amino acids, peptides and proteins in organic chemistry (origins and synthesis of amino acids) ||...

Part TwoProduction/Synthesis of Amino Acids

Amino Acids, Peptides and Proteins in Organic Chemistry. Vol.1 – Origins and Synthesis of Amino Acids.Edited by Andrew B. HughesCopyright � 2009 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32096-7

3Use of Enzymes in the Synthesis of Amino AcidsTheo Sonke, Bernard Kaptein, and Hans E. Schoemaker

3.1Introduction

Amino acids, natural as well as synthetic ones, are extensively used in the food, feed,agrochemical, and pharmaceutical industries. The total amino acid market has beenestimated at approximately US$4.5 billion in 2004 [1]. Many proteinogenic aminoacids are used as infusion solutions, whereas the essential amino acids (e.g., lysine,threonine, and methionine) are used as feed additives. Monosodium L-glutamate(more commonly knownasMSG) iswidespread as a taste enhancer/seasoning agent,the commercial production of which dates back to 1908 [2]. L-Aspartic acid andL-phenylalanine methyl ester together form the low-calorie sweetener aspartame(1) [3]. However, not only proteinogenic amino acids are applied; synthetic aminoacids are also intermediates in pharmaceuticals and agrochemicals production.D-Phenylglycine and D-p-hydroxyphenylglycine (D-HPG) are produced in quantitiesof several thousands of tons per year for the synthesis of the semisynthetic broad-spectrum antibiotics ampicillin (2a), amoxicillin (2b), and others [4, 5]. Otherpharmaceuticals containing unnatural amino acids are, for example, the antihyper-tensive drugs Aldomet [L-a-methyl- 3,4-dihydroxyphenylalanine (L-a-methyl-DOPA)](4) [6], the angiotensin-converting enzyme inhibitor ramipril (3) [7], and many ofthe HIV-protease inhibitors of which atazanavir (5) is an example [8]. D-Valine isused as a building block of the pyrethroid insecticide fluvalinate (6) [9], whereas thea-methyl-substituted amino acid a-methylvaline is a structural element in theherbicide arsenal (7) (currently marketed as its isopropylamine salt by BASF) andrelated herbicides [10, 11], and L-a-methylphenylglycine is a part of the fungicidefenamidone (8) [12, 13].Although many amino acids appear in living organisms, only a few of them

are actually isolated from nature for commercial purposes. L-Cysteine and L-4-hydroxyproline are some of the examples in which extraction of a natural protein-rich feedstock like hair, keratin, and feather is still a commercially viable process [2].For most proteinogenic amino acids fermentation is nowadays the preferredroute for their preparation [1]. Examples include L-lysine and L-glutamic acid,

j79

Amino Acids, Peptides and Proteins in Organic Chemistry. Vol.1 – Origins and Synthesis of Amino Acids.Edited by Andrew B. HughesCopyright � 2009 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32096-7

which are produced in amounts of multihundreds of kilotons per year. However,fermentation is also the production method of choice for proteinogenic aminoacids with a much smaller market volume, such as L-phenylalanine, L-aspartic acid,and L-isoleucine [2].

H2NO

HN CO2Me

Aspartame 1

N

S MeMe

OCO2H

HN

O

NH2

R

Ampicillin (R = H) 2a

Amoxycillin (R = OH) 2b

O

HN O

CNO

CH3H3C

Cl

F3C

Fluvalinate 6

NN

HNO

CO2H·NEt3

H3C

CH3

H3C

Arsenal 7

HO

HO H2N CO2H

CH3

L-α-MethylDOPA 4

NH

NHN

OH

ONH

O

Atazanavir 5

ONH

N

COOHO

Me

O

Ramipril 3

NN

OH3C

S

NH

CH3

Fenamidone 8

HN

OO

OCH3O

H3C

N

HO2C

Of the proteinogenic amino acids, D,L-methionine and glycine are the only onesmanufactured synthetically. Both L- and D,L-methionine have a similar effect as a feedadditive, because the animal organism is converting the D-enantiomer into itsnutritive optical antipode. As a consequence, methionine has been fed as a racemicmixture for more than 50 years [14].Only a few examples are known in which amino acids are produced by chemical

(catalytic) asymmetric synthesis. The asymmetric hydrogenation developed byMonsanto for L-phenylalanine is today only used for the production of L-DOPA [15].Many other asymmetric routes to enantiopure a-H- and a,a-disubstituted a-aminoacids have been developed on a laboratory scale, but to our knowledge none of themhas been scaled-up beyond pilot-plant scale [16, 17].

3.2Chemo-Enzymatic Processes to Enantiomerically Pure Amino Acids

Commercialization of the first biocatalytic method for enantiomerically pure aminoacids goes back to 1954, when Tanabe Seiyaku implemented an aminoacylase-basedresolution process for the production of several L-amino acids (Section 3.2.1) [18].

80j 3 Use of Enzymes in the Synthesis of Amino Acids

Since then many different biocatalytic procedures for the production of L- as well asD-amino acids have been described, but only a handful have been developed beyondthe laboratory bench and implemented at full industrial scale.Enzymatic resolution steps have an inherent maximum yield per cycle of 50%

unless the unwanted enantiomer can be racemized. A second category of chemo-enzymatic processes rely on an enzyme-catalyzed asymmetric synthesis as a key step.Consequently, these processes have a maximum theoretical yield of 100% per cycle.In the last decades a number of chemo-enzymatic processes for the production of

enantiomerically pure amino acids have been commercialized by different compa-nies [19]. In the following sections an overview is given of their most importantfeatures,with themain focus on typical process characteristics like scope, limitations,and different modes of operation through the years.

3.3Acylase Process

One of the most widespread methods for the biocatalytic synthesis of L-amino acidsis the resolution of N-acetyl-D,L-amino acids (9) by the enzyme N-acyl-L-aminoacid amidohydrolase (aminoacylase, acylase I; EC 3.5.1.14) [18, 20]. In this process,the enzyme selectively hydrolyses the N-acetyl-L-amino acid to the correspondingL-amino acid without touching the D-substrate (Scheme 3.1). Equimolar amounts ofacetic acid are formed as a side-product. After the reaction has gone to completion,the amino acid product can be separated from the nonreacted substrate through ionexchange chromatography or crystallization. The racemic N-acetylated amino acidsubstrates are fairly easily accessible through acetylation of D,L-amino acids withacetyl chloride or acetic acid anhydride in alkali in a Schotten–Baumann reaction [21]or via amidocarbonylation [22].

R CO2H

NH2

OO

O

R CO2H

HN

O

R CO2H

HN

O

R CO2H

NH2

N-acetyl-D-amino acidL-amino acid

Acylase I

H2OCH3COOH

Racemization

9

Scheme 3.1 Enantiospecific hydrolysis of N-acetyl-D,L-amino acids (9) by A. oryzae acylase I.

3.3 Acylase Process j81

A drawback of the use of acylases is the fact that the hydrolysis reaction ofN-acetylamino acids is equilibrium-limited, but this equilibrium is well on the side ofhydrolysis [23]. Typical equilibrium constants range from 3.7M for methionine to12.5M for norleucine (pH 7.5, 25 �C), which translates to equilibrium conversions of89–96% based on 0.5M of N-acetyl amino acid [24]. This incomplete conversionresults in a lower enantiomeric excess of the remainingN-acetyl-D-amino acid, whichcertainly complicates the cost-efficient production of D-amino acids by this process.After introducing the acylase process in 1954 using the acylase I from Aspergillus

oryzae, the Japanese company Tanabe Seiyake further improved its competitivenessby switching from a batch reaction with the native enzyme to a continuous mode ofoperation in a fixed-bed reactor in 1969. Successful immobilization of the A. oryzaeacylase I onto diethylaminoethyl cellulose DEAE-Sephadex at an industrial scale wasessential for the switch to this continuous enzymatic process. This first immobilizedenzyme reactor system led to 40% lower production costs compared to the batchprocess [18].Degussa implemented the acylase I process fromA. oryzae in 1982 [19]. Currently,

this process is operated in an enzyme membrane reactor using a hollow-fibermembrane with a cut-off value of 10 kDa. This results in over 99.9% retention ofthe 73 kDa acylase. Using this setup Degussa is producing several hundred tons peryear of enantiomerically pure L-amino acids, mostly L-methionine and L-valine [25].Since the acylase process is a typical example of an enzymatic kinetic resolution

many attempts have been undertaken to racemize the remaining N-acetyl-D-aminoacid. The high pKa of approximately 15 [19] of the N-acetyl amino acids necessitatesrather extreme conditions to effect this racemization [25, 26], precluding simul-taneous resolution and racemization steps. This problemwas solved by the discoveryof an N-acylamino acid racemase, in several actinomycetes strains, first by scientistsat Takeda [27–29] and later also by scientists at Degussa [30]. More recently, anN-acylamino acid racemase was also identified in the bacterium Deinococcus radio-durans [31]. Although the reaction conditions for these acylamino acid racemasesseem to be compatible with those for acylase I (e.g., metal ion dependency, pH,temperature) and proof-of-principle for a dynamic kinetic resolution (DKR) acylaseprocess has been demonstrated on the laboratory scale [31–33], there are no clearindications that such a combined process has already been implemented at thecommercial scale [19].Over the years several microbial amidohydrolases have been isolated that readily

and enantioselectively convert, for example,N-acetyl-L-proline and its derivatives [34,35], a class of substrates not accepted by the acylase I. Interestingly, the enzyme fromComamonas testosteroni can also resolve racemic N-acylated N-alkyl-amino acids,thereby opening a novel route to enantiomerically pure N-alkyl-amino acids [36].Several Japanese groups have invested in the isolation of suitable D-aminoacylases.Although such D-specific enzymes have been found in, for example, Streptomycessp. [37], Pseudomonas sp. [38], and Alcaligenes sp. [39], for obtaining enantiopureproducts purification from the L-aminoacylase background activity present in themicroorganisms is necessary. Such tedious and expensive purification complicatestheir commercial application [20].


