (Merck Sharp &Dohme, USA), Depen (Carter-Wallace, USA),Distamine (Dista, UK),Metalcaptase (Heyl, Ger-many), Trolovol (ASTA Medica AWD, Ger-many), Trisorcin (Merdele, Germany), Trolovol(Bayer, France), Pendramine (ASTA Medica,UK), Pemine (Lilly, Italy).

5.4. Cosmetics

Amino acids are a major component of the‘‘natural moisturizing factor’’ (NMF) that pro-tects the skin surface from dryness, brittleness,and a deleterious environment [295]. The epider-mis of the skin contains about 15%water, which,in the presence of amino acids, principally serine,citrulline, and alanine, forms a stable water-in-oilemulsion with the skin lipids in the form of a thinlayer. The amino acids simultaneously stabilizethe pH of the skin (the acidic layer), and can beabsorbed into the skin and hair. Because of theseproperties, amino acids [296, 297], protein hy-drolysates [298], and proteins [296] are widelyutilized in skin and hair cosmetics, e.g., in mildskin creams, skin cleansing lotions, and hairshampoos. It has been claimed that 60% of hairstrength is accomplished by three amino acids,i.e., histidine, lysine, and tyrosine, and supple-menting these in hair shampoos can reduce lossof hair strength and improve shine [299]. Methi-onine is absorbed into the scalp and absorbed bythe hair fibers as cysteine, which is responsiblefor the cross-linking of keratin [300]. A reportpublished in 2004 claims that amino acids com-prise the fastest growing area of new ingredientsin the moisturizers and humectants market [301].Amino acids manufactured by fermentation rath-er than extracted from animal protein have been

increasingly promoted as being more ‘‘natural’’,and this has helped to increase the market forthese products.

The sodium salts of the reaction products offatty acids with amino acids, such as glutamicacid [302–305], or short-chain peptides (proteinhydrolysates) [306, 307] are surfactants. Theyare effective, skin-compatible cleaners and emul-sifiers, which are used in shampoos, shower gels,baby baths, medicinal skin cleansers, cold-wavepreparations, etc. [303, 308, 309]. Arginine hasbeen used as the cation in soaps to improve thefoam volume, and the free amino acid hasbeen used in conditioners to improve sensoryproperties [310]. The sulfur-containing aminoacids exhibit a special normalizing effect on skinmetabolism, e.g., in cases of excess skin lipidproduction (seborrhea), dandruff, or acne.Substances utilized for this purpose includederivatives of cysteine (e.g., S-carboxymethyl-cysteine), homocysteine (2-amino-4-mercapto-butyric acid), andmethionine [311]. Amino acidsare also used in hair lotions, where they arereported to have a nutritive effect [312]. Cyste-ine, which acts as a reducing agent, is gainingimportance, especially in Japan, as a substitutefor thioglycolic acid in permanent wave prepara-tions that are less damaging to hair [313]. Use ofaluminum, tin, and zirconium complexes of ami-no acids, especially glycine, as deodorants [314]and antiperspirants [314, 315] has been reported.

The use of peptides as mimics of humanprotein fragments in cosmetics has been devel-oped since 2000. One such product is Matrixyl(Sederma), a pentapeptide (Palmitoyl-Lys-Thr-Thr-Lys-Ser), which is a mimic of a precollagenfragment [316]. Matrixyl is applied in anti-wrin-kle creams. Another example is the dipeptide N-acetyl-Tyr-Arg-cetyl ester, registered as Calmo-sensine (Sederma), which stimulates endorphinprecursor release in skin cells and acts as a mildanalgesic [317].

5.5. Agrochemicals

An increasing number of pesticides [318] arederivatives of natural or nonnatural amino acids.Important amino acids like Glyphosate or Glu-fosinate are major products in the agrochemicalmarket. The synthesis of the active ingredientmay start with an amino acid, but very often the


amino acid moiety is formed during the chemicalsynthesis.

5.5.1. Herbicides (! Weed Control, 2. In-dividual Herbicides)

All pesticide segments (i.e., herbicides, fungi-cides, insecticides) contain compoundswith ami-no acid substructures, but the major applicationof pesticidal amino acids is in the herbicidearea [319].

Glyphosate [1071-83-6] [320], Roundup,C3H8NO5P, Mr 169.1, mp 200 �C, is the isopro-pylamine salt of a phosphorus containing glycinederivative with a hybrid structure [321].

Its mode of action is unique; it inhibits differ-ent enzyme systems that leads to the blocking ofdifferent amino acid biosyntheses. The substanceis used as a nonselective contact herbicide tocontrol deep rooted weeds. The sales [322] in1997 grew to about $2180 � 106 and reachingthis figure it is the worlds top selling pesticide.The growth rates were double digits in the 1990s(17%) and for 2002 an annual production of180 000 t was estimated. Additional drivingimpact on the sales of glyphosate has beenachieved by the introduction of glyphosate resis-tant crops (1996: soy beans, 1997: canola andcotton, 1998: maize) and the consequent distri-bution of genetically engineered seeds in combi-nation with this herbicide.

The classical synthesis starts with iminodia-cetic acid, phosphorous trichloride and formal-dehyde, but also sequences using glycine anddimethyl phosphate have been applied. There areno confirmed reports of resistance. The expira-tion of the original patent and the advent ofgeneric producers guarantee an increase of pro-duction of the substance becoming a bulkchemical.

Sulfosate [81591-81-3] [323], Touchdown,C6H16NO5PS, Mr 245.2, is the trimethylsulfo-nium salt of Glyphosate also applied as anonselective herbicide that was introduced in1989. As in the case of Glyphosate, sales aregrowing well.

Glufosinate [324], Basta, Phosphinothri-cine [53369-07-6], C5H12NO4P, Mr 181.1, mp215 �C (for the ammonium salt). This phospho-rous analogue of glutamic acid was introduced in1984 as a nonselective herbicide in the specialitymarket, but meanwhile also resistant crops havebeen developed, which are distributed in combi-nation with the herbicide (Liberty Link). In 1997the sales were at $170 � 106.

The contact herbicide inhibits the plant spe-cific enzyme glutamine synthetase. The chemicalsynthesis requires acrolein, hydrocyanic acid andmethyl phosphinous ester, resulting in a racemicproduct [325]. As the L-enantiomer is 40 timesmore active than the D-compound, many asym-metric synthetic routes are under investigation.

Bialofos [35597-43-4] [326], Bilanafos,C11H22N3O6P, Mr 323.3, mp 160 �C (decomp.),is a natural occurring tripeptide of the sequence L-phosphinothricine-L-alanine-L-alanine isolatedfrom Streptomyces viridochromogenes, Strepto-myces hygroscopicus and others. The compoundwas launched as a nonselective contact herbicidein 1984. It has the same mode of action asGlufosinate, but has much lower sales.

Herbicides Based on 2-Methylvaline. In1985, the first member of a new and very impor-tant class of selective herbicides based on 2-methylvaline was introduced. These imidazoli-none herbicides are acetolactate synthase (ALS)inhibitors [327] meaning that the synthesis ofbranched amino acids is blocked [328].

The synthesis of the racemic active ingredi-ents starts frommethyl isopropyl ketone which isconverted to 2-methylvaline and finally cyclizedto an imidazolinone with an ortho-carboxysubstituted aromatic or heteroaromatic ring sys-tem. The sales of the imidazolinone herbicidesgrew with 6.8% per annum from 1992 to 1997.The sales for Imazethapyr, the star productagainst mono- and dicotyl weeds in soya wereabout $540 � 106 in 1997.

Imazaquin [81335-37-7], 2-(4-isopropyl-4-methyl-5-oxo-4,5-dihydro-1H-imidazol-2-yl)-


3-quinoline carboxylic acid, Scepter,C17H17N3O3, Mr 311.3, mp 219–224 �C, waslaunched in 1984.

Imazapyr [81334-34-1], 2-(4-isopropyl-4-methyl-5-oxo-4,5-dihydro-1H-imidazol-2-yl)-nicotinic acid, Arsenal, C13H15N3O3,Mr 261.3,mp 169–173 �C, was launched in 1985.

Imazamethabenz [81405-85-8], 2-(4-isopro-pyl-4-methyl-5-oxo-4,5-dihydro-1H-imidazol-2-yl)-5-methylbenzoic acid methyl ester,Assert, C15H18N2O3,Mr 274.3,mp 113–153

�C(of methyl ester), was launched in 1986.

Imazethapyr [81335-77-5], 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-4,5-dihydro-1H-imi-dazol-2-yl)nicotinic acid, Pursuit, C15H19N3O3,Mr 289.3,mp 169–173

�C,was launched in 1987.

Imazapic [104098-48-8], 5-methyl-2-(4-isopropyl-4-methyl-5-oxo-4,5-dihydro-1H-imi-dazol-2-yl)-nicotinic acid, Cadre, C14H17N3O3,Mr 275.3,mp 204–206

�C,was launched in 1997.

Imazamox [114311-32-9], 2-(4-isopropyl-4-methyl-5-oxo-4,5-dihydro-1H-imidazol-2-yl)-5-methoxymethylnicotinic acid, Raptor,C15H19N3O4, Mr 305.3, mp, was launched in1997.

Herbicides derived from AcylphenylAmino Acids. Acylphenyl amino acids are agroup of herbicides with decreasing importance.The development of enantiomerically pure com-pounds are new concepts for improving the sub-stance performance.

Benzoylprop [22212-56-2] [329], Suffix,C16H13C12NO3 (for ethyl ester), has an alaninesubunit, which is formed by reacting 3,4-dichlor-oaniline with racemic ethyl 2-chloropropionateand benzoyl chloride. This herbicide is mainlyused in wheat. The racemic active ingredient andthe analogue with an 3-chloro-2-fluoroanilinearomatic are more and more replaced by the D-alanine derivative (racemic switch).

Flamprop-M [63782-90-1] [330], MatavenL, Suffix BW, C19H19CIFNO3, Mr 363.8, mp72.5–74.5 �C (data for isopropyl ester), is syn-thesized from 3-chloro-4-fluoroaniline, benzoylchloride and methyl S-2-chloro propionate. Thiscompound is used as selective grass herbicide inwheat and barley.


Amino Acids in Protox Herbicides. Inhi-bitors of the protoporphyrinogene oxidase [331],also known as Protox, are important herbicidesblocking the biosynthesis of chlorophyll inplants. Phenyl substituted heterocycles have astrong impact on that enzyme [332]. These her-bicides can contain amino acids in two ways. Onthe one side, amino acids like proline, 4-oxapro-line [333] or pipecolinic acid [334] could be partof the heterocycle forming a hydantoin, on theother side amino acids like alanine can form thesubstituent at 5-position of the aromatic system.The preemergence activity and selectivity isincreased using the R-enantiomer instead of theracemic alanine.

Herbicides Based on Glycine. Two herbi-cides based on a glycine residue are the chlor-oacetanilide herbicide Diethatyl [38725-95-0],Antor, C14H18ClNO3 Mr 283.75, produced byHercules Agrochemicals [318]

and the triazine herbicide Eglinazine [68228-19-3], C7H10ClN5O2, Mr 231.64, introduced by Ni-trok�emia Ipartelepek [318]

5.5.2. Fungicides (! Fungicides,Agricultural, 2. Individual Fungicides)

Valinamide Fungicides. A lot of acylvalineanilides are claimed to be active against a broadspectrum of fungi, but especially the control ofPlasmopara and Phytophthora [335] is pointedout. The literature indicates [336] that the S-

compound is the more active enantiomer. Thisgroup of compounds may be divided into valinederivatives and nonvaline derivatives. The valineis usually acylated to form an urethane or amaleimide. The carboxylic group is condensedwith a substituted aniline, an alkyl benzylamineor an alkyl homobenzylamine [337, 338].