3.4Amidase Process

Another process for the production of enantiomerically pure a-H-a-amino acids wasdeveloped at DSM in the mid-1970s. This process, which is based on the enzymatickinetic resolution of racemic a-H-a-amino acid amides (10) with L-selective amidehydrolases, was commercialized by DSM in the mid-1980s for the production ofseveral L- and D-amino acids [4, 40, 41]. As a rule, the amide substrates are directlyaccessible by Strecker synthesis on their corresponding aldehydes followed byhydrolysis under mild basic conditions in the presence of catalytic amounts of analdehyde or ketone [42].Different from the acylase process, the amide substrate is thusa precursor of the amino acid tobeprepared. If needed, the racemic amino acid amidescan also be prepared on a laboratory scale by the alkylation of N-acetamidomalonateesters or of imino esters and amides derived from glyoxylic acid [43]. Anotheradvantage over the acylase process is the fact that the amide hydrolysis is notthermodynamically limited, which implies that the conversion can, in principle, bequantitative at every substrate concentrationapplied [19]. Consequently, D-aminoacidswith near absolute enantiomeric excess are also accessible through this processapplying L-selective amide hydrolases. Alternatively, D-selective amino acid amidehydrolases can be used for the production of D-amino acids. Although suitableD-selective hydrolases have among others been identified in Ochrobactrum anthropistrains SV3 [44] and C1-38 [45], their large-scale application is complicated by the factthat they need to be separated from the L-amide hydrolases that are present in almostall microorganisms.The amidase process at DSM has long been operated with permeabilized whole

cells of Pseudomonas putida ATCC 12 633. Because this biocatalyst has a nearly 100%enantioselectivity for the hydrolysis of most L-amides (enantiomeric ratio E > 200),both the L-amino acid and the D-amino acid amide are obtained in almost 100%e.e. at50% conversion. The hydrolysis reaction further furnishes 1 equiv. of ammonia(compared to the L-amino acid amide) as side-product. After completion of thehydrolysis reaction 1 equiv. of benzaldehyde is added to form the Schiff base of theunreacted D-amino acid amide, which precipitates from the solution. In this waythe L-acid and D-amide can easily be separated (Scheme 3.2). Alternatively, separationof acid and amide is possible with a basic ion-exchange resin.Apart from its exquisite enantioselectivity, the P. putida whole-cell biocatalyst is

characterized by its broad substrate specificity. So far over 100 differenta-H-a-aminoacid amides have been successfully resolved. The size of the side-chain may rangefrom the small methyl group in alanine to the very bulky group of lupinic acid [46].Furthermore, the alkyl or aryl side-chains may contain heteroatoms like sulfur,nitrogen, and oxygen. Also cyclic amino acid amides like proline amide andpiperidine-2-carboxyamide, and amino acid amides with alkenyl and alkynyl subs-tituents are substrates for this biocatalyst [47, 48]. Protein purification experimentsidentified an L-aminopeptidase that contributes to a considerable extent to the broadsubstrate specificity of P. putida ATCC 12 633 [49]. This enzyme performs optimallyat pH 9.0–9.5 and 40 �C in the presence of 0.2–20mM Mn2þ ions as an activating

3.4 Amidase Process j83

divalent metal ion. Cloning of the gene encoding this aminopeptidase by reversedgenetics and subsequent overexpression in E. coli led to a highly efficient whole-cellbiocatalyst [50]. Protein database searches revealed that this L-aminopeptidasebelongs to the leucine aminopeptidase family of proteins, which catalyze thehydrolysis of amino acids from the N-terminus of (poly)peptide chains. Their, ingeneral, broad substrate specificity correlates well with the fact that the purifiedP. putida L-aminopeptidase displays activity towards a broad range of a-H-a-aminoacid amides and dipeptides.The added value of this highly active recombinant E. coli based whole-cell

aminopeptidase biocatalyst became manifest in the preparation of a set of unsatu-rated a-H-a-amino acids (Scheme 3.3) [51]. In general, the resolution of theseunsaturated amino acid amides with the recombinant E. coli-based system as well as

H2N

R

O

NH2

10

H2N

R

O

OHH2N

R

O

NH2Pseudomonas putida

+

N

R

O

NH2Ph

pH ~ 8.537 °C

L-acid

1) OH– (racemization)

2) H+/H2O

D-amide

H2N

R

O

OH

D-acid

H+

∆

PhCHO

Scheme 3.2 Enzymatic resolution of a-H-a-amino acid amides (10) by P. putida.

H2N COX

H2N COX

H2N COX

H2N COX

H2N COX

H2N COX

H2N COX

H2N COX

11

12

13

14

15

16

17

18

a: X = OH ; b: X = NH2

Scheme 3.3 The unsaturated amino acids resolved by Wolfet al. [51] using a recombinant E. coli-based whole-cell biocatalystexpressing the P. putida L-aminopeptidase.


with the wild-type P. putida cells proceeded smoothly yielding the L-acid and D-amidein high enantiomeric excess (>95%) at 50% conversion. Owing to the increasedexpression of the L-aminopeptidase in the E. coli cells they were applied in acell : substrate ratio of 1 : 500 only, whereas this ratio was 1 : 10 with the P. putidacells. More interestingly, however, L-3-butynylglycine 15a and especially its methyl-ated homolog 17a were obtained in a moderate enantiomeric excess only (91 and70%, respectively) using P. putida cells, whereas the resolution reactions with therecombinant E. coli biocatalyst resulted in a superior enantiomeric excess of bothL-acids (97 and 99%, respectively). Further experiments made clear that this unsatis-factory low enantiomeric excess of both L-acids with P. putida cells as biocatalystoriginates from the presence of an amino acid racemase with narrow substratespecificity that is absent in the recombinant E. coli whole-cell biocatalyst [51].This latter biocatalyst was subsequently used for the preparation of the L- and

D-enantiomers of amino acids 11a–18a on the multigram scale using the proceduredepicted in Scheme 3.4. After standard work-up of the unreacted D-amides, thesewere hydrolyzed under very mild conditions by the nonselective amidase present inRhodococcus erythropolis NCIMB 11 540 cells. Chemical hydrolysis was not anoption for this type of unsaturated amide, since the harsh acidic conditions neededwould lead to decomposition of some of the side-chains. All L-acids were obtainedin above 98% e.e. except for 18a, which was obtained in an enantiomeric excess of96%. The enantiomeric excesses of the D-acids appeared to be excellent withoutexception.

H2NO

OHH2N

O

NH2

D-11b - 18b

H2NO

NH2

rec. E. coliR RR

+

PhCHO

NO

NH2

R

Ph

HClAcetone

-Cl+H3NO

NH2

R

R. erythropolisNCIMB 11540

pH 8, 37°CH2N

O

OHR

pH 9.2, 40°C

L-11a - 18a

D-11a - 18a

Scheme 3.4 Optimized amidase based process for themulti-gram synthesis of enantiomerically pure L- andD-unsaturated amino acids.


Like the acylase process, the amidase process is a resolution and thus it ishampered by a maximum yield of 50%. Racemization of the remaining D-aminoacid amide, which can be easily done via formation of the benzaldehyde Schiff baseunder basic conditions, can lead to 100% yield (Scheme 3.2) [52]. As formation of thisSchiff base is also the basis for the separation of the L-acid and D-amide, racemizationcan be performed without any additional step. Nevertheless, it was envisioned that afully enzymatic DKR process combining the action of an enantioselective amidaseand an a-H-a-amino acid amide racemase in one vessel would be a more cost-efficient process. Scientists at DSMrecently identified the requireda-H-a-amino acidamide racemase in a novel O. anthropi strain [53]. This amino acid amide racemase(AmaR), which appeared to be pyridoxal 50-phosphate (PLP)-dependent, combinesgood thermostabilitywith a broadpHoptimum(6–10),which are twoprerequisites forlarge-scale application. Apart from lactams, AmaR also racemizes a range of lineara-H-a-amino acid amides, althoughwith lower activity, especially in the case of aminoacid amides with a Cb branched side-chain. Furthermore, the racemase is stronglyinhibited by a couple of divalent metal ions, including Mn2þ , that is essential for theoptimal performance of the P. putida L-aminopeptidase. This conflict was solved bycareful tuning of theMn2þ concentration, which showed that 0.6mMof this divalentmetal ion is the best compromise [54]. The applicability of AmaR for a DKR amidaseprocess was demonstrated for the production of L-aminobutyric acid using a cell-freeextract of anE. coli strain expressingAmaR and the endogenous L-selective amino acidamide hydrolase. Whereas the blank reaction with solely the L-amide hydrolasestopped near 50% conversion, the presence of AmaR led to formation ofL-aminobutyric acid in 84% conversion and 96.3% e.e. [54].AmaR has the highest homology (52% identity) with the a-amino-e-caprolactam

(ACL) racemase from Achromobacter obae. This ACL racemase has been developedby scientists at Toray Industries for the industrial production of L-lysine fromD,L-ACL [26, 55]. In contrast to earlier reports [56], Asano and Yamaguchi recentlyreinvestigated this ACL racemase and found that it is also able to racemize lineara-H-a-amino acid amides, although with at least 35-fold lower specific activity thanfor ACL [57]. Furthermore, they showed that by combining this enzyme with theD-aminopeptidase from O. anthropi C1-38 [45], synthesis of near stoichiometricamounts of D-amino acids from L-amino acid amides is possible [58].

3.4.1Amidase Process for a,a-Disubstituted a-Amino Acids

Inspired by the advantages of the amidase process, DSM scientists developed asimilar enzymatic kinetic resolution process for the production of enantiopurea,a-disubstituted amino acids. Although alternative routes are available [59, 60], inthis case the Strecker synthesis also is the most direct way to prepare the racemicdisubstituted amino acid amides, but the hydrolysis of the aminonitrile intermediateneeds more harsh conditions (e.g., benzaldehyde/pH 14, concentrated H2SO4, orHCl-saturated formic acid) because of the increased steric hindrance [61]. Since theP. putida biocatalysts require an a-hydrogen atom for activity, novel amidase


biocatalysts needed to be identified. Screening led to the identification ofMycobacte-rium neoaurum ATCC 25 795, a biocatalyst that affords the (S)-a,a-disubstitutedamino acids and the corresponding (R)-amides in almost 100%e.e. at 50% conver-sion for most a-methyl-substituted compounds (E > 200) [62, 63]. Only for glycineamides with two small substituents at the chiral center is the enantioselectivitydecreased. Generally, a-methyl-substituted amino acid amides are hydrolyzed withhigh activity, but increasing the size of the smallest substituent to ethyl, propyl, or allyldramatically reduces the activity, especially if the largest substituent does not containa -CH2- spacer at the chiral carbon atom [62]. In line with this, a-H-a-amino acidamides are also good substrates and are hydrolyzed enantioselectively. The enzymethat was responsible for the enantioselective hydrolysis of D,L-a-methylvalineamide has been purified and characterized [64]. This enzyme, which was classifiedas an amino amidase, is active toward a rather broad range of both a-H- anda-alkyl-substituted amino acid amides, which are hydrolyzed with moderate to highL-selectivity – the lowest enantioselectivity was obtained toward alanine amide(E� 25). However, this biocatalyst is inactive toward dialkyl amino acid amides withvery bulky substituents and a-hydroxy acid amides.To close this gap in the range of compounds that can be produced by the amidase

technology, a novel amidase biocatalyst was identified in a classical screeningprogram [65]. This novel biocatalyst, O. anthropi NCIMB 40 321, is characterizedby its extremely broad substrate specificity including a-H- and (bulky) a,a-dialkyl-substituted amino acid amides, a-hydroxy acid amides, and N-hydroxyamino acidamides, its very good temperature stability and especially its relaxed pH profile.Although the amidase displays its highest activity at pH 8.5, 55% of this activity is stillretained at pH5.0. This enables the hydrolysis of substrates,which are only very poorlysoluble at weakly alkaline conditions, just by performing the hydrolysis reaction atslightly acidic conditions. This feature of theO. anthropi amidase biocatalyst appearedto be crucial in the resolution of thea-H-a-aminob-hydroxy acid amides 19, which areintermediates in novel routes to the antibiotics thiamphenicol (20a) and florfenicol(20b) [66]. In this case, the enzymatic resolution reactionwas performed at pH5.6–6.0to ensure a fair solubility of the otherwise insoluble amides. Also in the preparation of(S)-a-methyl-3,4-dichlorophenylalanine 21, an intermediate for cericlamine HCl, apotent and selective synaptosomal 5-hydroxytryptamine (serotonin) uptake inhibitorunder development [67, 68], the activity of theO. anthropi amidase at low pH (in thiscase pH 5.3 was used) was a decisive factor [69].

OH

NH2

NH2

O

H3CS(O)n

19

XHN

OH

CHCl2

OS

O

H3C O

(X=OH)20a(X=F)20b

H2NOH

O

ClCl

21

NOH

ClCl

CH3

H3C

Cericlamine

0,2=n


Recently, the purification, cloning, and heterologous expression in E. coli of themost important amidase (LamA) from O. anthropi NCIMB 40 321 has beenreported [70]. LamA displays activity toward a broad range of substrates consistingof a-hydrogen- and (bulky) a,a-disubstituted a-amino acid amides, a-hydroxy acidamides, and a-N-hydroxyamino acid amides, and is thus responsible on its own forthe extremely broad substrate specificity of the O. anthropi whole cells. For allsubstrates investigated, only the L-enantiomer was hydrolyzed (E > 150). LamAappeared to be a metallo-enzyme, as it was strongly inhibited by the metal-chelatingcompounds ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline.The activity of the EDTA-treated enzyme could be restored by the addition of Zn2þ

(to 80%), Mn2þ (to 400%), and Mg2þ (to 560%). The L-amidase gene encodes apolypeptide of 314 amino acids with clear homology to the acetamidase/formami-dase family of proteins including the stereoselective amidases from E. cloacae [71],Thermus sp. [72], and Klebsiella oxytoca [73]. The Enterobacter and Thermus amidases(67 and 53% identical to LamA) have been developed byMitsubishi researchers. Bothamidases are highly L-selective toward D,L-tert-leucine amide and are also activetoward lactate amide, implying that these amidases can also convert a-hydroxyacid amides. The amidase from K. oxytoca (28% identity to LamA) has beendeveloped by Lonza for the (R)-selective hydrolysis of racemic 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide [74].Altogether, theO. anthropiL-amidase has a unique set of properties for application

in the fine-chemicals industry.

3.5Hydantoinase Process

A third commercialized chemo-enzymatic production method for enantiomericallypure a-H-a-amino acids is based on the enantioselective hydrolysis of racemic5-monosubstituted hydantoins by D- or L-selective hydantoinases, often combinedwith D- or L-selectiveN-carbamoylases [25, 75, 76]. In 1979,Kanega implemented afirstgeneration of such processes for the production of D-HPG (24) [75] – the side-chain forthe semisynthetic b-lactam antibiotic amoxicillin and nowadays produced in severalthousands of tons annually using the hydantoinase process. This first-generationprocess involved one enzymatic and two chemical conversions (Scheme 3.5).In this process the D-hydantoin is selectively hydrolyzed to N-carbamoyl-D-HPG

(23) by a D-specific hydantoinase in immobilized whole resting cells of thebacterium Bacillus brevis [77]. The slightly alkaline conditions (pH 8.0) lead tospontaneous racemization of the remaining L-hydantoin (see below), resulting in aquantitative conversion intoN-carbamoyl-D-HPG. In the last step theN-carbamoyl-D-HPG is chemically hydrolyzed to the desired D-HPGusing an equimolar amountof HNO2.Alternatively, Recordati commercialized a similar process employing resting cells

of an Agrobacterium radiobacter strain, which contained both a D-hydantoinase and astrictly D-selective N-carbamoylamino acid amido hydrolase (D-N-carbamoylase)


activity [78]. Consequently, the initially formed N-carbamoyl-D-HPG is irreversiblyconverted in situ to D-HPG in nearly 100% yield [79]. The carbamoylase also broadensthe scope of the D-hydantoinase process to a-amino acids that cannot withstand theHNO2 treatment, like D-tryptophan, D-citrulline, or D-pyridylalanine [76]. Since theA. radiobacter cells have a very broad substrate specificity, Degussa in collaborationwith Recordati used theA. radiobacter biomass for the production in bulk amounts ofa broad array of D-amino acids [80].Industrial application of D-N-carbamoylases has long been complicated by their

limited stability and their inhibition by ammonium ions. The inhibition by ammo-nium ions has been tackled by in situ removal via absorption to a silicate complex [81,82] or via formation of a poorly soluble ammonium salt, for example, MgNH4PO4,by performing the reaction in the presence of MgHPO4 [83], making industrialapplication more viable. More stable D-N-carbamoylases could, for instance, beobtained by a combination of random mutagenesis and screening for increasedthermal [84, 85] or oxidative stability [86], or by immobilization [87]. Since 1995,Kanega has been applying a more thermostable variant of the D-N-carbamoylaseof Agrobacterium sp. KNK712 as an immobilized biocatalyst in combination with aD-hydantoinase in a second-generation D-HPG process [75].Chemical racemization of the hydantoins at slightly alkaline conditions (pH8–9) is

strongly dependent on the stabilizing effect of the side-chain in the 5-position. At pH8.5 and 40 �C, racemization half-lives of the hydantoins range from 0.21 h in the caseof the p-hydroxyphenyl side-chain to 120 h in the case of the tert-butyl side-chain [76].

Racemization

HOCO2HO

NH2

OH2N

22

HN

O

NH

O

HO

D-Hydantoinase

HN

O

NH2

CO2H

HO

HNO2orD-Carbamoylase

NH2

CO2H

HO

NH3 CO2

23

D-HPG 24

HN

O

NH

O

HO

L-Hydantoin

chemicalsynthesis

Scheme 3.5 Industrial production of D-HPG (24) by the D-hydantoinase process.

3.5 Hydantoinase Process j89

Therefore, for 5-monoalkyl-substituted hydantoins the rate of racemization canbecome the rate-limiting step in the overall process.Based on the observation that racemic 5-isopropylhydantoin was converted into

D-valine in around 75–85% conversion by resting A. radiobacter cells, Battilotti andBarberini suggested that an enzymemight be responsible for the racemization of theremaining L-hydantoin [88]. A few years later hydantoin racemases could indeed bepurified fromArthrobacter aurescensDSM 3747 [89] and Pseudomonas sp. NS671 [90],which were subsequently cloned and heterologously expressed in E. coli. [91, 92].Recently, more hydantoin racemases were characterized and cloned, for instance,from Agrobacterium tumefaciens [93], Sinorhizobium meliloti [94], andMicrobacteriumliquefaciens [95]. In particular, the new hydantoin racemase from an A. radiobacterstrain has industrial potential because it does not suffer from substrate inhibition – adrawback of many other hydantoin racemases [96].Next to the enzymes acting on D-hydantoins and N-carbamoyl-D-amino acids, a

number of strains have been identified that convert racemic 5-substituted hydantoinsinto L-amino acids. An example is the process for L-methionine using Pseudomonassp. NS671 [97], Bacillus stearothermophilus NS1122A [98], and Arthrobacter sp. DSM7330 [99]. L-Hydantoinases and L-carbamoylases fromdifferentmicroorganismshavebeen purified and characterized, and genes encoding these enzymes have beencloned and heterologously expressed [76]. Interestingly, the hydantoinases were notalways fully L-specific [100], but a strictly L-specific carbamoylase in these micro-organisms without exception resulted in the L-amino acids.The recombinant expression of the three required enzymes in an easy to ferment

microorganism not only leads to higher expression levels than in the wild-typemicroorganisms [101], but also enables the fine-tuning of the amount of each of thesethree enzymes securing an optimal flux through this mini-pathway without accumu-lation of intermediates [102]. This has also been achieved by reversing the enantios-electivity and improving the activity of the hydantoinase from an Arthrobacter sp. bydirected evolution [103]. These developments will lead to more productive biocatalystsand consequently lower costs. It is expected that this will drive the replacement of thewild-type strains by more active and tuned recombinant production strains as well asthe implementation of new processes. The availability of production hosts that areoptimized for the production of D-amino acids [104, 105], the straightforward synthesisof the racemic hydantoins from cheap raw materials via a number of differentroutes [76] as well as the low side-product formation will certainly stimulate this.

3.6Ammonia Lyase Processes

The second category of chemo-enzymatic processes to form a-H-a-amino acids relyon an enzyme-catalyzed asymmetric synthesis, which implies that they can theoreti-cally utilize up to 100% of the substrate per cycle. One such process platformmakesuse of ammonia lyases, a class of enzymes that catalyze the reversible addition ofammonia to carbon–carbon double bonds [106].


3.6.1Aspartase-Catalyzed Production of L-Aspartic Acid

L-Aspartate ammonia lyase (aspartase; EC 4.3.1.1) is one of the few ammonia lyasesthat is applied in industry, namely for the large-scale conversion of inexpensivefumaric acid into L-aspartic acid (25) via addition of ammonia (Scheme 3.6). Thisamino acid is widely used, for example, for food flavoring (mainly in Japan), inparenteral and enteral nutrition, as an acidulant, and as a precursor for the artificialsweetener aspartame (1) [26, 107].Anumber of companies are operating such an aspartase-based process, but each in

a somewhat different format. Although started in 1953 with a batchwise process [26],Tanabe Seiyaku switched to a continuous mode of operation in 1973 applying wholeE. coli cells immobilized in a polyacrylamide gel lattice [108, 109]. The highimmobilization yield (>70%) and excellent biocatalyst stability (half-life of 120 daysat 37 �C) reduced the production costs to about 60% compared to the conventionalbatch process [18, 110]. The process was further improved by the introduction ofk-carrageenan as immobilization matrix in 1978 [18, 111] and of a hyper-aspartase-producing E. coli variant in 1983 [112].Kyowa Hakko Kogyo, Mitsubishi Petrochemical, and W. R. Grace & Co. have

implemented different process formats after similar optimization trajectories [26,107, 113–117].All these processes have in common that they are carried out with a 2- to 3-fold

excess of ammonia to drive the reaction equilibrium from fumaric acid in thedirection of L-aspartic acid (Keq¼ [L-Asp]/([ fumaric acid]�[NH3])¼ 4.2� 102 M�1)[118]. This leads to almost stoichiometric conversion and excellent enantiomericpurity of greater than 99.9%.More active aspartase biocatalysts have been obtained by cloning and overexpres-

sion of the aspartase-encoding gene from Brevibacterium flavum MJ-233 [119] andE. coli [120, 121], and by protein engineering via site-directed mutagenesis anddirected evolution [122, 123]. A highly thermostable aspartase has been identified inBacillus sp. YM55-1 [124]. On-going engineering projects will certainly benefit fromthe recently elucidated X-ray crystal structures of the E. coli aspartase [125] and of thethermostable aspartase from Bacillus sp. YM55-1 [126].Production costs can be further reduced by combining aspartase with maleate

isomerase (EC 5.2.1.1) and using maleic acid, derived from cheap maleic anhydride,as a substrate [127, 128].The continuous optimization programs of the different players in this field have

made the aspartase-based processes some of the most efficient enzyme processes

fumaric acid

HO2CCO2H

L-aspartic acid 25

HO2C

NH2

CO2HAspartase

+ NH3

Scheme 3.6 Aspartase-catalyzed synthesis route to L-aspartic acid (25) from fumaric acid.

3.6 Ammonia Lyase Processes j91

known today [19] [space-time yield: 60 000 g/(l day); initial substrate concentration:1.5–2M [129]] and economically more attractive than fermentation.

3.6.2Production of L-Alanine from Fumaric Acid by an Aspartase–Decarboxylase Cascade

Although L-alanine (26) is applied in enteral and parenteral nutrition, and as a foodadditive because of its sweet taste and bacteriostatic properties, the annual worldproduction of this amino acid is approximately 500 tons only [130]. Although studieshave proved that L-alanine can be fermented in high yield and optical purity [131], it isstill produced enzymatically from L-aspartic acid by irreversible decarboxylation withL-aspartate b-decarboxylase (EC 4.1.1.12) (Scheme 3.7) [19]. L-Aspartate b-decarbox-ylase needs PLP as a prosthetic group and is allosterically activated by a-ketoacids [132].In 1965, Tanabe Seiyaku started production of L-alanine from L-aspartic acid as a

batch process applying whole cells of the strain Pseudomonas dacunhae, a strain withhigh L-aspartate b-decarboxylase activity [133]. To improve the productivity andcompetitiveness, a switch was made to a continuous process in 1982 [134], whichemployed sonicatedP. dacunhae cells immobilized in k-carrageenan in a pressurizedfixed-bed reactor [133, 134].In the L-alanine manufacturing process the decarboxylase reaction can be com-

bined with the aspartase-catalyzed synthesis of L-aspartic acid from fumaric acid asdepicted in Scheme 3.7 [135, 136]; however, because of the different pHoptima of thetwo enzymes a two-step production process is more efficient [118]. Such a two-stepprocess has been operated by Tanabe Seiyaku since 1982 [137].Since the aspartate b-decarboxylase is highly L-enantioselective it can also be used

for the production of D-aspartic acid (27), together with L-alanine (26), from racemicaspartic acid via a kinetic resolution (Scheme 3.8). Racemic aspartic acid is easilyprepared by the chemical additionof ammonia to thea,b-doublebond in fumaric acid.