Two valinamide fungicides have been intro-duced into the market.

Iprovalicarb [140923-17-7], Melody,C18H28N2O3,Mr 320.43, has been introduced byBayer CropScience for the prevention of downymildew and Phomopsis viticola in vines [339].The valine moiety is in the L-form, but thestereochemistry of the second asymmetric centeris not defined.

Benthiavalicarb [413615-35-7],C15H18FN3O3S, Mr 339.38, has been developedby Kumiai Chemicals Industry for treatment oftomatoes and potato late blight [340].

Anilide Fungicides. A number of anilidefungicides with a market volume of approx.$610 � 106 in 1997 have been established sincethe 1990s. Three amino acid derivatives are soldas fungicides with systemic activity.


Metalaxyl [57837-19-1] [341], Ridomil,C15H21NO4, Mr 279.3, mp 71.8–72.3 �C (datafor isopropyl ester). This most important fungi-cide of its class is synthesized by condensation of2,6-dimethylaniline with methoxyacetyl chlo-ride and subsequent coupling with racemicmeth-yl 2-chloropropionate.

This fungicide is mainly used for the seedtreatment business, in wine and in vegetables.The compound is sold as a stand-alone product orin a variety of mixtures with other fungicides. Itwas introduced in the market in 1978. The (R)-isomer, based on D-alanine, is known as Meta-laxyl-M [70630-17-0] or Ridomil Gold, and wasintroduced by Syngenta in the USA in 1996.

Furalaxyl [57646-30-7], Fongarid,C17H19NO4,Mr 301.3, mp 70 and 84

�C (dimor-phic) (data for isopropyl ester). Furalaxyl is acondensation product of furan-2-carboxylic acidand 2,6-dimethylaniline which is subsequentlycoupled with methyl 2-chloropropionate. Thecompound was introduced in 1984.

Benalaxyl [71626-11-4] [342], Galben,C20H23NO3, Mr 325.4, mp 78–80 �C. In thecommercial synthesis again 2,6-dimethylanilineis coupled with phenylacetyl chloride and subse-quently methyl 2-bromopropionate. The singleenantiomer product Benalaxyl-M [98243-83-5],based on D-alanine, has also been registered.

Hydantoins. Some amino acids being mar-keted as fungicides are sold as their hydantoins.One established product is iprodione.

Iprodione [36734-19-7], Rovral,C13H13Cl2N3O3,Mr 330.2, mp 134

�C. The syn-thesis uses 3,5-dichloroaniline which is coupled

with glycine and phosgene. The urea substructureis formed by reaction with isopropylamine andphosgene. Many fungicide mixtures with Ipro-dione are on the market.

Perfuazoate [101903-30-4], Healthied,C18H23N3O4, Mr 345.4. This rice fungicide hasbeen introduced by Showa Denko for the treat-mentof riceseedlings inJapan [318]. It isbasedona racemic homoalanine pentenyl ester backbone.

5.5.3. Insecticides (! Insect Control)

Only a few amino acid based insecticides aredistributed in the market.

tau-Fluvalinate [102851-06-9] [343], Klar-tan, Mavrik, C26H22CIF3N2O3, Mr 502.9, bp164 �C (9.33 Pa) is the most important com-pound. This substance, preferably used incotton fields, but also in corn, rape tomatoesand vegetables, is a pyrethroid with contactand stomach action. The market of that classhad a volume of $1530 � 106 in 1997. Thechemical synthesis starts with 3,4-dichlorotri-fluoromethylbenzene and D-valine. The carbox-ylic acid is esterified with the cyanohydrin of3-phenoxybenzaldehyde. The compound is mar-keted with the defined stereochemistry at thevaline moiety, but the cyanohydrin subunit is aR/S mixture.


Imiprothrin, Pralle [72693-72-5],C17H22N2O4, Mr 318.4, has been introduced bySumitomo as a household insecticide againstcockroaches and other crawling insects, and isa pyrethroid based on glycine hydantoin. It is soldas a mixture of cis- and trans-isomers [318]. Thelong synthetic pathway leads to the vinyl substi-tuted cyclopropane carboxylate, which forms anester with 3-hydroxymethyl hydantoin. The re-sulting substance is N-alkylated with 3-bromo-propyne.

5.5.4. Plant Growth Regulators (! PlantGrowth Regulators)

Two compounds with amino acid structure arehighlighted because of their direct chemical re-lationship to Glyphosate.

Glyphosine [2439-99-8] [344], Polaris,C4H11NO8P2 was introduced in 1973 and ispreferably used in sugar beets. The synthesisstarting with glycine uses two equivalents offormaldehyde and of phosphorous trichlorideunder oxidative conditions.

A second derivative of Glyphosate is a cyclicphosphaoxazole being formed from Glyphosateand formaldehyde. This compound is claimed tocontrol the growth of some grasses without dis-coloration or phytotoxicity.

Aviglycine [49669-74-1], aminoethoxyvi-nylglycine, ReTain, C6H12N2O3,Mr 160.2, is pro-duced by Streptomyces spp. and was isolated andpurified by Abbott. It is marketed by Valent Bios-ciences for control of fruit ripeningas it inhibits theproduction of endogenous ethylene [318].

5.6. Industrial Uses

Polyfunctional compounds, such as amino acidsand their derivatives, have been applied in a widevariety of industrial areas. This is particularlybecauseof their physicochemicalproperties, suchas high thermal stability, low volatility, ampho-terism, buffering capacity, and the ability to formcomplexes. However, properties of amino acidssuch as environmental acceptability and low tox-icity, are becoming increasingly important.A fewof the recorded uses have been listed below, andinclude acylamino acid monomers for epoxy re-sins [345]; amino acid dispersing agents for pig-ments in coloring polyester fibers [346]; N-acy-lamino acid dispersants for polyurethanes in wa-ter [347]; amino acid setting retarders for cement[348]; zinc salts of N-acyl-derivatives of basicamino acids and N-acylamino acids for the ther-mal stabilizationofPVC[349];polyglutamicacidesters and polyaspartic acid esters coatings fornatural and synthetic leather [350]; amino acidhardening agents for methacrylate resins [351];N-acylamino acid, amino acid ester, and aminoacid amide gel-forming agents for oils [352];basic amino acid vulcanization accelerators fornatural rubber [353]; amino acid [354] and N-acylamino acid [355] corrosion inhibitors formetals; amino acids to stabilize the latent imageof photographic emulsions [356]; and amino acidbrighteners in galvanic baths [357, 358].

6. Chemical Analysis

Amino acids do not have defined melting pointsbut decompose over a broad range between 250and 300 �C. Therefore, theymust be transformedinto derivatives before melting points are usefulfor identification. Phenylisothiocyanate is usedto yield the phenylthiohydantoin amino acid(PTH amino acid) [8], or 2,4-dinitrofluoroben-zene (Sanger’s reagent) is used to yield thedinitrophenyl amino acid [8].

Spectroscopic methods for the identificationof amino acids include infrared [359], Raman,1H-NMR, and 13C-NMR spectroscopy [360] offree amino acids or PTH derivatives and massspectrometry of PTH derivatives [361]. Ultravi-olet spectroscopy is important only for aromaticamino acids. The different methods for qualita-tive and quantitative analysis of amino acids,


particularly of mixtures from protein hydrolysisand biological fluids, have been reviewed [362].Separation methods are described in [363].

Various staining methodsmay be used for thequalitative identification of a-amino acids [364].Some of these dye-forming reactions are suitablefor quantitative analysis within the validity rangeof the Lambert–Beer law. By far the most impor-tant is the reaction with ninhydrin, which yields ared-violet to blue-violet dye (lmax ¼ 570 nm).

The imino acids proline and hydroxyprolineform a structurally different dye, with an absorp-tion maximum at 440 nm.

Fluorescent reagents also have been usedsuccessfully for quantitative analysis. The aminoacid is converted into a strongly fluorescentderivative, which increases the sensitivity byorders of magnitude. Typical fluorescent re-agents are o-phthalaldehyde–2-mercaptoethanol[365], 1-dimethylaminonaphthalene-5-sulfonylchloride (dansyl chloride) [366], and 4-phenyl-spiro [furan-2(3H)-10-phthalan]-3,30-dione(fluorescamine) [367].

The separation of amino acid mixtures ispossible by electrophoresis or chromatography.The latter is especially useful, techniques includ-ing paper, thin layer, ion-exchange, high-pressure liquid, and gas chromatography. Thedifferent techniques have been reviewed andcompared in [368]. Paper chromatography isgenerally carried out in two dimensions. A num-ber of eluents are available for this purpose.Quiteoften the amino acid is converted to its dinitro-phenyl derivative.

More common than paper chromatographyis thin layer chromatography (TLC) of eitherthe free amino acids [369] or their PTHderivatives [370]. Silica gel, aluminum oxide,cellulose powder, or polyacrylamide may be

used as carrier, but silica is preferred. An indica-tor reagent, most often ninhydrin, is used fordetection [371]. For detection of very smallquantities the amino acids separated on an TLCplate can be converted to the fluorescaminederivatives [372]. The detection limit is 10 pmol.The time required for the analysis of dinitrophe-nyl amino acids can be reduced by using high-pressure thin layer chromatography [373]. TLCis often specified as an analytical test to showthe levels of foreign amino acids in a sample,particularly when the amino acid is from naturalsources.

Ion-exchange chromatography [374, 375] onorganic resins (Dowex,Amberlite, etc.) has prov-en to be the most exact and reliable method forthe separation and quantitative analysis of aminoacids. Before the automatic analysis techniquewas introduced [376], complete analysis of anamino acid mixture required 24 h. Today 2 h isthe rule. Sodium citrate or lithium citrate buffersolutions are the eluents. The eluate is reactedwith ninhydrin [374, 377] or o-phthalaldehyde[378]. With ninhydrin, 1–20 nmol amino acidcan be measured with an accuracy of � 1–5%.The detection limit with o-phthalaldehyde is inthe picomole range. Ion-pair chromatography ona porous graphitic carbon stationary phase hasalso been applied. Detection is by evaporativelight scattering, or by electrospray mass spec-trometry [379].

Ion-exchange chromatography is the methodof choice for analyzing amino acids in feeds,foodstuffs, and biologic fluids [380]. In general,analysis is preceded by a hydrolysis, which de-grades the proteins and peptides to their compo-nent amino acids. However, acidic hydrolysiscan destroy Cys, Met, and Trp residues, andspecial techniques may be necessary to analyzefor these amino acids. Figure 10 shows a sampleaminogram of broiler feed.

Utilizationofhigh-pressureliquidchromatog-raphy (HPLC) allows a further reduction in anal-ysis time. Originally, the amino acids were con-verted to derivatives, e.g., to dansyl amino acids[381], PTH amino acids [382], or dinitrophenylderivatives [383], before analysis. Reversedphases are the preferred stationary phases. Nin-hydrin, o-phthalaldehyde–mercaptoethanol[384], or fluorescamine [385] are the usual re-agents fordetection.AnHPLCmethodwithdirectUV detection has also been described [386] for


analysis of infusion solutions.Modern automatedamino acid analyzers are now available, that usequaternary gradient elution to separate all thecommon amino acids efficiently without priorderivatization. These machines generally incor-porate postcolumn visualization with ninhydrinand photometric quantification. Today this meth-od rivals ion-exchange chromatography as thepreferred analytical method.