Scheme 3.7 Biocatalytic cascade for the production of L-alanine(26) from fumaric acid by the combined action of aspartaseand L-aspartate b-decarboxylase.

DL-aspartic acid

HO2C

NH2

CO2H

L-alanine 26

HO2C

NH2

Aspartate-β-decarboxylase

CO2 D-aspartic acid 27

HO2C

NH2

CO2H OC++ 2

Scheme 3.8 Aspartate b-decarboxylase-catalyzed kineticresolution process to D-aspartic acid (27) and L-alanine (26).


Both products can subsequently be separated via selective crystallization atdifferent pH [26]. The continuous version of this process to D-aspartic acid andL-alanine using the pressurized plug-flow reactor and P. dacunhae cells in immo-bilized form is operated by Tanabe Seiyaku since 1989 [18].

3.6.3Phenylalanine Ammonia Lyase-Catalyzed Production ofL-Phenylalanine and Derivatives

Apart from aspartase, L-phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is a secondlyase of commercial importance. This enzyme is found ubiquitously in plants andalso in specific microorganisms, especially yeasts and fungi. It catalyzes the non-oxidative deamination of L-phenylalanine (28) into ammonia and trans-cinnamic acid(29), which is the committed step in the phenylpropanoid pathways, which lead to agreat variety of lignins, flavonoids, and coumarins [138].The use of PAL-containing yeast cells (mostly Rhodotorula rubra, Rhodotorula

glutinis, and Rhodosporidium toruloides) in the reverse reaction [139, 140] has beenconsidered for the production of L-phenylalanine (Scheme 3.9). More than 12 000metric tonnes [141] of this is applied as a building block for the sweetener aspartameannually.The conversion of trans-cinnamic acid and ammonia into L-phenylalanine is

thermodynamically quite unfavorable (Keq¼ 4.7M) [118], necessitating high con-centrations of ammonia and an elevated pH to drive the reaction into the direction ofL-phenylalanine formation [142]. Further drawbacks of the PAL process are lowenzyme-specific activity, low enzyme stability, and strong substrate inhibition [141].The specific activity of the PALs from the yeasts R. toruloides [143, 144] andR. glutinis [145, 146] at 30 �C is 2.5–5U/mg only. In addition, the low PAL expressionlevel of below 1% led to a relatively low cellular activity (e.g., for R. glutinis 35U/g celldryweight) [147] and consequently high cell loading in the bioconversion step.Owingto the profound lack of stability, the reuse of PAL is not a feasible option [147].Fed-batch operation has been used to circumvent inhibition of PAL by trans-

cinnamic acid above 50mM [147, 148] and has actually been commercialized byGenex to L-phenylalanine titers of 43 g/l [space-time yield: 8 g L-phenylalanine/(l�day)] [26, 149].The PAL-mediated bioconversion to L-phenylalanine has been improved over the

last decades using both anaerobic (byN2-sparging) and static conditions [149, 150], orby applying reducing conditions, for example, by the addition of 2-mercaptoethanolor thioglycolic acid [151, 152], as well as by addition of other types of compounds [142,150, 151] and the omission of chlorine ions [150, 153]. All these stabilizing factors

PhenylalanineAmmonia Lyase

+ NH3

CO2H

NH2

CO2H

cinnamic acid 29 L-phenylalanine 28

Scheme 3.9 Phenylalanine ammonia lyase-catalyzed production of L-phenylalanine (28).

3.6 Ammonia Lyase Processes j93

could successfully be combined, enabling the reuse of the yeast cells in six to eightconsecutive L-phenylalanine synthesis runs [150, 151].In a different approach to improve the PAL-catalyzed L-phenylalanine synthesis,

mutants of Rhodotorula graminis with increased cellular activity and an improvedstability during fermentation and bioconversion have been obtained by classicalstrain improvement [154, 155]. Alternatively, a superior R. rubra strain was obtainedby enrichment [156].More recently, PALs from plants [157] and prokaryotic origin [145, 158, 159] have

becomemore readily available for bioconversion reactions through their overexpres-sion in efficient enzyme production systems. Diversa recently isolated a set of 18 newPALs by a sequence-based approach [160], 16 of which were from bacterial specieswhose genome sequence was already publicly available and erroneously had beenannotated as genes encoding a histidine ammonia lyase.Owing to its relaxed substrate specificity nowadays PAL is only used for the

synthesis of non-natural L-phenylalanine analogs, characterized by small volumesand high prices, by Mitsui [161], Great Lakes [162], and others [162–165].

3.7Aminotransferase Process

A second enzyme-catalyzed asymmetric process relies on the action of aminotrans-ferases (EC 2.6.1.X), also frequently named transaminases, which catalyze thereversible transfer of an amino group from a (preferably cheap) amino acid donorto an a-keto acid acceptor yielding a new amino acid along with an a-keto acid side-product (Scheme 3.10). Aminotransferases belong to the large and diverse group ofPLP-dependent enzymes. The PLP prosthetic group serves as an amine acceptor inthe first of two distinct half-reactions and subsequently transfers the amino group tothe a-keto acid in the second half-reaction.Aminotransferases are ubiquitous enzymes in nature, playing an essential role in

the biosynthesis of most proteinogenic amino acids, and in the supply of specificD-amino acids for peptidoglycan and secondary metabolite biosynthesis [166–168].Aminotransferases are generally quite active with typical specific activities up to

400U/mg of protein [169], and they are highly enantioselective and therefore verysuitable as biocatalysts for the production of enantiomerically pure a-H-a-aminoacids [170–172]. Both highly selective D- and L-aminotransferases are known.Furthermore, the substrate specificity of these enzymes is rather broad, which alsoenables their application in the synthesis of enantiopure non-natural amino acids.Finally, in contrast to amino acid dehydrogenases (AADHs) (Section 3.8), they do notrequire external cofactor regeneration.

R CO2H

O

R CO2H

NH2

L-Aminotransferase

L-amino acidα-keto acid

R1 CO2H

O

α-keto acid

R1 CO2H

NH2

L-amino acid + +

Scheme 3.10 General reaction catalyzed by an L-selective aminotransferase.


The application of aminotransferases for synthetic purposes, however, suffersfrom one major disadvantage – the equilibrium constant of the reaction is generallynear unity. For example, the apparent equilibriumconstantsK 0

eq for the synthesis of L-alanine, L-valine, L-leucine, and L-tert-leucine using L-glutamate as the amino donorappear to be 1.86, 0.53, 0.37, and 0.16, respectively [170, 173], resulting in a reducedyield of the desired product and a complex productmixturewith severe complicationsfor downstreamprocessing.Over the years different approaches have beendevelopedto overcome this incomplete conversion, of which the use of an excess of a cheapamino acid donor ismaybe themost obvious one. Schulz et al., for instance, used thehighly stable immobilized E. coli 4-aminobutyrate : 2-ketoglutarate transaminase(EC 2.6.1.19) and a 4-fold molar excess of L-glutamate to convert the 2-keto acidprecursor 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (31) to L-phosphino-thricin (30), the active ingredient of the herbicide Basta (Bayer CropScience), in over90% yield [174]. Space-time yields of above 50 g/(l h) have been reached in acontinuous production process.Precipitation of the amino acid product is a second option to drive the equilibrium

towards product formation, for instance, as applied in the synthesis of L-homo-phenylalanine (pH 2–9: solubility 2mM) in greater than 99%e.e. and 94% conver-sion from 840mM 2-oxo-4-phenylbutyric acid using 900mM L-aspartic acid as theamine donor and the aromatic amino acid aminotransferase from Enterobacter sp.BK2K-1 overexpressed in E. coli as the catalyst [175]. A similar precipitation-drivenapproach has been reported by Tosoh for the synthesis of 3-(2-naphthyl)-L-alanine in93% yield and greater than 99%e.e. from its a-keto acid (180mM) applyingL-glutamic acid (360mM) as amino donor and the hyper-thermostable aminotrans-ferase from Thermococcus profundus [176].A more generally applicable and economically attractive approach to shift the

equilibrium is the use of aspartic acid as the amine donor. The formation ofoxaloacetate and its essentially irreversible decarboxylation to pyruvate and carbondioxide shifts the equilibrium of the aminotransferase reaction almost completely inthe direction of the desired amino acid (Scheme 3.11). Although oxaloacetate

L-amino acid

CO2H

NH2

HO2C

L-Asp

CO2HHO2C

oxaloacetate

O

R CO2H

NH2

R CO2H

O

α-keto acid

CO2H

pyruvate

O

CO2AT

Scheme 3.11 Coupled reaction system to drive the equilibrium ofL-aspartic acid-dependent aminotransferase (AT) reactions tocompletion. The decarboxylation of oxaloacetate can beaccelerated by multivalent metal ions as well as by the enzymeoxaloacetate decarboxylase.

3.7 Aminotransferase Process j95

decarboxylates spontaneously under physiological conditions [177], a real efficientaminotransferase process requires a further rate enhancement by the addition ofmultivalent metal ions [178] or by applying the enzyme oxaloacetate decarboxylase(EC 4.1.1.3) [179]. Use of the E. coli aspartate aminotransferase AspC (EC 2.6.1.1)and the oxaloacetate decarboxylase from P. putida ATCC 950 for the production ofL-phenylalanine fromphenylpyruvate and L-aspartic acid resulted in a conversion of noless than 97%, whereas the identical reaction without the oxaloacetate decarboxylasefurnished L-phenylalanine in 42% conversion only [170, 172].Not all amino acid aminotransferases can utilize L-aspartic acid as the amino

donor. In those cases the aminotransferase can be coupled with AspC to regene-rate the intermediate amine donor, such as L-glutamic acid in the case of thebranched-chain aminotransferase IlvE (EC 2.6.1.42) (Scheme 3.12) [170, 180].This principle of coupled aminotransferases was, amongst others, appliedby Rozzell et al. for the branched-chain a-H-a-amino acids L-valine, L-leucine,and L-isoleucine [180], and by Bartsch et al. for an improved process toL-phosphinothricin (30) [181].Although this enzyme-coupled process concept was a major improvement, its

application is still complicated by the formation of significant amounts of L-alaninefrom pyruvate, catalyzed by a side-activity of many of the broad-spectrum amino-transferases. This inherent drawback has been elegantly solved by scientists of NSCTechnologies by engineering of novel biosynthetic pathways. These pathways containthe enzyme acetolactate synthase, which dimerizes the pyruvate side-product toacetolactate (33), that undergoes spontaneous decarboxylation resulting in theoverall formation of acetoin (34) as the final volatile side-product (see Scheme 3.13

L-phosphinothricin 30

CO2H

NH2

HO2C

L-Asp

CO2HHO2C

oxaloacetate

O

CO2H

O

HOOC CO2HHOOC

NH2L-Gluα-ketoglutarate

CO2H

O

CO2H

pyruvate

O

CO2AT1

AT2OP

HOCO2H

OP

HONH2

PPO 31

Scheme 3.12 AgrEvo process for the productionof the herbicide ingredient L-phosphinothricin asexample of a coupled aminotransferase processto drive the equilibrium of an L-glutamate-dependent aminotransferase reaction (AT2) in

the synthesis direction by decarboxylation ofoxaloacetate. AT1: AspC (glutamate :oxaloacetate aminotransferase, EC 2.6.1.1); AT2:for example, 4-aminobutyrate : 2-ketoglutarateaminotransferase (EC 2.6.1.19).


for similar reaction artificial pathway) [182]. The strength of this approachwas demonstrated by Fotheringham et al. for the efficient ton-scale production ofL-2-aminobutyric acid from cheap L-threonine applying a single recombinant E. colistrain expressing the genes encoding the E. coli aromatic aminotransferase (TyrB),Bacillus subtilis acetolactate synthase (AlsS), and E. coli threonine deaminase(IlvA) [183, 184].In an attempt to completely eliminate alanine formation, novel aspartic acid-

independent reaction pathways have recently been developed. These pathways relyon w-aminotransferases for the regeneration of L-glutamate from a-ketoglutarateby transfer of the side-chain amino group from the donors L-lysine or L-ornithine.The subsequent spontaneous cyclization of the aldehydes drives the formation ofthe a-H-a-amino acid product to near completion. This system was appliedfor the preparation of L-aminobutyric acid from 2-ketobutyric acid in 92% yieldusing L-ornithine as the amino group donor [185, 186]. A similar approach wassuccessful in the preparation of L-tert-leucine (35) from trimethylpyruvic acid andL-ornithine by combining the ornithine d-aminotransferase with the E. coli branched-chain aminotransferase (IlvE).

3.7.1Aminotransferase-Catalyzed Production of D-a-H-a-Amino Acids

As already briefly referred to above, bacteria can also contain D-amino acid amino-transferases (EC 2.6.1.21) for the biosynthesis of the peptidoglycan amino acidsD-alanine and D-glutamic acid and of a broad range of D-amino acids used in thesynthesis of secondary metabolites, such as peptide-based antibiotics. However, thenumber of known D-amino acid aminotransferases is limited. These enzymes alsocontain PLP as a coenzyme [187] and show a similar broad substrate specificity astheir L-selective counterparts [168, 188, 189].The D-amino acid aminotransferase from Bacillus sp. YM-1 has been studied in

most detail; its gene has been cloned and overexpressed in E. coli [190], and its crystalstructure has been solved [191, 192]. These studies showed that this enzyme has aunique fold not seen for any other PLP-dependent enzyme, except for the structure ofthe L-branched-chain amino acid aminotransferase from E. coli [193], suggesting acommon ancestral gene [190].For D-aminotransferase reactions D-amino acids are required as amine donors. As

these D-amino acids are expensive, the process is combined with highly specificracemases, such as for aspartic acid (EC 5.1.1.13), glutamic acid (EC 5.1.1.3), andalanine (EC 5.1.1.1), to generate these amine donors in situ from L-amino acids. As anexample, the D-amino acid aminotransferase from Bacillus sphaericus [194] and theaspartic acid racemase from Streptococcus thermophilus [195] have been combinedwith the threonine deaminase and acetolactate synthase, alreadymentioned above, ina recombinant microorganism for the production of D-2-aminobutyric acid (32)(Scheme 3.13) [171, 183, 196]. This process to make D-amino acids can also beoperated with living whole cells under fermentative conditions, as was exemplifiedby the production of 4.2 g/l D-phenylalanine of greater than 99% enantiomeric purity

3.7 Aminotransferase Process j97

in a fed-batch fermentation with feeding of glucose and D,L-alanine as the aminogroup donor [182, 197]. Other types of multistep D-aminotransferase reactionpathway using D-alanine as the amino group donor have also been described [196,198, 199].The strict stereo-conservation of the D-amino acid aminotransferases mentioned

above urges for D-amino acids as the amino group donor [191, 192, 200], whereas inthe 1980s van den Tweel et al. identified an unusual aminotransferase from P. putidastrain LW-4 [201], able to catalyze the formation of D-HPG (24) from p-hydroxyphe-nylglyoxylate with L-glutamic acid as the amino donor [202, 203]. This novel type ofstereo-inverting aminotransferase (EC 2.6.1.72), of which the gene was only recentlycloned and sequenced [204], can also be applied for the formation of D-phenylglycine,but has no activity for the other D-a-H-a-amino acids [205]. Due to the low cost ofthese two aromatic D-amino acids and the expensivea-keto acid precursors, this novelbiocatalyst is still of limited commercial relevance [196]. However, this may change

Scheme3.13 Synthetic biochemical pathway for theproductionofD-aminobutyric acid (32) employing a D-amino acidaminotransferase (D-AT). Both substrates are synthesized in situ(i.e., a-ketobutyrate from L-threonine by the action of a threoninedeaminase and D-aspartic acid from L-aspartic acid by the action ofthe enzyme aspartate racemase).


once the scope of this aminotransferase is broadened to other classes of D-aminoacids by protein engineering – a process which will certainly benefit from thestructural information that recently became available [206].

3.8AADH Process

AADHs (EC 1.4.1.X) form a second class of enzymes that catalyze the reductiveamination of a-keto-acids to a-amino acids, in this case with the concomitantoxidation of the cofactor NAD(P)H (Scheme 3.14). These enzymes are found inprokaryotes and eukaryotes, catalyzing the oxidative deamination of amino acids,which is the first step in their degradation for use as carbon and nitrogen sources aswell as for energy [207, 208].In contrast to aminotransferase-catalyzed reactions, the thermodynamics of the

AADH reactions dictates that the equilibrium is usually far on the side of theaminated products with a typical Keq of 9� 1012M�2 (leucine) and 2.2� 1013M�2

(phenylalanine) [208, 209].Only a few of themany AADHs identified are of synthetic interest, amongst which

are alanine dehydrogenase (EC 1.4.1.1), glutamate dehydrogenase (EC 1.4.1.2-4), andparticularly phenylalanine dehydrogenase (EC 1.4.1.20) and leucine dehydrogenase(EC 1.4.1.9) [207, 210–212]. Except for the alanine dehydrogenase from Phormidiumlapideum [210], all AADHs are L-selective.Most of the synthetic work with leucine dehydrogenases has been performed with

the enzymes from Thermoactinomyces intermedius [213] and from several Bacillussp. [214, 215], which appeared to be NADH-specific. The Bacillus sp. leucinedehydrogenases accept a-keto acids with hydrophobic, aliphatic, branched, andunbranched carbon chains from four to six carbon atoms and some alicyclic a-ketoacids, but not aromatic substrates, like L-phenylalanine [212, 214, 215]. On the otherhand, the NADH-specific phenylalanine dehydrogenase enzymes found in a Brevi-bacterium strain [216], aRhodococcus sp. [208, 217], and fromT. intermedius [218] showa very broad substrate specificity, accepting a-keto acids with aromatic as well asaliphatic side-chains. The substrate ranges of phenylalanine dehydrogenase andleucine dehydrogenase are thus complementary.Implementation of AADHs as a competing L-amino acid production technology

requires an efficient system to regenerate the oxidized cofactor, NADþ . This isachieved, for example, by the simultaneous oxidation of glucose to gluconicacid using the glucose dehydrogenase from Bacillus megaterium [219]. Another,

R CO2H

O

R CO2H

NH2

+ NADH + H+ + NH3

L-Amino aciddehydrogenase


+ NAD+ + H2O

Scheme 3.14 General reaction catalyzed by L-selective amino acid dehydrogenases.

3.8 AADH Process j99

more frequently used regeneration system is based on the formate dehydrogenasefrom the yeast Candida boidinii (EC 1.2.1.2) [220] introduced by Whitesides et al.(Scheme 3.15) [221, 222]. Formate dehydrogenase catalyzes the NADþ -dependentoxidation of formate to carbon dioxide. The low-cost availability of formate dehydro-genase and formate [223, 224], the irreversibility of the reaction, and the scalabledownstream processing add to the distinct advantages for industrial application ofthis system [222], despite low specific activity of 6.5U/mg and instability of formatedehydrogenase under process conditions [223].By using an enzyme-membrane reactor (EMR) concept in which both enzymes

are completely retained [225], Kragl et al. developed a cost-efficient process, eitherin a continuous or in a repetitive batch mode [226, 227]. Further improvementswere made by the use of polyethylene glycol (PEG) enlarged NADþ to preventleakage of the expensive cofactor from the EMR. In this way the efficiency of useof this cofactor (total turnover number) could be maximized [221, 225, 228].The EMR has been used in a continuous mode in the synthesis of the unnaturalamino acid L-tert-leucine (35) from trimethylpyruvic acid by the action of theBacillus cereus leucine dehydrogenase and C. boidinii formate dehydrogenaseand applying PEG-enlarged NADþ – a process that has been commercializedby Degussa [225].It has to be noted here that because of the considerable price reduction in recent

years native NADþ can now be used instead of the PEG-enlarged cofactor inthis type of continuous process without additional costs [226]. This led Kragl et al.to develop a new continuous L-tert-leucine (35) process in the EMR with nativeNADþ as cofactor, reaching an average conversion of 93% and a space-time yieldof 366 g/(l�day) [227].Krix et al., in collaborationwithDegussa, developed a repetitive batch EMRprocess

using native NADþ and partially purified leucine dehydrogenase from B. cereus orB. stearothermophilus and formate dehydrogenase from C. boidinii for the gram-scalesynthesis of several unnatural aliphatic a-H-a-amino acids with bulky side-chains [215]. The average enzyme recovery was above 95% (often >99%) forthe leucine dehydrogenases and 70% for the less stable formate dehydrogenase. Inall cases the L-amino acids were obtained in greater than 99.9% e.e. This process was

R CO2H

O

R CO2H

NH2

NADH + H+

HCO2-NH4

+CO2

L-Amino acid dehydrogenase

Formate dehydrogenase


NAD+

Scheme 3.15 Reductive amination process to enantiomericallypure L-amino acids with formate dehydrogenase-based cofactorregeneration.


scaled to 400 l for the synthesis of 30 kg of L-neopentylglycine (36) to prove itscommercial-scale feasibility [215, 229].

L-tert-leucine 35

CO2H

NH2

L-neopentylglycine 36

CO2H

NH2

CO2H

NH2

O

O

(S)-allysine ethylene acetal 37

Apart from processes relying on the use of isolated enzymes, whole-cell processesare increasingly used. Asano et al., for instance, reported on the use of permeabilizedB. sphaericus and C. boidinii whole cells as a source of phenylalanine dehydrogenaseand formate dehydrogenase, respectively, for the synthesis of L-phenylalanine fromphenylpyruvate [230]. At Bristol-Myers Squibb a reductive amination route toL-allysine ethylene acetal (37) has been successfully run on semi-production scaleusing phenylalanine dehydrogenase from T. intermedius ATCC 33 205, in combina-tion with the formate dehydrogenase from C. boidinii SC13 822 [231]. Since theT. intermedius cells quickly lysed at the end of the large-scale fermentation process, itsphenylalanine dehydrogenase gene was expressed in E. coli. Using this recombinantE. coli strain in combination with the heat-dried C. boidinii cells, almost 200 kg ofproduct 37 was prepared with an average yield of 91% and greater than 98%e.e.A further decrease in the cost price of this process appeared to be possible bydevelopment of a recombinant Pichia pastoris strain coexpressing the T. intermediusphenylalanine dehydrogenase gene and the endogenous formate dehydrogenasegene. Using this strain in heat-dried form, 15 kg of 37was produced in 97% yield andgreater than 98%e.e. [231].Two single, E. coli based whole-cell biocatalysts for the asymmetric reductive

amination of a-keto acids have been constructed by Galkin et al. by expression of theformate dehydrogenase fromMycobacterium vaccae [232] in combination with eitherthe leucine dehydrogenase [213] or the phenylalanine dehydrogenase [233] fromT. intermedius. With these cells L-leucine, L-valine, L-norvaline, L-methionine (combi-nation leucine dehydrogenase–formate dehydrogenase), and L-phenylalanine andL-tyrosine (combination phenylalanine dehydrogenase–formate dehydrogenase)were produced in high chemical yields (>88%) and excellent enantioselectivity(>99.9%) [198]. Recently, Degussa also reported on the construction of an E. coliwhole-cell biocatalyst that is based on the coexpression of the gene encoding astabilized formate dehydrogenase mutant [223] and the B. cereus leucine dehydroge-nase gene [234, 235]. Application of this newwhole-cell biocatalyst for the synthesis ofL-tert-leucine (35) and L-neopentylglycine (36) showed that this reaction proceededeven without the addition of an external cofactor [235, 236]. Only at substrateconcentrations exceeding 0.5M did addition of a low amount of additional cofactor(1–10mM) appear to be necessary to get full conversion. Alternatively, near quantita-tive conversion of the substrate trimethylpyruvic acid could be obtained with fed-batch operation of the process.Until recently, synthesis of D-a-H-a-amino acids via enzymatic reductive amina-

tion was impossible because a suitable D-AADH had not been identified. In 2006,

3.8 AADH Process j101

however, engineering of the enzyme meso-2,6-diaminopimelic acid D-hydrogenasefrom Corynebacterium glutamicum led to the first known highly stereoselectiveD-AADH [237]. Application of this mutant D-AADH in the reductive aminationdirection in combination with glucose dehydrogenase to regenerate the NADPþ

cofactor led to the synthesis of D-amino acids in an enantiomeric excess of 95% orhigher. The sole exception was D-alanine, which was obtained in 77%e.e. onlybecause of the presence of an alanine racemase in the partially purified enzymepreparation used.