Gas chromatographic methods [387] are use-ful for the analysis of complex amino acid mix-tures. However, the amino acids must be con-verted into volatile derivatives, e.g., PTH aminoacids [388], methyl esters of N-trifluoroacetyla-mino acids [389], n-butyl esters of N-trifluoroa-cetylamino acids [390], N-trimethylsilylaminoacids [391], or N,O-bis-trimethylsilylaminoacids [392]. Gas chromatography coupled withelectron-capture negative-ionization mass spec-trometry (GC–ECNI–MS) has been used to de-termine amino acids at the femtomole scale,using 2H or 13C labeled amino acids as referencestandards [393].

Electrophoresis [394], which employs thediffering rates of migration in an electric field,is becoming more important [395]. The tech-nique is already well-established for proteinanalysis, and has been coupled with electrosprayionization mass spectrometry (CE–ESI–MS) to

determine underivatized amino acids at micro-molar level [396]. Capillary isotachophoresis is anew high-resolution electrophoresis techniquefor amino acids.

The chromatographic separation of aminoacid enantiomers is the subject of intensive in-vestigation. Separation is currently possible bygas chromatography [397] and high-pressureliquid chromatography [398], using opticallyactive phases or chiral solvents [399]. A widerange of chiral stationary phases is now availablefor analytical or preparative separations. Anothermethod is to use a chiral precolumn derivatizingagent. Marfey’s reagent (1-fluoro-2,4-dinitro-phenyl-5-L-alanine amide) has been successfullyapplied to separate complex mixtures of aminoacids into their separate enantiomers, and iseffective at the nanomolar scale [400, 401].Marfey’s reagent forms diastereomeric salt pairswith the amino acids, that are particularly wellseparated by HPLC.

The microbiological analysis of amino acidsis based on the fact that several L-amino acids areessential for certain bacteria strains. The growthof the bacteria cultures under standardconditions can be quantitatively evaluated (acidi-metry or turbidimetry) and related to the aminoacid concentration. Lactic acid bacteria [402](Lactobacteriaceae) can be used to analyze 19

Figure 10. Amino acid chromatogram of a broiler feedInternal standard: norleucine. Solid curve: UV detection at l ¼ 570 nm. Dotted curve: UV detection at l ¼ 440 nm for Pro(and Hyp)


L-amino acids. Typical test microbes includeLeuconostoc mesenteroides (ATCC 8042), Lac-tobacillus arabinosus 17�5 (ATCC 8014), andStreptococcus faecalis R (ATCC 9790, 8043).The conventional microbiological methodsare quite complicated but can be simplified byautomation [403].

A series of L- or D-amino acids can be ana-lyzed by enzymatic methods. L-Amino acids, orthe enantiomeric purity of D-amino acids, can bedetermined with bacteria decarboxylases bymeasurement of CO2 formed. D-Amino acids, orthe enantiomeric purity of L-amino acids, can bedetermined with kidney D-amino acid oxidasesby measurement of the O2 consumption. En-zymes that react only with a single amino acidallow determination of that amino acid, e.g.,arginine using liver arginase. An improved en-zymatic method consists of the use of enzymeelectrodes that contain the enzymes [404], ormicroorganisms having a special enzyme in afixed form. Enzyme electrodes, however, arerelatively unstable [405].

The quantitative determination of crystallizedamino acids is carried out by acidimetric titrationin nonaqueousmedium [406, 407]. Glacial aceticacid is a suitable solvent. Formic acid may beadded to improve solubility. The titrant is per-chloric acid. Formol titration by the method ofS€orensen can be used for aqueous solutions but isless accurate.

Standards of purity for individual L- and D,L-amino acids that are used in drugs or as foodadditives are published in pharmacopeias [406,408] and food codices [409].

7. Economic Significance

The 2005 world market for amino acids is esti-mated at more than 3.3 � 106 t/a (Table 19).The ‘‘big three’’ amino acids, sodium L-gluta-mate, D,L-methionine, and L-lysine � HCl, ac-count for approximately 95% of the volume(Table 20). The other amino acids play only asmall role. The dominant amino acid, sodiumglutamate, is used almost exclusively as ataste enhancer. D,L-Methionine and L-lysineHCl are used almost exclusively to improve thenutritive value of animal feeds. L-Threonineis also used mainly in animal feeds. L-Asparticacid and L-phenylalanine are used principally

for the manufacture of the intense sweeteneraspartame. The other amino acids have diversi-fied applications. With the exception of glycine,they are more expensive than the big threeamino acids. In terms of volume, feed additivesused about 1 500 000 t of amino acids, pharma-ceuticals 17 000 t and sweeteners around 17 000 t(2005) [410]. Table 21 shows the marketvalue for amino acids in 2004 broken down byfield of use [52]. The total value of the globalamino acids market in 2004 was estimated at $4.5 � 109 [52].

The main manufacturers of amino acidsare located in Japan (e.g., Ajinomoto, KyowaHakko, Tanabe) and in Europe (Degussa, Wac-ker, Amino, Flamma). Recently there has beenan increasing trend towards production in

Table 19. Amino acid production, 2005

Amino acid Quantity, t/a [410–413]

L-Alanine 1500

L-Arginine* 3000

L-Asparagine 200

L-Aspartic acid 15 000

L-Cysteine* 7000

L-Glutamic acid* 1 690 000

L-Glutamine 2000

Glycine 16 000

L-Histidine 300

L-Hydroxyproline 100

L-Isoleucine 500

L-Leucine 800

L-Lysine* 850 000

D,L-Methionine 600 000

L-Methionine 400

L-Phenylalanine 14 000

L-Proline 800

L-Serine 300

L-Threonine 85 000

L-Tryptophan 2000

L-Tyrosine 150

L-Valine 1100

D-Phenylglycine þD-p-hydroxyphenylglycine

9000

Others 5000

Total 3 300 000

*Free amino acid and salts.*Free amino acid and derivatives.

Table 20. Percentage of individual amino acids as a part of the total

market, 2005

Amino acid Quantity, %

L-Glutamic acid (Na) 51

D,L-Methionine 18

L-Lysine (HCl) 26

Other amino acids 5


China, with established manufacturers setting upplants there.

8. Toxicology

Excess amino acids are rapidly disposed of byincreased metabolic degradation. Should theamino acid dose be suddenly increased, e.g., byextremely high protein consumption, withinabout two days the liver adaptively increases thelevels of amino acid-catabolizing enzymes –transaminases, enzymes of the urea cycle, cy-stathionase, tryptophan pyrrolase, etc. The ex-cess amino acids are to a large extent used toprovide energy. The nitrogen is eliminated asurea. A smaller portion is used in protein synthe-sis, mainly liver protein and plasma albumin.

When too little protein or no protein is con-sumed or when the component amino acids areimbalanced, alteration of the ribosome profileoccurs in the liver, and ribonucleic acids arecatabolized. Themanifestation of chronic proteindeficiency is known as marasmus (slight defi-ciency) and kwashiorkor (extreme deficiency).Protein deficiency is usually coupledwith caloriedeficiency (protein–calorie malnutrition). Thismanifests itself on the biochemical level as anegative nitrogen balance, indicating a reductionin protein inventory. Initially, the labile enzymeand plasma proteins are consumed, the greatestlosses occurring first in the liver, then in themusculature. Brain, heart, and kidneys sufferonly minimal protein loss. Symptoms of proteinsynthesis disorders include disturbances inwound healing and bone growth, lowered resis-tance to infection and stress, loss of fertility andappetite, and anorexia. The urinary excretion of3-methylhistidine is a common indicator of thecatabolism of muscle protein.

The total absence of an essential amino acid inthe diet ismore serious than protein deficiency. Inthis case the proteins or amino acids in the diet aretotally worthless because protein synthesis can

occur only by degradation of body protein. Ageneral interruption of the protein synthesis re-sults. This manifests itself rapidly as a drop inenzyme activity and an impoverishment of theplasma proteins. Noticeable symptoms are lossof appetite and weight, alteration of cornea andlens, anatomic organ alterations, and an in-creased rate ofmortality. In addition there appearspecific deficiency symptoms characteristic ofthe missing amino acid or acids.

The metabolic disturbances brought about bygross divergences from the optimal amino acidpattern have three different causes [414, 415]:imbalance, antagonism, and toxicity.

Amino acid imbalancemanifests as an appear-ance of deficiency symptoms for the first limitingamino acid when the other amino acids areconsumed in great excess. The symptoms ofimbalance are eliminated by administration ofthe first limiting amino acid in sufficient quanti-ties. The main symptom is a severe reduction offood or feed assimilation and depression ofgrowth. The depression of growth in rats hasbeen investigated intensively by adding individ-ual L- and D,L-amino acids to basal diets ofvarious protein levels [416, 417].

Amino acid antagonismis caused by competi-tion for common transport systems. An exampleis antagonism of the branched-chain amino acidsisoleucine, leucine, and valine. The symptomsare reversible. In a study with young rats, addi-tion of 5% L-leucine to a low-protein diet (9%casein) reduced the plasma levels of isoleucineand valine, depressed growth, and reduced feedconsumption [418]. These effects were eliminat-ed after a latent period of three days by smalldoses of L-isoleucine (0.16%) and L-valine(0.15%).

Amino acid toxicity occurs when very largequantities of one or more amino acids are con-sumed and is characterized by total failure of theadaptive mechanisms. The toxic level has beenstudied by adding increasing quantities of indi-vidual amino acids to a protein basal diet [415].The toxicity of individual amino acids dependson the total protein consumption. Imbalance,antagonism, and toxicity are less pronouncedwhen overall protein consumption is sufficientbut becomemore severe at lower levels of proteinconsumption. The consumption of toxic amountsof amino acids increases their concentration inthe plasma and brain. Because of the blood–brain

Table 21. Market value by field of use, 2004

Use Value, %

Animal nutrition 56

Human nutrition 32

Specialty* 12

*Pharmaceuticals, cosmetics, agrochemicals, and industrial uses.


barrier, however, the increase in the brain is notas great [419]. Failure of the adaptation mechan-isms during consumption of large excess of anamino acid can lead to accumulation of the aminoacids or certain metabolites in the organism,leading directly or indirectly (e.g., by influencinghormone secretion) to anatomic or functionaldamage.

The toxicity of amino acids has been reviewed[415, 418]. The acute toxicities of most L-aminoacids and some derivatives have been determined[420–422]. The toxicology of D-amino acids isdiscussed in review articles [415] and otherpublications [421, 422]. There is no evidence todate that the D-enantiomers of the a-amino acidsfound in proteins exhibit specific toxic effects.Their LD50 values are generally higher than thoseof the L-amino acids.

References

General References1 J. P. Greenstein, M. Winitz: Chemistry of the Amino

Acids, vol. 1, 2, and 3, J. Wiley & Sons, New York–

London 1961.

2 A. Meister: Biochemistry of the Amino Acids, 2nd ed.,

vol. 1, Academic Press, New York 1965.

3 I. Wagner, H. Musso: ‘‘Neue nat€urliche Aminos€auren’’

Angew. Chem. 95 (1983) , 827–920; Angew. Chem. Int.

Ed. Eng. 22 (1983) 816 (review of the literature since

1956).

4 IUPAC and IUB Joint Commission on Biochemical

Nomenclature, Pure Appl. Chem., 56 (1984) 595.

5 G. C. Barrett (ed.): Amino Acid Derivatives, Oxford

University Press, Oxford 1999.

6 Houben-Weyl, E16d, 1992; Science and Synthesis, v.

26, 2004, p. 1255.

7 S. E.Wolkenberg, R.M.Garbaccio, Science of Synthesis

20a (2006) 385.

8 Th. Wieland et al.: ‘‘Methoden zur Herstellung und

Umwandlung von Aminos€auren und Derivaten,’’ Hou-

ben-Weyl, 11/2.

9 H. D. Jakubke, H. Jeschkeit: Aminos€auren, Peptide,

Proteine, Verlag Chemie, Weinheim, Germany 1982.

10 B. Weinstein (ed.): Chemistry and Biochemistry of

Amino Acids, Peptides and Proteins, vol. 1–5, Marcel

Dekker, New York 1971 –1978.

11 D. Barton, W. D. Ollis: Comprehensive Organic Chem-

istry, vol. 2, Chap. 9.6, p. 815; vol. 5 Pergamon Press,

New York 1979.

12 G. Kr€ugers: ‘‘Aminocarbons€auren’’ in: Methodicum

Chimicum, vol. 6, p. 611.

13 E. W€unsch: ‘‘Synthese von Peptiden,’’ Houben-Weyl,

15/1 and 15/2.

Specific References14 S. K. Bhattacharyya, A. B. Banerjee, Folia Microbiol.

(Prague) 19 (1974) 43.

15 T. Robinson, Life Sci. 19 (1976) 1097.

16 R. E. Dickerson, Spektrum der Wissenschaften 1979,

no. 9, 98.

17 R. S. Cahn, C. Ingold, V. Prelog, Angew. Chem. 78

(1966) 413; Angew. Chem. Int. Ed. Engl. 5 (1966) 385,

511.

18 K. L€ubke, E. Schr€oder, G. Kloss:Chemie und Biochemieder Aminos€auren, Peptide und Proteine, vol. I and II,

Thieme Verlag, Stuttgart 1975.

19 A. Kleemann, Chem. Ztg. 106 (1982) 151–167.

20 K.H. Drauz, A. Kleemann, J.Martens,Angew. Chem. 94

(1982) 590; Angew. Chem. Int. Ed. Engl. 21 (1982) 584.

21 A. Berkessel, H.w Gr€oger: Asymmetric Organocataly-

sis, Wiley-VCH, Weinheim 2005.

22 Drugs of the Future 23 (1998) 1057.

23 R. P. Nargund, A. A. Patchett, M. A. Bach, M. G.

Murphy, R. G. Smith, J. Med. Chem. 41 (1998) 3103.


25 H. J. Federsel, Chirality 15 (2003) 128.

26 A.M€uller, E.Busker, J. Engel,B.Kutscher,M.Bernd,A.

V. Schally, Int. J. Peptide Prot. Res. 43 (1994) 264.

27 Europ. Pat. Appl. EP 0 530 167A 1, 1993 (B. Atrash, D.

M. Jones, M. Szelke).




31 J. A. Robl et al., J. Med. Chem. 40 (1997) 1570.

32 S. Budavari (ed.): The Merck Index, 12th ed., Merck &

Co., Inc., Whitehouse Station, NJ 1996, p. 97.

33 A. S. Bommarius, M. Schwarm, K. Stingl, M. Kotten-

hahn, K. Huthmacher, K. Drauz, Tetrahedron: Asymme-

try 6 (1995) 2851.




37 J. F. Atkins, R. Gesteland, Science 296 (2002) 1409.





42 S. Budavari (ed.): The Merck Index, 12th ed., Merck &

Co., Inc., Whitehouse Station, NJ 1996, p. 99.






48 V. N. Gladyshev, D. L. Hatfield, J. Biomed. Sci. 6

(1999) 151.

49 G. N. Schrauzer, J. Nutrition 130 (2000) 1653.


51 R.Meister (ed.):FarmChemical Handbook 1990, Meis-

ter Publishing Co., Willoughby, OH 1990, p. C136.

52 W. Leuchtenberger, K. Huthmacher, K. Drauz, Appl.

Microbiol. Biotech. 69 (2005) 1.


53 T. Hermann, J. Biotechnol. 104 (2003) 155.

54 M. Breuer et al., Angew. Chem. Int. Ed. 43 (2004) 788.

55 J. Jones: Amino Acid and Peptide Synthesis, Oxford

University Press, Oxford 2002.

56 M. Ikeda, in T. Scheper, R. Faurie, J. Thommel (eds.):

Advances in Biochemical Engineering/Biotechnology

79 (2003) 1.

57 S. Hashimoto, R. Katsumata, L-Alanine production by

alanine racemase-deficient mutant of Arthrobacter

oxydans in: Proc. Ann. Meet. (Agric. Chem. Soc. Jpn.)

(1994) 341.

58 S. Yahata, H. Tsutsui, K. Yamada, T. Konehara:

‘‘Fermentative production of D-alanine:’’ in: Proc. Ann.

Meet. Agric. Chem. Soc. Jpn., (1993), 92.

59 Tanabe Seiyaku, JP-Kokai 58-9692, 1983 (I. Chibata,M.

Kisumi, T. Tagaki).

60 Ajinomoto, US 5 164 307, 1992 (Y. Yoshihara, Y.

Kawahara, Y. Yamada).

61 M. Sugiura, S. Suzuki,M. Kisumi,Agric. Biol. Chem. 51

(1987) 371–377.

62 Kyowa Hakko, EP-A 557 996, 1993 (K. Kino,

K. Okamoto, Y. Takeda, Y. Kuratsu).

63 C. Keilhauer, L. Eggeling, H. Sahm, J. Bacteriol. 175

(1993) 5595–5603.

64 Cheil Sugar, FR 2 645 172, 1990 (J.W.Oh, S. J. Kim,Y.

J. Cho, N. H. Park, L. H. Lee).

65 University Odessa, SU 1 730 152, 1992 (I. I. Brown et

al.).

66 Sci. Res. Technol Inst. Amino Acids, USSR, DE-OS

3 612 077, 1986 (S. M. Kocarian et al.).

67 All Union Sci.-Res. Inst. Genetics andMicroorg. Gen.þSelec Univ. Odessa, WO 90/04636, 1990 (V. G. Deba-

bov et al.).

68 M. Ikeda, K. Nakanishi, K. Kino, R. Katsumata, Biosci.

Biotech. Biochem. 58 (1994) 674–678.

69 M. Furui, K. Yamashita, J. Ferment, Technol. 61 (1983)

587–591.

70 H. Yoshida: ‘‘Arginine, Citrulline, and Ornithine,’’ in K.

Aida et al. (eds.): Biotechnology of Amino Acid Produc-

tion, Kodansha, Tokyo/Elsevier, Amsterdam 1986,

pp. 131–143.

71 I. Chibata, T. Tosa, T. Sato: ‘‘Aspartic Acid,’’ in H. W.

Blanch, S. Drew, D. I. C. Wang (eds.): Comprehensive

Biotechnology, vol. 3, Pergamon Press, Oxford

1985.

72 Y. Yokote et al., J. Solid-Phase Biochem. 3 (1978) 247–

261.

73 M. Terasawa, H. Yukawa, Y. Takayama, Process Bio-

chem. 20 (1985) 124–128.

74 T. Sato et al., Biotechnol. Bioeng. 17 (1975) 1779–1804.

75 I. Chibata, T. Tetsuya, T. Sato, Appl. Biochem. Biotech-

nol. 13 (1986) 231–240.

76 M. C. Fusee, W. E. Swann, G. J. Calton, Appl. Environ.

Microbiol. 42 (1981) 672–676.

77 Genex, EP-A 197 784, 1986 (W. E. Swann, A. C. Nolf).

78 J. Michelet, A. Deschamps, J. M. Lebeault, 3rd Eur.

Congr. Biotechnol., vol. 2, Verlag Chemie, Weinheim,

Germany 1984, pp. 133–138.

79 M. Mori, I. Shiio, Agric. Biol. Chem. 48 (1984) 1189–

1197.

80 N. Nishimura, S. Komatsubara, M. Kisumi, Appl. Envi-

ron. Microbiol. 53 (1987) 2800–2803.

81 Maggi, DE 2 449 711, 1979 (P. Hirsbrunne, R.

Bertholet).

82 Mitsubishi Chemical Ind., US 3974031, 1976 (H. Ya-

mada, K. Gumagai, H. Ohkishi).

83 Consortium f€ur Elektrochemische Industrie GmbH,WO

01/27307, 2001 (T. Maier, C. Winterhalter).

84 T. H. P Maier, Nat. Biotechnol. 21 (2003) 422.

85 K. Sano, K.Mitsugi,Agric. Biol. Chem. 42 (1978) 2315–

2321.

86 S. Kinoshita, S. Ukada, M. Shimono, J. Gen. Appl.

Microbiol. 3 (1957) 139–205.

87 S. Kinoshita, K. Tanaka: ‘‘Glutamic acid,’’ in K. Yama-

da, et al. (eds.): The Microbial Production of Amino

Acids, John Wiley & Sons, New York 1972, pp. 263–

324.

88 L. Eggeling: Handbook of Corynebacterium Glutami-

cum, CRC Press, Boca Raton, FL 2005.

89 B. J. Eikmans, M. Kircher, D. J. Reinscheid, FEMS

Microbiol. Lett. 82 (1991) 203–208.

90 W. Liebl, M. Ehrmann, W. Ludwig, K. H. Schleifer, Int.

J. Syst. Bacteriol. 41 (1991) 225–235.

91 Y. Hirose, H. Enei, H. Shibai: ‘‘L-Glutamic acid fer-

mentation,’’ in H. W. Blanch, S. Drew, D. I. C. Wang

(eds.):Comprehensive Biotechnology, vol. 3, Pergamon

Press Ltd., Oxford 1985.

92 M. Kikuchi, Y. Nakao: ‘‘Glutamic acid,’’ in K. Aida, I.

Chibata, K. Nakayama, K. Takinami, H. Yamada (eds.):

Biotechnology of Amino Acid Production, Kodansha,

Tokyo/Elsevier, Amsterdam 1986, pp. 101–116.

93 R. Kr€amer, BioEngineering 9 (1993) 51–61.

94 R. Kr€amer, FEMS Microbiol. Rev. 13 (1994) 75–93.

95 I. Shiio, K. Ujigawa,Agric. Biol. Chem. 42 (1980) 1897–

1904.

96 E. R. B€ormann, B. J. Eikmanns, H. Sahm, Molecular

Microbiol. 6 (1992) 317–326.

97 T. E. Walker et al., J. Biol. Chem. 257 (1982) 1189–

1195.

98 H. Yoishii et al.,NipponNogei KagakuKaishi 67 (1993)

949–954.

99 H. Yoishii, M. Yoshimura, S. Nakamori, S, Inoue,

Nippon Nogei Kagaku Kaishi 67 (1993) 955–960.

100 Ajinomoto, JP 56 148 295, 1981 (T. Tsuchida,K.Miwa,

S. Nakamori, H. Mimose).

101 M. Shirasuchi et al., Biosci. Biotechnol. Biochem. 59

(1995) 83–86.