3.9Conclusions

In this chapter an overview is given of the main chemo-enzymatic platforms for theproduction of enantiomerically pure amino acids that have been commercialized bydifferent companies. Depending on the critical step, these platforms can be dividedinto resolution-based processes (acylase, amidase, and hydantoinase processes) andprocesses that rely on asymmetric synthesis (ammonia lyase-, aminotransferase-,and AADH-based processes). Many factors determine the commercial attractivenessof a biocatalytic process, such as substrate costs and availability, biocatalyst produc-tivity, space-time yield, and costs for downstream processing. As a consequence, it isvery difficult to agree on the most preferred process platform. Resolution-basedprocesses have the intrinsic drawback that the maximum yield is 50% only and theremaining substrate is wasted, but this is of less importance in the case of cheaplyavailable raw materials or simple external recycling loops. Furthermore, futuredevelopments of DKR processes of the acylase- and amidase-based processes will,in principle, lead to a near-quantitative conversion of the racemic substrate. Also, thehydantoinase-based processes, although based on a kinetic resolution, have amaximum yield of 100% due to spontaneous or enzymatic racemization of thenonhydrolyzed hydantoin.Processes relying on an asymmetric synthesis, on the other hand, intrinsically

have a theoretical yield of 100% per cycle. However, this advantage maybe counterbalanced by much higher substrate costs or, even worse, limitedsubstrate availability at large-scale. The AADH route is certainly one of the mostattractive methods to produce L-a-H-a-amino acids today, but it is not suitable forthe production of a,a-disubstituted amino acids. Ammonia lyase-based methods,on the other hand, seem to be especially suited for niche applications because of thegenerally limited substrate spectrum of these enzymes and thermodynamiclimitations. Nevertheless, the L-aspartate ammonia lyase-based synthesis ofL-aspartic acid is one of the most efficient biocatalytic processes currently known.Thus, each of these process platforms has its specific advantages and disadvan-tages. The attractiveness of the different platforms concepts will further depend oncompany-specific knowledge, intellectual property rights, and equipment. It istherefore expected that each of these processes will continue to be in operation, atleast for the next decade.


References

1 Leuchtenberger,W., Huthmacher, K., andDrauz, K. (2005) Biotechnologicalproduction of amino acids andderivatives: current status and prospects.AppliedMicrobiology and Biotechnology, 69,1–8.

2 Ikeda, M. (2003) Amino acidproduction processes. Advances inBiochemical Engineering/Biotechnology,79, 1–35.

3 Oyama, K. (1992) The industrialproduction of aspartame,in Chirality in Industry (eds A.N.Collins, G.N. Sheldrake, and J. Crosby),John Wiley & Sons, Ltd, Chichester,pp. 237–247.

4 Kamphuis, J., Boesten, W.H.J., Kaptein,B., Hermes, H.F.M., Sonke, T.,Broxterman, Q.B., Van den Tweel, W.J.J.,and Schoemaker, H.E. (1992)The production and uses of opticallypure natural and unnatural aminoacids, in Chirality in Industry (eds A.N.Collins, G.N. Sheldrake, and J. Crosby),John Wiley & Sons, Ltd, Chichester,pp. 187–208.

5 Wegman, M.A., Janssen, M.H.A., vanRantwijk, F., and Sheldon, R.A. (2001)Towards biocatalytic synthesis of b-lactamantibiotics. Advanced Synthesis andCatalysis, 343, 559–576.

6 Kleemann, A., Engel, J., Reichert, D.,and Kutscher, B. (1999) PharmaceuticalSubstances: Syntheses, Patents,Applications, Thieme, Stuttgart,pp. 1213–1215.

7 Teetz, V., Geiger, R., Henning, R., andUrbach, H. (1984) Synthesis of a highlyactive angiotensin converting enzymeinhibitor: 2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S,3S,5S)-2-azabicyclo[3.3.0]octane-3-carboxylic acid(Hoe 498). Arzneimittelforschung, 34,1399–1401.

8 Bold, G., F€assler, A., Capraro, H.-G.,Cozens, R., Klimkait, T., Lazdins, J.,Mestan, J., Poncioni, B., R€osel, J., Stover,

D., Tintelnot-Blomley, M., Acemoglu, F.,Beck, W., Boss, E., Eschbach, M.,H€urlimann, T., Masso, E., Roussel, S.,Ucci-Stoll, K., Wyss, D., and Lang, M.(1998) New aza-dipeptide analogues aspotent andorally absorbedHIV-1proteaseinhibitors: candidates for clinicaldevelopment. Journal of MedicinalChemistry, 41, 3387–3401.

9 Henrick, C.A. and Garcia, B.A. (1973)Esters and thiolesters of amino acids,processes for their production, andcompositions including them, GB1,588,111 to Zoecon Corporation.Chemical Abstracts, 78, 123297.

10 Stepek, W.J. and Nigro, M.M. (1986)Novel process for the preparation ofaminonitriles useful for the preparationof herbicides, EP 0,123,830 to AmericanCyanamid Company. Chemical Abstracts,105, 148198.

11 Tomlin, C.D.S. (2003) The PesticideManual, British Crop Protection Council,Alton, pp. 555–556.

12 Genix, P., Guesnet, J.-L., and Lacroix, G.(2003) Chemistry and stereo-chemistry offenamidone. Pflanzenschutz –

Nachrichten Bayer, 56, 421–434.13 Lacombe, J.-P., Patty, L., and Steiger, D.

(2001) Fenamidone. An antimildewcompound for grapevine and potatoes.Phytocoenologia, 535, 42–44.

14 Mueller, U. and Huebner, S. (2003)Economic aspects of amino acidsproduction. Advances in BiochemicalEngineering/Biotechnology, 79, 137–170.

15 Knowles, W.S. (2004) Asymmetrichydrogenations – the Monsanto L-DOPAprocess, in Asymmetric Catalysis onIndustrial Scale: Challenges, Approachesand Solutions (eds H.-U. Blaser and E.Schmidt), Wiley-VCH Verlag GmbH,Weinheim, pp. 23–38.

16 N�ajera, C. and Sansano, J.M. (2007)Catalytic asymmetric synthesis ofa-amino acids. Chemical Reviews, 107,4584–4671.

References j103

17 Vogt, H. and Br€ase, S. (2007) Recentapproaches towards the asymmetricsynthesis of a,a-disubstituted a-aminoacids.Organic andBiomolecularChemistry,5, 406–430.

18 Chibata, I., Tosa, T., and Shibatani, T.(1992) The industrial production ofoptically active compounds byimmobilized biocatalysts, in Chirality inIndustry (edsA.N.Collins,G.N. Sheldrake,and J. Crosby), John Wiley & Sons,Chichester, Ltd, pp. 351–370.

19 Bommarius, A.S., Schwarm, M., andDrauz, K. (2001) Comparison of differentchemoenzymatic process routes toenantiomerically pure amino acids.Chimia, 55, 50–59.

20 Bommarius, A.S. (2002) Hydrolysis ofN-acylamino acids, in Enzyme Catalysis inOrganic Synthesis (eds K. Drauz and H.Waldman), Wiley-VCH Verlag GmbH,Weinheim, pp. 741–760.

21 Sonntag, N.O.V. (1953) The reactions ofaliphatic acid chlorides.Chemical Reviews,52, 237–416.

22 Beller, M., Eckert, M., and Moradi, W.A.(1999) First amidocarbonylation withnitriles for the synthesis of N -acyl aminoacids. Synlett, 108–110.

23 Wandrey, C. and Flaschel, E. (1979)Process development and economicaspects in enzyme engineering. AcylaseL-methionine system. Advances inBiochemical Engineering, 12, 147–218.

24 Galaev, I.Y. and Švedas, V.K. (1982) Akinetic study of hog kidney aminoacylase.Biochimica et Biophysica Acta, 701,389–394.

25 Bommarius, A.S., Drauz, K., Groeger, U.,and Wandrey, C. (1992) Membranebioreactors for the production ofenantiomerically pure a-amino acids,in Chirality in Industry (eds A.N.Collins, G.N. Sheldrake, and J. Crosby),John Wiley & Sons, Ltd, Chichester,pp. 371–397.

26 Liese, A., Seelbach, K., and Wandrey,C. (2000) Industrial Biotransformations,Wiley-VCH Verlag GmbH, Weinheim.

27 Tokuyama, S. and Hatano, K. (1995)Purification and properties ofthermostable N-acylamino acid racemasefrom Amycolatopsis sp. TS-1-60. AppliedMicrobiology and Biotechnology, 42,853–859.

28 Tokuyama, S., Hatano, K., and Takahashi,T. (1994) A novel enzyme, N-acylaminoacid racemase, in actinomycetes. Part 1.Discovery of a novel enzyme, N-acylamino acid racemase in anActinomycete: screening, isolation andidentification. Bioscience, Biotechnology,and Biochemistry, 58, 24–27.

29 Tokuyama, S., Miya, H., Hatano, K., andTakahashi, T. (1994) Purification andproperties of a novel enzyme, N-acylamino acid racemase, fromStreptomyces atratus Y-53. AppliedMicrobiology and Biotechnology, 40,835–840.

30 May, O., Verseck, S., Bommarius, A.,and Drauz, K. (2002) Development ofdynamic kinetic resolution processesfor biocatalytic production of naturaland nonnatural L-amino acids. OrganicProcess Research & Development, 6,452–457.

31 Hsu, S.-K., Lo, H.-H., Kao, C.-H.,Lee, D.-S., and Hsu, W.-H. (2006)Enantioselective synthesis of L-homophenylalanine by whole cells ofrecombinantEscherichia coli expressingL-aminoacylase and N-acylamino acidracemase genes from Deinococcusradiodurans BCRC12827. BiotechnologyProgress, 22, 1578–1584.

32 Tokuyama, S. (2001) Discovery andapplication of a new enzymeN-acylaminoacid racemase. Journal of MolecularCatalysis B – Enzymatic, 12, 3–14.