102 T. Tsuchida et al., Agric. Biol. Chem. 51 (1987) 2089–

2094.

103 K. Araki: ‘‘Histidine,’’ in K. Aida, I. Chibata, K. Na-

kayama, K. Takinami, H. Yamada (eds.): Biotechnology

of Amino Acid Production, Kodansha, Tokyo/Elsevier,

Amsterdam 1986, pp. 247–256.

104 M. B. Vickery, J. Biol. Chem. 143 (1942) 77.

105 KyowaHakko, EP-A 759 472, 1996 (A. Ozaki, H.Mori,

T. Shibasaki).


106 J. Ogawa, S. Shimizu,Curr. Opin. Biotechnol. 13 (2002)

367.

107 I. Eggeling, C. Cordes, L. Eggeling, H. Sahm, Appl.

Microbiol. Biotechnol. 25 (1987) 346–351.

108 E. Scheer, L. Eggeling, H. Sahm, Appl. Microbiol.

Biotechnol. 28 (1988) 474–477.

109 C. Wilhelm et al., Appl. Microbiol. Biotechnol. 31

(1989) 458–462.

110 Ajinomoto, Patent 5 164 307, 1992 (Y. Yoshihara, Y.

Kawahara, Y. Yamada).

111 S. Komatsubara, M. Kisumi, I. Chibata, J. Gen. Micro-

biol. 119 (1980) 51–61.

112 Ajinomoto, EP-A 519 113, 1992 (V. A. Livshits et al.).

113 Kyowa Hakko, EP-A 595 163, 1994 (T. Nakano,

T. Azuma, Y. Kuratsu).

114 Forsch. Z. J€ulich, DE 44 00 926, 1995 (B. M€ockel,

L. Eggeling, H. Sahm).

115 Kyowa Hakko, US 5 362 637, 1994 (K. Kino,

Y. Kuratsu).

116 S. Morbach, H. Sahm, L. Eggeling, Appl. Environ.

Microbiol. 61 (1995) 4315–4320.

117 Ajinomoto, EP-A 685 555, 1995 (K. Hashiguchi,

H. Kishino, N. Tsujimoto, H. Matsui).

118 U. Groeger, H. Sahm, Appl. Microbiol. Biotechnol. 25

(1987) 352–236.

119 S. Komatsubara, M. Kisumi: ‘‘Histidine,’’ in K. Aida,

I. Chibata, K. Nakayama, K. Takinami, H. Yamada

(eds.): Biotechnology of Amio Acid Production,

Kodansha, Tokyo/Elsevier, Amsterdam 1986,

pp. 233–246.

120 Ajinomoto, US 5 164 307, 1992 (Y. Yoshihara,

Y. Kawahara, Y. Yamada).

121 M.Karasawa,O. Tosaka, S. Ikeda, H. Yoshi,Agric. Biol.

Chem. 50 (1986) 339–346.

122 Kyowa Hakko, US 4 623 623, 1986 (T. Nakanashi

et al.).

123 Kawahara et al., Appl. Microbiol. Biotechnol. 34 (1990)

87–90.

124 Ajinomoto, US 5 250 423, 1993 (Y. Murakami,

H. Miwa, S. Nakamori).

125 A. Erdmann, B. Weil, R. Kr€amer, Appl. Microbiol.

Biotechnol. 42 (1994) 604–610.

126 A. Erdmann, B. Weil, R. Kr€amer, Biotechnol. Lett. 17

(1995) 927–932.

127 R. Kr€amer, J. Biotechnol. 45 (1996) 1–21.

128 M. S. N. Jetten, A. J. Sinskey, Crit. Rev. in Biol. 15

(1995) 73–103.

129 Kyowa Hakko, EP-A 197 335, 1986 (R. Katsumata,

T. Mitzukami, T. Oka).

130 Forsch. Z. J€ulich, EP-A 435 132, 1991 (J. Cremer,

L. Eggeling, H. Sahm).

131 Y.-C. Liu, W.-T. Wu, J.-H. Tsao, Bioprocess. Eng. 9

(1993) 135–139.

132 Eurolysine, EP-A 534 865, 1993 (P. Lucq, C. Domont).

133 Rhone-Polenc, EP 122 163, 1984 (N. Rouy).

134 Degussa, EP 533 039, 1993 (W. Binder et al.).

135 Degussa, DE 2 421 167, 1974 (T. L€ußling, K. M€uller,

G. Schreyer, F. Theissen).

136 W. Leuchtenberger, U. Pl€ocker: ‘‘Amino acids and

hydroxycarboxylic acids,’’ in W. Gerhartz (ed.):

Enzymes in Industry, Production and Applications,

VCH, Weinheim, Germany 1990.

137 T. Ishikawa et al., Biosci. Biotech. Biochem. 57 (1993)

982–986.

138 Genencor, WO 93/17112, 1993 (J. C. Lievense).

139 Genex, US 4 598 047, 1986 (J. C. McGuire).

140 C. T. Evans, C. Coma, W. Petreson, M. Misawa,

Biotechnol. Bioeng. 30 (1987) 1067–1072.

141 G. J. Calton et al., Biotechnology 4 (1986) 317–320.

142 C. T. Evans et al., Biotechnology 5 (1987) 818–923.

143 A. J. Pittard, Amer. Soc. Microbiol. Conf. Proc. (1987)

368–394.

144 I. Shiio: ‘‘Tryptophan, phenylalanine, and tyrosine,’’ in

K. Aida, I. Chibata, K. Nakayama, K. Takinami,

H. Yamada (eds.): Biotechnology of Amino Acid Pro-

duction, Kodansha, Tokyo/Elsevier, Amsterdam 1986,

pp. 188–206.

145 S. O. Hwang et al., Appl. Microbiol. Biotechnol. 22

(1985) 108–113.

146 H. Ito et al., Agric. Biol. Chem. 54 (1990) 707–713.

147 B. K. Konstantinov, N. Nishio, T. Yoshida: ‘‘Glucose

feeding strategy accounting for the decreasing oxidative

capacity of recombinant Escherichia coli in fed-batch

cultivation for phenylalanine production, ’’ J. Ferment.

Bioeng. 70 (1990) 253.

148 Ajinomoto, US 4 584 400, 1986 (M. Ootani, S. Sano, I.

Kusumotu).

149 A. M. Vasconcellos, A. L. Neto, D. M. Grassiano, C. P.

De Oliveira, Biotechnol. Bioeng. 33 (1989) 1324–1329.

150 Ajinomoto, US 5 362 635, 1994 (T. Hirose et al.).

151 F. Yoshinaga: ‘‘Proline,’’ in K. Aida, I. Chibata, K.

Nakayama, K. Takinami, H. Yamada (eds.):Biotechnol-

ogy of Amino Acid Production, Kodansha, Tokyo/Else-

vier, Amsterdam 1986, pp. 117–120.

152 M. Sugiura, T. Takagi, M. Kisumi, Abst. 31st Symp.

Amino Acid and Nucleic Acid (Japan), 1982, p. 10.

153 Y. Imai, T. Takagi, M. Sugiura, M. Kisumi, Abst. Ann.

Meet Agr. Chem. Soc. (Japan), 1984, p. 100.

154 Y. Izumi et al., Appl. Microbiol. Biotechnol. 39 (1993)

427–432.

155 T. Yoshida, T. Mitsunaga, Y. Izumi, J. Fermentation

Bioeng. 75 (1993) 405–408.

156 H.-Y. Hsiao, T. Wie, Biotechnol. Bioeng. 28 (1986)

1510–1518.

157 Mitsui Toatsu, US 5 382 517, 1995 (D. Ura, T. Hashi-

mukai, T. Matsumoto, N. Fukuhara).

158 K. Shimura: ‘‘Threonine,’’ in H.W. Blanch, S. Drew, D.

I. C.Wang (eds.):ComprehensiveBiotechnology, vol. 3,

Pergamon Press, Oxford 1985.

159 S. Nakamori: ‘‘Threonine and Homoserine,’’ in K. Aida,

I. Chibata, K. Nakayama, K. Takinami, H. Yamada

(eds.): Progress in Industrial Microbiology, Kodansha,

Tokyo/Elsevier, Amsterdam 1986, pp. 173–182.

160 Genetics Ind., US 4 278 765, 1981 (V. G. Debabov

et al.).

161 Ajinomoto, EP-A 593 792, 1994 (V. G. Debabov et al.).


162 Eurolysine, FR 2 627 508, 1989 (F. Richaud et al.).

163 Ajinomoto, FR 2 588 016, 1987 (M. Ootani, T. Kita-

hara, K. Akashi).

164 A. Yokota, T. Takao, Agric. Biol. Chem. 48 (1984)

2663–2668.

165 Mitsui Toatsu, EP-A 341 674, 1989 (K. Ishiwata et al.).

166 Mitsui Toatsu, EP-A 438 591, 1991 (S. Ogawa, S.

Iguichi, S. Morita, H. Kuwamoto).

167 B. K. Hamilton et al., Trends Biotechnol. 3 (1985)

64–68.

168 J. Plachy, S. Ulbert, Acta Biotechnol. 10 (1990)

517–522.

169 Showa Denko, JP 62 186 786, 1987 (E. Takinishi, H.

Takamatsu, K. Sakimoto, Y. Yajima).

170 A. N. Mayeno, G. J. Gleich, TIBTECH 12 (1994)

346–352.

171 K. Sakimoto, Y. Torigoe, Curr. Prospects Med. Drug

Saf. (1994) 295–311.

172 K. Nakayama: ‘‘Tryptophan,’’ in M. Moo-Young (ed.):

Comprehensive Biotechnology, vol. 3, Pergamon Press,

Oxford 1985, 621–631.

173 I. Shiio: ‘‘Tryptophan, Phenylalanine, and Tyrosine,’’ in

K. Aida, I. Chibata, K. Nakayama, K. Takinami,

H. Yamada (eds.): Biotechnology of Amino Acid

Production, Kodansha, Tokyo/Elsevier, Amsterdam

1986, pp. 188–206.

174 T. K. Maiti, S. P. Chatterje, Hindustan Antibiotics

Bulletin 33 (1991) 26–61.

175 M. Ikeda, Appl. Microbiol. Biotech. 69 (2006) 615.

176 S. Komatsubara, M. Kisumi: ‘‘Isoleucine, Valine, and

Leucine,’’ in K. Aida, I. Chibata, K. Nakayama,

K. Takinami, H. Yamada (eds): Biotechnology of Amino

Acid Production, Kodansha, Tokyo/Elsevier, Amster-

dam 1986, pp. 131–143.

177 Ajinomoto, EP 287 123, 1993 (N. Katsurada, H. Uchi-

bori, T. Tsuchida).

178 D. A. Bender: Amino Acid Metabolism, J. Wiley &

Sons, London–New York–Sydney–Toronto 1975, p. 2.

179 H. N. Munro, Drug Intell. Clin. Pharm. 6 (1972) 216.

180 M. L. Sunde, Poult. Sci. 51 (1972) 44.

181 R. F. Barker, D. A. Hopkinson, Ann. Hum. Genet. 41

(1977) 27.

182 L. D. Stegink: Clinical Nutrition Update: Amino Acids,

Am. Med. Assoc. Publ., Chicago 1977, pp. 198–205.

183 K. Schreier, €Arztl. Fortbildung 19 (1971) 107.

184 U. Porath, K. Schreier, Med. Ern€ahr. 11 (1970) 229.

185 H. Bickel, S. Kaiser-Grubel, Dtsch. Med. Wochenschr.

96 (1971) 1415.

186 Y. Izumi, I. Chibata, T. Itoh, Angew. Chem. 90 (1978)

187; Angew. Chem. Int. Ed. Eng. 17 (1978) 176.

187 M. S. Sadovnikova, V. M. Belikov, Russ. Chem. Rev.

(Engl. Transl.) 47 (1978) 199.

188 K. Drauz, A. Kleemann, J. Martens, Angew. Chem. 94

(1982) 590; Angew. Chem. Int. Ed. Engl. 21 (1982) 584.

189 Winnacker-K€uchler, 5th ed., vol. 8, 715.

190 FAO:Nutritional Studies No. 24, Amino Acid Content of

Foods and Biological Data on Proteins, Interprint (Mal-

ta), Rome 1970.

191 Degussa, unpublished

192 W. C. Rose, Nutr. Abstr. Rev. 27 (1957) 631.

193 D. M. Hegsted, Fed. Proc. Fed. Am. Soc. Exp. Biol. 22

(1963) 1424.

194 Report of a Joint FAO/WHOAdHocExpert Committee:

Energy and Protein Requirements, Rome 1973.

195 E. Kofr�anyi, Ern€ahr. Umsch. 23 (1976) 205.

196 Dtsch. Ges. f€ur Ern€ahrung: Empfehlungen f€ur die

N€ahrstoffzufuhr, 3rd ed., Umschau-Verlag, Frankfurt

1975.

197 Dtsch. Ges. F€ur Ern€ahrung: Referenzwerte f€ur die

N€ahrstoffzufuhr, Umschau Buchverlag, Frankfurt 2000.

198 Recommended Dietary Allowances, 9th ed., Nat. Acad.

of Sci., Washington, D.C., 1980.

199 J. J. Otten, J. P. Hellwig, L. D. Meyers (eds.): Dietary

Reference Intakes: the Essential Guide to Nutrient Re-

quirements, National Academies Press, Washington DC

2006.

200 G. K. Parman, J. Agric. Food Chem. 16 (1968) 169.

201 H.H. Ottenheym, P. J. Jenneskens, J. Agric. FoodChem.

18 (1970) 1010.

202 N. S. Scrimshaw, A. M. Altschul: Amino Acid Fortifica-

tion of Protein Foods, MIT Press, Cambridge,MA1971.

203 E. E. Howe, G. R. Jansen, E. W. Gilfilian, Am. J. Clin.

Nutr. 16 (1965) 315.

204 A. M. Altschul, Nature (London) 248 (1974) 643.

205 J. Kato, N. Muramatsu, J. Am. Oil Chem. Soc. 48 (1971)

415.

206 J. Mauron, Z. Ern€ahrungswiss. Suppl. 23 (1979) 10.

207 D. M. Hegsted, Am. J. Clin. Nutr. 21 (1968) 688.

208 L. D. Satterlee, H. F. Marshall, J. M. Tennyson, J. Am.

Oil Chem. Soc. 56 (1979) 103.

209 H. W. Staub, Food Technol. (Chicago) 32 (1978) 57.

210 F. Jekat, Fette Seifen Anstrichm. 79 (1977) 273.

211 J. C. Somogyi in:Die Bedeutung der Eiweiße in unserer

Ern€ahrung, Issue 48 a, Schriftenreihe der Schweizer

Vereinigung f€ur Ern€ahrung, 1982, p. 3.

212 G. G. Graham et al., Am. J. Clin. Nutr. 22 (1969) 1459.

213 G. R. Jansen, C. F. Hutchison, M. E. Zanetti, Food

Technol. (Chicago) 20 (1966) 323.

214 R. Bressani et al., J. Nutr. 79 (1963) 333.

215 J. L. Iwan, Cereal Sci. Today 13 (1968) 202.

216 D. Rosenfield, F. J. Stare, Mod. Gov. 11 (1970) 47.

217 S. N. Gershoff et al., Am. J. Clin. Nutr. 30 (1977) 1185.

218 H. N. Parthasarathy et al., Can. J. Biochem. 42 (1964)

385.

219 S. J. Fomon et al., Am. J. Clin. Nutr. 32 (1979) 2460.

220 A. L. Jung, S. L. Carr, Clin. Pediatr. (Philadelphia) 16

(1977) 982.

221 Procter & Gamble, US 3878305, 1975 (R. A. Damico,

R. W. Boggs).

222 S. Wallrauch, Fl€uss. Obst 44 (1977) 386.

223 H. D. Pruss, I. P. G. Wirotama, K. H. Ney, Fette Seifen

Anstrichm. 77 (1975) 153.

224 C. Ambrosino et al., Minerva Pediatr. 18 (1966) 759.

225 C. A. Masschelein, J. Van de Meerssche, Tech. Q.

Master Brew. Assoc. Am. 13 (1976) 240.

226 T. Take, H. Otsuka, Chem. Abstr. 70 (1969) 46270 m.


227 J. Koch, Fl€uss. Obst 46 (1979) 212.

228 G. Baumann, K. Gierschner, Dtsch. Lebensm. Rundsch.

70 (1974) 273.

229 A. Askar, H. J. Bielig, Alimenta 15 (1976) 3.

230 W. Hashida, Food Trade Rev. 44 (1974) 21.

231 Food Technol. (Chicago) 34 (1980) 49.

232 B. Lindemann, Nat. Neurosci. 3 (2000) 99.

233 S. Schwimmer, D. G. Guadagni, J. Food Sci. 32 (1967)

405.

234 Riken Kagaku, JP 7249707, 1972 (H. Watanabe et al.);

Chem. Abstr. 79 (1973) 114219 q.

235 Pillsbury Comp., US 3510310, 1970.

236 C. Colburn, Am. Soft Drink J. 126 (1971) 16.

237 R. S. Shallenberger, T. E. Acree, C. Y. Lee, Nature

(London) 221 (1969) 556.

238 J. Solms, L. Vuataz, R. H. Egli, Experientia 21 (1965)

693.

239 H. Wieser, H. Jugel, H.-D. Belitz, Z. Lebensm. Unters.

Forsch. 164 (1977) 277.

240 R. H. Mazur, J. M. Schlatter, A. H. Goldkamp, J. Am.

Chem. Soc. 91 (1969) 2684.

241 L. A. Pavlova et al., Russ. Chem. Rev. (Engl. Transl.) 50

(1981) 316.

242 W. J. Herz, R. S. Shallenberger, Food Res. 25 (1960)

491.

243 Lever Brothers, US 3922365, 1975 (K. H. Ney et al.).

244 H. Tanaka, Y. Obata, Agric. Biol. Chem. 33 (1969) 147.

245 M. Giaccio, L. Surricchio, Quad. Merceol. 16

(1977) 151.

246 H. Valaize, G. Dupont, Ind. Agric. Aliment. 68 (1951)

245.

247 E. L. Wick, M. DeFigueiredo, D. H. Wallace, Cereal

Chem. 41 (1964) 300.

248 A. A. M. El-Dash, Dissertation, Kansas State University

1969 (Univ. Microfilms Inc., Ann Arbor, Michigan,

No. 69-21123).

249 W. Baltes, Ern€ahr. Umsch. 20 (1973) 35.

250 M. Angrick, D. Rewicki, Chem. Unserer Zeit 14 (1980)

149.

251 H. E. Nurstein, Food Chem. 6 (1980) 263.

252 T. A. Rohan, Food Technol. (Chicago) 24 (1970) 29.

253 Maggi, DE 2246032, 1973 (R. J. Gasser). Z.Mielniczuk

et al., Acta Aliment. Pol. 2 (1976) 213. Chas. Pfizer &

Co., US 3365306, 1968 (M. A. Perret). Y.-P. C. Hsieh

et al., J. Sci. Food Agric. 31 (1980) 943. R. A.Wilson, J.

Agric. Food Chem. 23 (1975) 1032. P. van de Rovaart,

J.-J. Wuhrmann, US 3930044, 1975. R. Schroetter, G.

Woelm, Nahrung 24 (1980) 175.

254 H. Kisaki, Chem. Abstr. 69 (1968) 9824 d. Ajinomoto,

FR 2005896 (1969). G. Rubenthaler, Y. Pomeranz, K.

F. Kinney, Cereal Chem. 40 (1963) 658. I. R. Hunter,

M. K.Mayo, US 3425840, 1969. Y. H. Liau, C. C. Lee,

Cereal Chem. 47 (1970) 404. Y.-Y. Linko, J. A. John-

son, B. S. Miller, Cereal Chem. 39 (1962) 468. G. L.

Bertram, Cereal Chem. 30 (1953) 126. Research Corp.,

US 3268555, 1966 (L.Wiseblatt). Hoffmann-La Roche,

US 3547659, 1970 (W. Cort, L. Neck). M. Rothe, Nah-

rung 24 (1980) 185. A. A. El-Dash, A. A. Johnson,

Cereal Chem. 47 (1970) 247. L. Wiseblatt, H. F. Zou-

mut, Cereal Chem. 40 (1963) 162. M. Rothe, Ern€ah-

rungsforschung 5 (1960) 131.

255 G. Ziegleder, D. Sandmeier, Dtsch. Lebensm. Rundsch.

78 (1982) 315.W. Mohr, E. Landschreiber, Th. Severin,

Fette Seifen Anstrichm. 78 (1976) 88. S. Turos,

US 4346121, 1982.

256 E. Cremer, M. Riedmann, Monatsh. Chem. 96 (1965)

364.

257 Yuki Gosei Kogyo, US 3478015, 1969 (I. Onishi, A.

Nishi, T. Kakizawa).

258 Fuji Oil Co., GB 1357511, 1974; GB 1488282, 1977.

259 Naarden Int., NL 7712745, 1979.

260 J. A. Newell,M. E.Mason, R. S.Matlock, J. Agric. Food

Chem. 15 (1967) 767.

261 S.-C. Lee, B. R. Reddy, S. S. Chang, J. Food Sci. 38

(1974) 788. Research Corp., US 3814818, 1974 (S. S.

Chang, B. R. Reddy). T. Y. Fan, M. H. Yueh, J. Food

Sci. 45 (1980) 748. P. T. Arroyo, D. A. Lillard, J. Food

Sci. 35 (1970) 769.

262 Yuki Gosei Kogyo, DE 1593733, 1972 (I. Onishi et al.).

Japan Monopoly, Tanabe Seiyaku, US 3722516,

1973 (K. Suwa et al.). Philip Morris, US 4306577,

1981 (D. L. S. Wu, J. W. Swain). Reynolds Tobacco,

US 3996941, 1976 (C. W. Miller, J. P. Dickerson,

C. E. Rix).

263 R. G. Henika, N. E. Rodgers, Cereal Chem. 42 (1965)

397.

264 J. M Bruemmer, W. Seibel, H. Stephan, Getreide Mehl

Brot 34 (1980) 173. Patent Technology, US 3803326,

1974. J. Geittner, Gordian 79 (1979) 202.

265 R. Marcuse, Fette Seifen Anstrichm. 63 (1961) 940.

266 H. Iwainsky, C. Franzke, Dtsch. Lebensm. Rundsch. 52

(1956) 129. H. Lingnert, C. E. Eriksson, J. Food

Process. Preserv. 4 (1980) 161.

267 N. Watanabe et al., JP 7314042, 1973; Chem. Abstr. 79

(1973) 145052 j.