33 Tokuyama, S. and Hatano, K. (1996)Overexpression of the gene for N-acylamino acid racemase fromAmycolatopsis sp. TS-1-60 in Escherichiacoli and continuous produciton ofoptically active methionine by abioreactor. Applied Microbiology andBiotechnology, 44, 774–777.


34 Groeger, U., Leuchtenberger, W., andDrauz, K. (1991) Substantially purifiedN-acyl-L-proline acylase from Comamonastestosteroni DSM 5416 and Alcaligenesdenitrificans DSM 5417, US 5,120,652 toDegussa AG. Chemical Abstracts, 115,130669.

35 Kikuchi, M., Koshiyama, I., andFukushima, D. (1983) A new enzyme,proline acylase (N-acyl-L-prolineamidohydrolase) from Pseudomonasspecies. Biochimica et Biophysica Acta,744, 180–188.

36 Groeger, U., Drauz, K., and Klenk, H.(1992) Enzymatic preparation ofenantiomerically pure N-alkyl aminoacids. Angewandte Chemie (InternationalEdition in English), 31, 195–197.

37 Sugie, M. and Suzuki, H. (1980) Opticalresolution of DL-amino acids with D-aminoacylase of Streptomyces.Agriculturaland Biological Chemistry, 44, 1089–1095.

38 Sakai, K., Oshima, K., and Moriguchi, M.(1991) Production and characterization ofN-acyl-D-glutamate amidohydrolase fromPseudomonas sp. strain 5f-1. Applied andEnvironmental Microbiology, 57,2540–2543.

39 Yang, Y.-B., Lin, C.-S., Tseng, C.-P., Wang,Y.-J., and Tsai, Y.-C. (1991) Purificationand characterization of D-aminoacylasefrom Alcaligenes faecalis DA1. Applied andEnvironmental Microbiology, 57,1259–1260.

40 Kamphuis, J., Boesten, W.H.J.,Broxterman, Q.B., Hermes, H.F.M., vanBalken, J.A.M., Meijer, E.M., andSchoemaker, H.E. (1990) Newdevelopments in the chemo-enzymaticproduction of amino acids. Advances inBiochemical Engineering/Biotechnology, 42,133–186.

41 Kamphuis, J., Meijer, E.M., Boesten,W.H.J., Broxterman, Q.B., Kaptein, B.,Hermes, H.F.M., and Schoemaker, H.E.(1992) Production of natural andsynthetic L- and D-amino acids byaminopeptidases and amino amidases, inBiocatalytic Production of Amino Acids and

Derivatives (eds J.D. Rozzell and F.Wagner), Hanser, Munich,pp. 177–206.

42 Boesten, W.H.J. (1977) Process forpreparing a-amino-acid amides, GB1,548,032 to DSM/Stamicarbon BV.Chemical Abstracts, 87, 39839.

43 Hyett, D.J., Didon�e, M., Milcent, T.J.A.,Broxterman, Q.B., and Kaptein, B. (2006)A new method for the preparation offunctionalized unnatural a-H-a-aminoacid derivatives. Tetrahedron Letters, 47,7771–7774.

44 Asano, Y.,Mori, T.,Hanamoto, S., Kato, Y.,and Nakazawa, A. (1989) A newD-stereospecific amino acid amidase fromOchrobactrum anthropi. Biochemical andBiophysical Research Communications,162, 470–474.

45 Asano, Y., Nakazawa, A., Kato, Y., andKondo, K. (1989) Properties of a novelD-stereospecific aminopeptidase fromOchrobactrum anthropi. The Journal ofBiological Chemistry, 264, 14233–14239.

46 Shadid, B., van der Plas, H.C., Boesten,W.H.J., Kamphuis, J., Meijer, E.M., andSchoemaker, H.E. (1990) The synthesis ofL-(�)- and D-(þ )-lupinic acid.Tetrahedron,46, 913–920.

47 Rutjes, F.P.J.T. and Schoemaker, H.E.(1997) Ruthenium-catalyzed ring closingolefin metathesis of non-natural a-aminoacids. Tetrahedron Letters, 38, 677–680.

48 Wolf, L.B., Tjen, K.C.M.F., Rutjes,F.P.J.T., Hiemstra, H., and Schoemaker,H.E. (1998) Pd-catalyzed cyclizationreactions of acetylene-containing a-amino acids. Tetrahedron Letters, 39,5081–5084.

49 Hermes, H.F.M., Sonke, T., Peters,P.J.H., van Balken, J.A.M., Kamphuis, J.,Dijkhuizen, L., and Meijer, E.M. (1993)Purification and characterization of anL-aminopeptidase from Pseudomonasputida ATCC 12633. Applied andEnvironmental Microbiology, 59,4330–4334.

50 Sonke, T., Kaptein, B., Boesten, W.H.J.,Broxterman, Q.B., Kamphuis, J.,

References j105

Formaggio, F., Toniolo, C., Rutjes,F.P.J.T., and Schoemaker, H.E. (2000)Aminoamidase-catalyzed preparationand further transformations ofenantiopure a-hydrogen- anda,a-disubstituted a-amino acids, inStereoselective Biocatalysis(ed. R.N. Patel), Marcel Dekker,New York, pp. 23–58.

51 Wolf, L.B., Sonke, T., Tjen, K.C.M.F.,Kaptein, B., Broxterman, Q.B.,Schoemaker, H.E., and Rutjes, F.P.J.T.(2001) A biocatalytic route toenantiomerically pure unsaturated a-H-a-amino acids. Advanced Synthesis andCatalysis, 343, 662–674.

52 Boesten, W.H.J., Schoemaker, H.E., andDassen, B.H.N. (1987) Process forracemizing an optically activeN-benzylidene amino-acid amide, EP0,199,407 to Stamicarbon BV. ChemicalAbstracts, 107, 40327.

53 Boesten, W.H.J., Raemakers-Franken,P.C., Sonke, T., Euverink, G.J.W., andGrijpstra, P. (2003) Polypeptides havinga-H-a-amino acid amide racemaseactivity and nucleic acids encodingthe same, WO 2003/106691 to DSMIP Assets BV. Chemical Abstracts, 140,55597.

54 Sonke, T. (2008) Novel developments inthe chemo-enzymatic synthesis ofenantiopure a-hydrogen- anda,a-disubstituted a-amino acids andderivatives, PhD Thesis, University ofAmsterdam.

55 Fukumura, T. (1977) Conversion of D- andDL-a-amino-e-caprolactam into L-lysineusing both yeast cells and bacterial cells.Agricultural and Biological Chemistry, 41,1327–1330.

56 Ahmed, S.A., Esaki, N., Tanaka, H., andSoda, K. (1983) Properties of a-amino-e-caprolactamracemasefromAchromobacterobae. Agricultural and Biological Chemistry,47, 1887–1893.

57 Asano, Y. and Yamaguchi, S. (2005)Discovery of amino acid amides as newsubstrates for a-amino-e-caprolactam

racemase from Achromobacter obae.Journal of Molecular Catalysis B –

Enzymatic, 36, 22–29.58 Asano, Y. and Yamaguchi, S. (2005)

Dynamic kinetic resolution of amino acidamide catalyzed by D-aminopeptidase anda-amino-e-caprolactam racemase. Journalof the American Chemical Society, 127,7696–7697.

59 Kaptein, B., Boesten,W.H.J., Broxterman,Q.B., Schoemaker, H.E., and Kamphuis,J. (1992) Synthesis of a,a-disubstituteda-amino acid amides by phase-transfercatalyzed alkylation. Tetrahedron Letters,33, 6007–6010.

60 Roos, E.C., López, M.C., Brook, M.A.,Hiemstra, H., Speckamp, W.N., Kaptein,B., Kamphuis, J., and Schoemaker, H.E.(1993) Synthesis of a-substituteda-amino acids via cationic intermediates.The Journal of Organic Chemistry, 58,3259–3268.

61 Becke, F., Fleig, H., and P€assler, P. (1971)General method for the preparation ofamides from their corresponding nitriles.II.AnnalenDerChemie– Justus Liebig, 749,198–201.

62 Kaptein, B., Boesten,W.H.J., Broxterman,Q.B., Peters, P.J.H., Schoemaker, H.E.,and Kamphuis, J. (1993) Enzymaticresolution of a,a-disubstituted a-aminoacid esters and amides. Tetrahedron:Asymmetry, 4, 1113–1116.

63 Kruizinga, W.H., Bolster, J., Kellogg,R.M., Kamphuis, J., Boesten, W.H.J.,Meijer, E.M., and Schoemaker,H.E. (1988) Synthesis of optically pure a-alkylated a-amino acids and a single-stepmethod for enantiomeric excessdetermination. The Journal of OrganicChemistry, 53, 1826–1827.

64 Hermes, H.F.M., Tandler, R.F., Sonke, T.,Dijkhuizen, L., and Meijer, E.M. (1994)Purification and characterization of an L-amino amidase from Mycobacteriumneoaurum ATCC 25795. Applied andEnvironmental Microbiology, 60, 153–159.

65 Van den Tweel, W.J.J., van Dooren,T.J.G.M., de Jonge, P.H., Kaptein, B.,


Duchateau, A.L.L., and Kamphuis, J.(1993) Ochrobactrum anthropi NCIMB40321: a new biocatalyst with broad-spectrum L-specific amidase activity.Applied Microbiology and Biotechnology, 39,296–300.

66 Kaptein, B., van Dooren, T.J.G.M.,Boesten, W.H.J., Sonke, T., Duchateau,A.L.L., Broxterman, Q.B., and Kamphuis,J. (1998) Synthesis of 4-sulfur-substituted(2S,3R)-3-phenylserines by enzymaticresolution. Enantiopure precursors forthiamphenicol and florfenicol.Organic Process Research & Development,2, 10–17.

67 Gouret, C.J., Porsolt, R., Wettstein, J.G.,Puech, A., Soulard, C., Pascaud, X., andJunien, J.L. (1990) Biochemical andpharmacological evaluation of the novelantidepressant and serotonin uptakeinhibitor (2-(3,4-dichlorobenzyl)-2-dimethylamino-1-propanolhydrochloride.Arzneimittelforschung, 40, 633–640.

68 Gouret, C.J., Wettstein, J.G., Porsolt,R.D., Puech, A., and Junien, J.L. (1990)Neuropsychopharmacologial profileof JO 1017, a new antidepressant andselective semtonin uptake inhibitor.European Journal of Pharmacology, 183,1478.

69 Kaptein, B., Moody, H.M., Broxterman,Q.B., and Kamphuis, J. (1994) Chemo-enzymatic synthesis of (S)-(þ )-cericlamine and related enantiomericallypure 2,2-disubstituted-2-aminoethanols.Journal of the Chemical Society, PerkinTransactions 1, 1495–1498.

70 Sonke, T., Ernste, S., Tandler, R.F.,Kaptein, B., Peeters, W.P.H., vanAssema, F.B.J., Wubbolts, M.G., andSchoemaker, H.E. (2005) L-Selectiveamidase with extremely broad substratespecificity from Ochrobactrum anthropiNCIMB 40321. Applied andEnvironmental Microbiology, 71,7961–7973.

71 Nakamura, T. and Yu, F. (2000) Amidasegene, US 6,617,139 to Mitsubishi RayonCo. Ltd. Chemical Abstracts, 133, 319048.

72 Katoh, O., Akiyama, T., and Nakamura, T.(2003) Novel amide hydrolase gene, EP1,428,876 to Mitsubishi Rayon Co. Ltd.Chemical Abstracts, 138, 233991.

73 Shaw, N.M., Naughton, A., Robins, K.,Tinschert, A., Schmid, E., Hischier,M.-L.,Venetz, V., Werlen, J., Zimmermann, T.,Brieden, W., de Riedmatten, P., Roduit,J.-P., Zimmermann, B., and Neum€uller,R. (2002) Selection, purification,characterisation, and cloning of a novelheat-stable stereo-specific amidase fromKlebsiella oxytoca, and its application inthe synthesis of enantiomerically pure(R)- and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide. Organic ProcessResearch & Development, 6, 497–504.