268 A. G. Castellani, Appl. Microbiol. 1 (1953) 195. Nisshin

Flour Milling, JP 7319945, 1973(G. Ogawa, K. Tagu-

chi); Chem. Abstr. 81 (1974) 76689 z. Nippon Kayaku,

JP-Kokai 81109580, 1981; Chem. Abstr. 95 (1981)

202313 b.

269 US Functional Beverages Market, Frost & Sullivan,

London, 2004.

270 Ajinomoto Co. Inc.: Amino Acid Link News, No. 14

(May 2006) . Available at http://www.ajinomoto.com/

aminoacid/link/E/pdf/Vol.14E.pdf.

271 M. Pack: Amino Acids in Animal Nutrition, Coral Sani-

vet, Bucharest 2002.

272 M. Kirchgeßner: Tierern€ahrung, 11th ed., DLG-Verlag,

Frankfurt 2004.

273 Nutrient Requirements of Poultry, 9th revised ed., Na-

tional Academies Press, Washington DC 1994.

274 Nutrient Requirements of Swine, 10th revised ed., Na-

tional Academies Press, Washington DC 1998. P. L. M.

Berende, H.-L. Bertram, Z. Tierphysiol. Tierern€ahr.

Futtermittelk. 49 (1983) 30. H.-L. Bertram, P. L. M.

Berende, Kraftfutter (1983) 46.


275 Degussa Feed Additives: available at http://www.ami-

noacidsandmore.com.

276 M. L. Scott, M. C. Nesheim, R. J. Young: Nutrition of

the Chicken, 3rd ed., M. L. Scott, Ithaka, New York,

1982.

277 W. Prinz, A. Becker, Arch. Gefl€ugelk. 29 (1965) no. 2,

135.

278 R. Chernoff, J. Am. Diet. Assoc. 79 (1981) 426. M. R.

Polk, Am. Pharm. NS 22 (1982) 25.

279 W. F. Caspary, Dtsch. €Arztebl. 1978 (2. Febr.) 243. H.

Kasper, Aktuel. Ern€ahrung 1 (1978) 22.

280 D. M. Matthews, S. A. Adibi, Gastroenterology 71

(1976) 151. M. T. Lis, R. F. Crampton, D.M.Matthews,

Br. J. Nutr. 27 (1972) 159.

281 Mead Johnson, US 3091569, 1963; US 3184505, 1965.

282 H. A. Smith, G. Gorin, J. Org. Chem. 26 (1961) 820.

283 A. Kleemann, J. Engel: Pharmaceutical Substances, 4th

ed., Thieme Verlag, Stuttgart 2001.

284 Rech. et Propagande Scientif., FR 1288907, 1962.

285 Degussa, US 4129593, 1978.


287 M. Bergmann, G. Michalis, Ber. Dtsch. Chem. Ges. 63

(1930) 987.

288 Degussa, DE 1239697, 1963.

289 May & Baker, GB 845 034, 1957.

290 B. Witkop et al., J. Am. Chem. Soc. 76 (1954) 5579.

291 A. J. Morris et al., J. Org. Chem. 22 (1957) 306.

292 E. A. Bell et al., Nature (London) 210 (1966) 529.

293 Distillers, GB 854339, 1957. Squibb, US 3281461,

1966. Heyl & Co., DE 2114329, 1971; DE 2413185,

1974.

294 Degussa, DE 1795299, 1968; DE 1795297, 1968; DE

2032952, 1970; DE2123232, 1971; DE2156601, 1971;

DE 2335990, 1973; DE 2138122, 1971; DE 2258411,

1972; DE 2304055, 1973.

295 K. Schrader, Am. Cosmet. Perfum. 87 (1972) 49. S.

Tatsumi, Am. Cosmet. Perfum. 87 (1972) 61. O. K.

Jacobi, Am. Cosmet. Perfum. 87 (1972) 35. G. Hopf, J.

K€onig, G. Padberg, Kosmetologie 4 (1971) 132. A.

Szakall, Arch. Klin. Exp. Dermatol. 201 (1955) 331. K.

Laden, R. Spitzer, J. Soc. Cosmet. Chem. 18 (1967) 351.

H. W. Spier, G. Pascher, Klin. Wochenschr. 31 (1953)

997.

296 Y. Kumano et al., J. Soc. Cosmet. Chem. 28 (1977) 285.

297 L’Ore�al, DE 2807607, 1978 (J.-C. Ser et al.). Unilever,

DE 2337342, 1973 (G. F. Johnston et al.). Orlane Paris,

DE2524297, 1975 (A.Meybeck,H.Noel). Shiseido,US

4035513, 1977 (Y. Kumano).

298 E. S. Cooperman, Am. Cosmet. Perfum. 87 (1972) 65.

Colgate Palmolive, GB 1573529, 1980.

299 Proctor and Gamble: Press Release, 23 August 2004;

available at http://www.uk.pg.com.

300 C. R. Robbins: Chemical and Physical Behaviour of

Human Hair, Springer Verlag, Berlin 1994.

301 European Markets for Active Ingredients in Personal

Care, Frost & Sullivan, London, 2004.

302 M. Takehara et al., J. Am. Oil Chem. Soc. 50 (1973) 227;

51 (1974) 419.

303 Ajinomoto, Kawaken Fine Chemicals, US 4273684,

1981 (T. Nagashima et al.)

304 Ajinomoto, GB 1483500, 1977.

305 Kawaken Fine Chemicals. JP-Kokai 75117806, 1975

(K. Nakazawa); Chem. Abstr. 84 (1976) 8858 r.

306 G. Schuster, H. Modde, E. Scheld, Seifen Ole Fette

Wachse 38 (1965) 477. G. Schuster, H. Modde, Parf€umKosmet. 45 (1964) 337.

307 Estee Lauder, US 4005210, 1977 (J. Gubernick).

308 American Cyanamid, US 3988438, 1976. Ajinomoto,

DE 2010303, 1970 (R. Yoshida et al.).

309 R. Yoshida, M. Takehara, Chem. Abstr. 84 (1976)

6843 h.

310 M. Koyama, J. Dispersion Sci. Tech. 26 (2005) 785.

311 Dominion Pharmacal., US 4176197, 1979 (B. N. Olson).

L’Or�eal,US4002634, 1977 (G.Kalopissis, C.Bouillon).

L’Or�eal, GB 1397623, 1975 (G. Kalopissis, G. Man-

oussos). L’Or�eal, DE 1492071, 1965 (G. Kalopissis).

312 Mare Corp., US 4201235, 1980 (V. G. Ciavatta). Uni-

lever, EP 8171, 1981 (G. P. Mathur et al.).

313 Kyowa Hakko Kogyo, US 4139610, 1979 (Y. Miyazaki

et al.) Hans Schwarzkopf, DE 958501, 1957 (J. Saphir,

E. Kramer). K. Yoneda et al., DE 2951923, 1979.

314 Schuylkill Chem., GB 1516890, 1978.

315 Procter & Gamble, EP 47650, 1982. Unilever, GB

1597498, 1981 (K. Gosling, M. R. Hyde).

316 Sederma, US 6974799, 2003 (K. Lintner).

317 Sederma, WO 98/07744, 1998 (D. Greff).

318 C. Tomlin (ed.): The Pesticide Manual, 13th ed., British

Crop Protection Council, Farnham 2003.

319 B. Hock, C. Fedtke, R. R. Schmidt: Herbizide, 1st ed.,

Georg Thieme Verlag Stuttgart 1995.

320 Proc. North. Cent. Weed Control Conf. 26 (1971) 64.

321 P. Knuuttila, H. Knuuttila, Acta Chem. Scand. 33 (1979)

623.

322 Agrochemical Service, Wood Mackenzie Consultants

Limited, Edinburgh 1996.

323 Zeneca, US 4 315 765.

324 Z. Pflanzenkr. Pflanzenschutz (1981) Sonderheft IX,

431.

325 Hoechst, DE-OS 2 717 440, 1976.

326 K. Tachibana et al., Abstr. 5th Int. Congr. Pestic. Chem.,

IVa Abstract 19.

327 D. W. Ladner, Pestic. Sci. 29 (1990) 341–356.

328 M. J. Muhitch, D. L. Shaner, M. A. Stidham, Plant

Physiol. 83 (1987) 451–456.

329 T.Chapman et al.,Symp.New.Herbic. 3rd ed. (1969) 40.

330 R. M. Scott et al., Proc. Br. Crop. Prot. Conf.–Weeds 2

(1976) 723.

331 S. O. Duke et al., Pestic. Sci. 40 (1994) 265–277.

332 N. Mito, R. Sato, M. Miyakado, H. Oshio, S. Tanaka,

Pestic. Biochem. Physiol. 40 (1991) 128–135.

333 Degussa, WO 94/03458, 1994 (M. Sch€afer, H. Baier, K.

Drauz, H.-P. Krimmer, S. Landmann).

334 Sumitomo, JP 62 158 280.

335 BASF, DE 409 432, 1994.

336 BASF, DE 407 023, 1994.

337 American Cyanamid Co., EP 101 098, 1993.


338 Bayer, DE 030 062, 1990.

339 S. Sauza et al., Phytoma–la d�efense des vegetaux 558

(2003) 51.

340 Y. Miyake et al., Nippon Noyaku Gakkaishi 30 (2005)

390.

341 P. A. Urech, Proc. Br. Crop. Prot. Conf.–Pests Dis. 2

(1977) 623.

342 Garavaglia et al.: Atti Simp. Chim. Antiparassitari, 3rd,

Piacenza 1981.

343 C. A. Henrick et al., Pestic. Sci. 11 (1980) 224.

344 C. A. Porter, L. E. Ahlrichs, Hawaii Sugar Tech. Rep.

1971 30 (1972) 71.

345 Ciba Geigy, EP 18948, 1980.

346 Emori Shoji, JP 78097022, 1978.

347 Dainichi Seika Kogy, JP 78079990, 1978.

348 L. Mueller, Zem. Kalk Gips 27 (1974) 69.

349 Ajinomoto, DE 2533136, 1975 (R. Yoshida et al.).

350 Kyowa Fermentation, GB 1400741, 1975. Honny Che-

micals, GB 1402758, 1975 (Y. Nakagoshi). Kyowa

Hakko Kogyo, DE 2229488, 1972 (Y. Fujimoto et al.).

Toyo Cloth, US 3676206, 1972 (K. Nishitani et al.).

351 Sanyo Trading Co., DE 2716758, 1977 (M. Onizawa,

S. Ohmiya).

352 Ajinomoto, JP 81035179, 1981. Ajinomoto, JP-Kokai

7522801, 1975 (T. Saito et al.); Chem. Abstr. 84 (1976)

33494 b.

353 Sanyo Trading Co., DE 2602988, 1976; US 4069213,

1978; DE 2604053, 1976 (M. Onizawa); DE 2658693,

1976 (M. Onizawa, S. Ohmiya).

354 R.M. Saleh, A.M. Shams El Din,Corros. Sci. 12 (1972)

688.

355 Ajinomoto, JP-Kokai 7426145, 1974 (Y. Kita et al.);

Chem. Abstr. 81 (1974) 53195 w.

356 Ilford, GB 1378354, 1974 (A. D. Ezekiel). Ilford, DE

2316632, 1973 (R. Jefferson).

357 Fr. Blasberg & Co., DE 2050870, 1973 (W. Immel, W.

Adams). Yokozawa Chemical Ind., JP 7410572, 1974

(K. Aoya et al.); Chem. Abstr. 81 (1974) 57631 h. Sony

Corp., DE 2325109, 1973 (S. Fueki et al.).

358 A. Steponavicius, R.Visomirskis,Electrodepos. Surface

Treat. Lausanne 1 (1972) 37.

359 F. S. Parker, D.M. Kirschenbaum, Spectrochim. Acta 16

(1960) 910. M. Tsuboi, T. Takenishi, A. Nakamura,

Spectrochim. Acta 19 (1963) 271. J. F. Pearson, M. A.

Slifkin, Spectrochim. Acta Part A 28 (1972) 2403.

360 C. S. Tsai et al.,Can. J. Biochem. 53 (1975) no. 9, 1005.

361 H. Hagenmeyer et al., Z. Naturforsch. B: Anorg. Chem.

Org. Chem. 25 B (1970), 681.

362 C. Cooper, W. Packer, K. Williams: Amino Acid Analy-

sis Protocols, Methods in Molecular Biology Series,

159, Humana Press, Totowa, NJ 2000.

363 W. S. Hancock: Handbook for the Separation of Amino

Acids, Peptides and Proteins, vol. 1, CRC Press, Boca

Raton, FL, 1984.

364 E. Scoffone, A. Fontana,Mol. Biol. Biochem. Biophys. 8

(1970) 185.

365 M. Roth, Anal. Chem. 43 (1971) 880. J. R. Benson, P. E.

Hare, Proc. Natl. Acad. Sci. USA 72 (1975) 619.

366 E. Bayer et al., Anal. Chem. 48 (1976) 1106.

367 S. Udenfried et al., Science (Washington, D.C.) 178

(1972) 871.

368 I. Moln�ar-Perl:Quantitation of Amino Acids and Amines

by Chromatography, Elsevier, Oxford, 2005.

369 C. Haworth, R. W. A. Oliver, J. Chromatogr. 64 (1972)

305. E. Stahl: D€unnschichtchromatographie, SpringerVerlag, Berlin 1967, p. 701. A. R. Fahmy et al., Helv.

Chim. Acta 44 (1959) 245.

370 M. Kubota et al., Anal. Biochem. 64 (1975) no. 2, 494.

K. D. Kulbe, Anal. Biochem. 44 (1970) 548. P. A.

Laursen, Biochem. Biophys. Res. Commun. 37 (1969)

663.

371 A. Wolf, Prax. Naturwiss. Chem. 23 (1974) no. 3, 74.

372 H.Nakamura, J. J. Pisano, J. Chromatogr.121 (1976) 33.

373 K.Macek, Z. Deyl, M. Smr�z, J. Chromatogr. 193 (1980)

421.

374 P. B. Hamilton, Anal. Chem. 35 (1963) 2055.

375 S. Blackburn: Amino Acid Determination, Marcel

Dekker, New York 1978.

376 D. H. Spackman,W. H. Stein, S. Moore, Anal. Chem. 30

(1958) 1190.

377 S. Moore, W. H. Stein, J. Biol. Chem. 211 (1954) 907.

378 H.-M. Lee et al., Anal. Biochem. 96 (1979) 298.

379 P. Chaimbault et al., J. Chromatogr. A, 870 (2000) 245.

380 J. Weiss: Handbook of Ion Chromatography, Wiley-

VCH, Weinheim 2004.

381 E. Bayer et al., Anal. Chem. 48 (1976) 1106. J. M.

Wilkinson, J. Chromatogr. Sci. 16 (1978) 547. K.-T.

Hsu, B. L. Currie, J. Chromatogr. 166 (1978) 555. T.

Jamabe, N. Takei, H. Nakamura, J. Chromatogr. 104

(1975) 359. A. Khayat, P. K. Redenz, L. A. Gorman,

Food Technol. (Chicago) 36 (1982) 46.

382 A. P. Graffeo,A.Haag, B. L. Karger,Anal. Lett. 6 (1973)

no. 6, 505. J. K. De Vries, R. Frank, C. Birr, FEBS Lett.

55 (1975) no. 1, 65. P. Frankhauser et al., Helv.

Chim. Acta 57 (1974) 271. C. C. Zimmermann,

E. Appella, J. J. Pisano, Anal. Biochem. 77 (1977)

569. G. Frank, W. Strubert, Chromatographia 6

(1973) no. 12, 522.

383 H. Beyer, U. Schenk, J. Chromatogr. 89 (1969) 483.

384 T. A. Kan, W. F. Shipe, J. Food Sci. 47 (1981) 338.

H. Umagat, P. Kucera, L. F. Wen, J. Chromatogr. 239

(1982) 463.

385 W. Voelter, K. Zech, J. Chromatogr. 112 (1975) 643.

386 R. Schuster, Anal. Chem. 52 (1980) 617.

387 A.Darbre,Biochem. Soc. Trans. 2 (1974) 70. B.M.Nair,

J. Agric. Food Chem. 25 (1977) 614.

388 J. J. Pisano, T. J. Bronzert, H. B. Brewer,Anal. Biochem.

45 (1972) 43.

389 A. Darbre, A. Islam, Biochem. J. 106 (1968) 923.

390 C. W. Gehrke, R. W. Zumwalt, K. Kuo, J. Agric. Food

Chem. 19 (1971) 605.

391 K. Ruhlmann, W. Giesecke, Angew. Chem. 73 (1961)

113.

392 D. L. Stalling, C. W. Gehrke, R. W. Zumwalt, Biochem.

Biophys. Res. Commun. 31 (1968) 4.

393 M. W. Duncan, A. Poljak, Anal. Chem. 70 (1998) 890.


394 W. Grassmann, K. Hannig, Houben-Weyl, 1/1, 708.

395 V Poinsot et al., Electrophoresis 27 (2006) 176.

396 T. Soga, D. N. Heiger, Anal. Chem. 72 (2000) 1236.

397 I. Abe, S. Musha, J. Chromatogr. 200 (1980) 195. G. J.

Nicholson, H. Frank, E. Bayer, HRC CC J. High

Resolut. Chromatogr. Chromatogr. Commun. 2 (1979)

411. W. A. K€onig, G. J. Nicholson, Anal. Chem. 47(1975) 951.

398 W. Lindner, Chimia 35 (1981) 294. V. A. Davankov

et al., Chromatographia 13 (1980) no. 11, 677.

399 P. E. Hare, E. Gil-Av, Science (Washington, D.C.) 204

(1979) 1226.

400 C.B’Hymer,M.Montes-Bayon, J. A.Caruso, J. Sep. Sci.

26 (2003) 7.

401 R. Bhushan, H. Bruckner, Amino Acids 27 (2004)

231.

402 L. M. Henderson, E. E. Snell, J. Biol. Chem. 172 (1947)

15. I. Grote, M€uhle Mischfuttertech. 116 (1979) 465.

403 H. Itoh, T. Morimoto, I. Chibata, Anal. Biochem. 60

(1974) 573.

404 Ch. Calvot et al., FEBS Lett. 59 (1975) no. 2, 258. S. J.

Updike, G. P. Hicks, Nature (London) 214 (1967) 986.

405 Ajinomoto, FR 2421380, 1979.

406 European Pharmacopoeia, 5th ed., European Director-

ate for the Quality of Medicine, Strasbourg 2005.

407 W. Seaman, E. Allen, Anal. Chem. 23 (1951) no. 4, 592.

H. P. Deppeler, G. Witthans, Fresenius Z. Anal. Chem.

305 (1981) 273.

408 United States Pharmacopeia, 29th ed.,USPharmcopeial

Convention, Inc., Rockville, MD 2006.

409 Food Chemicals Codex, 5th ed., National Academies

Press, Washington, D.C. 2003.

410 Ajinomoto: Amino Acids Business, 2005 (available at

http://www.ajinomoto.com).

411 E. O. Camara Greiner, R. Gubler, K. Yokose: Market

Research Report: Amino Acids, SRI International, Men-

lo Park, CA, 2003.

412 European Amino Acids Market, Frost & Sullivan, Lon-

don, 2006.

413 K. Brown: Amino Acids: Highlighting Synthesis Appli-

cations, Business Communications Co. Inc., Norwalk,

CT 2005.

414 H. N. Munro, Adv. Exp. Med. Biol. 105 (1978) 119.

415 A. E. Harper, N. J. Benevenga, R. M. Wohlhueter,

Physiol. Rev. 50 (1970) 428.

416 R. G. Daniel, H. A. Waisman, Growth 32 (1968) 255.

417 H. E. Sauberlich, J. Nutr. 75 (1961) 61.

418 K. Lang: Biochemie der Ern€ahrung, 4th ed., Steinkopff,

Darmstadt 1979.

419 Y. Peng et al., J. Nutr. 103 (1973) 608.

420 Degussa, unpublished, 1973 and 1983. R. J. Breglia,

C. O.Ward, C. I. Jarowski, J. Pharm. Sci. 62 (1973) 49.

P. Gullino et al.,Arch. Biochem.Biophys.58 (1955) 253.

O. Strubelt, C.-P. Siegers, A. Sch€utt, Arch. Toxicol. 33(1974) 55. W. Braun, Strahlentherapie 108 (1959) 262.

H. Gutbrod et al., Acta Hepatol. 5 (1957) no. 1/2, 1.

I. Petersone et al., Eksp. Klin. Farmakoter. 3 (1972) 5.

D. G. Gallo, A. L. Sheffner, DE 2018599, 1971. Trans-

bronchin, Homburg Pharma, Frankfurt 1971. L. Bona-

nomi, A. Gazzaniga, Therapiewoche 30 (1980) 1926.

W. F. Riker, H. Gold, J. Am. Pharm. Assoc. 31 (1942)

306.

421 G. Maffii, G. Schott, M. G. Serralunga, Res. Prog. Org.

Biol. Med. Chem. 2 (1970) 262.

422 Y. Kawaguchi et al., Iyakuhin Kenkyu 11 (1980) 635.

Kaken Chemical, JP 52083940, 1976 (S. Suzuki). P.

Gullino et al., Arch. Biochem. Biophys. 64 (1956)

319. Degussa, unpublished, 1981 and 1982. E.-J. Kirn-

berger et al., Arzneim. Forsch. 8 (1958) 72.

Further Reading

K. Araki, T. Ozeki: Amino Acids, Kirk Othmer Encyclopedia

of Chemical Technology, 5th ed., vol. 2, p. 554–618,

John Wiley & Sons, Hoboken, NJ, 2004, online: DOI:

10.1002/0471238961.1921182201180111.a01.pub2 (Ju-

ly 2003).

M. A. Blaskovich: Handbook on Syntheses of Amino Acids,

Oxford Univ. Press, New York 2010.

L. A. Cynober (ed.): Metabolic and Therapeutic Aspects of

Amino Acids in Clinical Nutrition, 2nd ed., CRC Press,

Boca Raton, FL 2004.

J. P. D’Mello (ed.):Amino Acids in Animal Nutrition, 2nd ed.,

CABI Publ, Wallingford 2003.

R. Faurie, J. Thommel (eds.): Microbial Production of L-

Amino Acids, Springer, Berlin 2002.

A. B. Hughes (ed.): Amino Acids, Peptides and Proteins in

Organic Chemistry, Vols. 1–6, Wiley-VCH, Weinheim

2009ff.

V. F. Wendisch (ed.): Amino Acid Biosynthesis, Springer,

Berlin 2007.


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