74 Brieden, W., Naughton, A., Robins, K.,Shaw, N.M., Tinschert, A., andZimmermann, T. (1998) Method ofpreparing (S)- or (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, US6,773,910 to Lonza AG. ChemicalAbstracts, 128, 153206.

75 Ogawa, J. and Shimizu, S. (2000)Stereoselective synthesis usinghydantoinases and carbamoylases, inStereoselective Biocatalysis (ed. R.N. Patel),Marcel Dekker, New York, pp. 1–21.

76 Pietzsch, M. and Syldatk, C. (2002)Hydrolysis and formation of hydantoins,in Enzyme Catalysis in Organic Synthesis(eds K. Drauz and H. Waldman),Wiley-VCH Verlag GmbH, Weinheim,pp. 761–799.

77 Baldaro, E.M. (1993) Chemo-enzymaticproduction of D-amino acids.Pharmaceutical ManufacturingInternational, 137–139.

78 Olivieri, R., Fascetti, E., Angelini, L., andDegen, L. (1979) Enzymatic conversion ofN-carbamoyl-D-amino acids to D-aminoacids. Enzyme andMicrobial Technology, 1,201–204.

79 Olivieri, R., Fascetti, E., Angelini, L., andDegen, L. (1981) Microbialtransformation of racemic hydantoins to

References j107

D-amino acids. Biotechnology andBioengineering, 23, 2173–2183.

80 Drauz, K., Kottenhahn, M., Makryaleas,K., Klenk, H., and Bernd, M. (1991)Chemoenzymatic syntheses of w-ureidoD-amino acids. Angewandte Chemie(International Edition in English), 30,712–714.

81 Kim, G.J. and Kim, H.S. (1994)Adsorptive removal of inhibitorybyproduct in the enzymatic production ofoptically active D-p-hydroxyphenylglycinefrom 5-substituted hydantoin.Biotechnology Letters, 16, 17–22.

82 Kim, G.-J. and Kim, H.-S. (1995)Optimization of the enzymatic synthesisof D-p-hydroxyphenylglycine from DL-5-substituted hydantoin using D-hydantoinase and N-carbamoylase.Enzyme and Microbial Technology, 17,63–67.

83 Boesten, W.H.J. and Kierkels, J.G.T.(2002) Process for the preparation ofenantiomer-enriched amino acids, WO2002/061107 to DSM N.V. ChemicalAbstracts, 137, 139489.

84 Ikenaka, Y., Nanba, H., Yajima, K.,Yamada, Y., Takano, M., and Takahashi, S.(1998) Increase in thermostability ofN-carbamyl-D-amino acid amidohydrolaseon amino acid substitutions. Bioscience,Biotechnology, and Biochemistry, 62,1668–1671.

85 Ikenaka, Y., Nanba, H., Yajima, K.,Yamada, Y., Takano, M., and Takahashi,S. (1998) Relationship between anincrease in thermostability and aminoacid substitutions inN-carbamyl-D-aminoacid amidohydrolase. Bioscience,Biotechnology, and Biochemistry, 62,1672–1675.

86 Oh, K.-H., Nam, S.-H., and Kim, H.-S.(2002) Improvement of oxidative andthermostability of N-carbamyl-D-aminoacid amidohydrolase by directedevolution. Protein Engineering, 15,689–695.

87 Nanba, H., Ikenaka, Y., Yamada, Y.,Yajima, K., Takano, M., Ohkubo, K.,

Hiraishi, Y., Yamada,K., andTakahashi, S.(1998) Immobilization of N-carbamyl-D-amino acid amidohydrolase. Bioscience,Biotechnology, and Biochemistry, 62,1839–1844.

88 Battilotti, M. and Barberini, U. (1988)Preparation of D-valine from D,L-5-isopropylhydantoin by stereoselectivebiocatalysis. Journal ofMolecular Catalysis,43, 343–352.

89 Syldatk, C., M€uller, R., Pietzsch, M.,and Wagner, F. (1992) Microbial andenzymatic production of L-amino acidsfrom DL-5-monosubstituted hydantoins,in Biocatalytic Production of Amino Acids &Derivatives (eds D. Rozzell and F.Wagner), Hanser, Munich, pp. 129–176.

90 Watabe, K., Ishikawa, T., Mukohara, Y.,and Nakamura, H. (1992) Purificationand characterization of the hydantoinracemase of Pseudomonas sp. strainNS671 expressed in Escherichia coli.Journal of Bacteriology, 174, 7989–7995.

91 Watabe, K., Ishikawa, T., Mukohara, Y.,and Nakamura, H. (1992) Identificationand sequencing of a gene encoding ahydantoin racemase from the nativeplasmid of Pseudomonas sp. strain NS671.Journal of Bacteriology, 174, 3461–3466.

92 Wiese, A., Pietzsch, M., Syldatk, C.,Mattes, R., and Altenbuchner, J. (2000)Hydantoin racemase from Arthrobacteraurescens DSM 3747: heterologousexpression, purification andcharacterization. Journal of Biotechnology,80, 217–230.

93 Mart�ınez-Rodr�ıguez, S., Las Heras-V�azquez, F.J., Clemente-Jim�enez, J.M.,and Rodr�ıguez-Vico, F. (2004)Biochemical characterization of a novelhydantoin racemase from Agrobacteriumtumefaciens C58. Biochimie, 86, 77–81.

94 Mart�ınez-Rodr�ıguez, S., Las Heras-V�azquez, F.J., Mingorance-Cazorla, L.,Clemente-Jim�enez, J.M., and Rodr�ıguez-Vico, F. (2004) Molecular cloning,purification, and biochemicalcharacterization of hydantoin racemasefrom the legume symbiont Sinorhizobium


meliloti CECT 4114. Applied andEnvironmental Microbiology, 70,625–630.

95 Suzuki, S., Onishi, N., and Yokozeki, K.(2005) Purification and characterizationof hydantoin racemase fromMicrobacterium liquefaciens AJ 3912.Bioscience, Biotechnology, and Biochemistry,69, 530–536.

96 Boesten, W.H.J., Kierkels, J.G.T., vanAssema, F.B.J., Ruiz Perez, L.M.,Gonzales Pacanowska, D., GonzalesLopez, J., and De La Escalera Huesco, S.(2003) Hydantoin racemase, WO 2003/100050 to DSM N.V. Chemical Abstracts,140, 14387.

97 Watabe, K., Ishikawa, T., Mukohara, Y.,and Nakamura, H. (1992) Cloning andsequencing of the genes involved in theconversion of 5-substituted hydantoins tothe corresponding L-amino acids from thenative plasmid of Pseudomonas sp. strainNS671. Journal of Bacteriology, 174,962–969.

98 Ishikawa, T., Mukohara, Y., Watabe, K.,Kobayashi, S., and Nakamura, H. (1994)Microbial conversion of DL-5-substitutedhydantoins to the corresponding L-aminoacids by Bacillus stearothermophilusNS1122A. Bioscience, Biotechnology, andBiochemistry, 58, 265–270.

99 Wagner, T., Hantke, B., and Wagner, F.(1996) Production of L-methionine fromD,L-5-(2-methylthioethyl) hydantoin byresting cells of a new mutant strain ofArthrobacter species DSM 7330. Journal ofBiotechnology, 46, 63–68.

100 May,O., Siemann,M., Pietzsch,M.,Kiess,M., Mattes, R., and Syldatk, C. (1998)Substrate-dependent enantioselectivity ofa novel hydantoinase from Arthrobacteraurescens DSM 3745: purification andcharacterization as new member of cyclicamidases. Journal of Biotechnology, 61,1–13.

101 Nozaki, H., Takenaka, Y., Kira, I.,Watanabe, K., and Yokozeki, K. (2005)D-Amino acid production by E. colico-expressed three genes encoding

hydantoin racemase, D-hydantoinase andN-carbamoyl-D-amino acidamidohydrolase. Journal of MolecularCatalysis B – Enzymatic, 32, 213–218.

102 Wilms, B., Wiese, A., Syldatk, C., Mattes,R., and Altenbuchner, J. (2001)Development of an Escherichia coli wholecell biocatalyst for the production ofL-amino acids. Journal of Biotechnology, 86,19–30.

103 May, O., Nguyen, P.T., and Arnold, F.H.(2000) Inverting enantioselectivity bydirected evolution of hydantoinase for theimproved production of L-methionine.Nature Biotechnology, 18, 317–320.

104 May, O., Buchholz, S., Schwarm, M.,Drauz, K., Turner, R.J., andFotheringham, I. (2003) Mutants for thepreparation of D-amino acids, WO 2004/042047 to Degussa AG. ChemicalAbstracts, 140, 405577.

105 Turner, R.J., Aikens, J., Royer, S.,DeFilippi, L., Yap, A., Holzle, D., Somers,N., and Fotheringham, I.G. (2004)D-Amino acid tolerant hosts forD-hydantoinase whole cell biocatalysts.Engineering in Life Sciences, 4, 517–520.

106 Wubbolts, M.G. (2002) Addition ofamines to C¼ C bonds, in EnzymeCatalysis in Organic Synthesis (eds K.Drauz and H. Waldman), Wiley-VCHVerlag GmbH, Weinheim, pp. 866–872.

107 Calton, G.J. (1992) The enzymaticproduction of L-aspartic acid, inBiocatalytic Production of Amino Acids andDerivatives (eds J.D. Rozzell and F.Wagner), Hanser, Munich, pp. 3–21.

108 Chibata, I., Tosa, T., and Sato, T.(1973) Process for the productionof L-aspartic acid, US 3,791,926 toTanabe Seiyaku Co. Chemical Abstracts,79, 30499.

109 Tosa, T., Sato, T., Mori, T., and Chibata, I.(1974) Basic studies for continuousproduction of L-aspartic acid byimmobilized Escherichia coli cells. AppliedMicrobiology, 27, 886–889.

110 Sato, T., Mori, T., Tosa, T., Chibata, I.,Furui, M., Yamashita, K., and Sumi, A.

References j109

(1975) Engineering analysis ofcontinuous production of L-aspartic acidby immobilized Escherichia coli cells infixed beds. Biotechnology andBioengineering, 17, 1797–1804.

111 Sato, T., Nishida, Y., Tosa, T., and Chibata,I. (1979) Immobilization ofEscherichia colicells containing aspartase activity withk-carrageenan. Enzymic properties andapplication for L-aspartic acid production.Biochimica et Biophysica Acta, 570,179–186.

112 Umemura, I., Takamatsu, S., Sato, T.,Tosa, T., and Chibata, I. (1984)Improvement of production of L-asparticacid using immobilized microbial cells.Applied Microbiology and Biotechnology, 20,291–295.

113 Sato, T. and Tosa, T. (1993) Production ofL-aspartic acid, in Industrial Application ofImmobilized Biocatalysts (eds A. Tanaka,T. Tosa, and T. Kobayashi), Marcel Dekker,New York, pp. 15–24.

114 Terasawa,M., Yukawa, H., and Takayama,Y. (1985) Production of L-aspartic acidfrom Brevibacterium by the cell re-usingprocess.Process Biochemistry, 20, 124–128.

115 Wood, L.L. and Calton, G.J. (1984) A novelmethod of immobilization and its use inaspartic acid production. NatureBiotechnology, 2, 1081–1084.

116 Wood, L.L. and Calton, G.J. (1983)Immobilization of cells with apolyazatidine prepolymer, US 4,732,851to Purification Engineering Inc. ChemicalAbstracts, 99, 211194.

117 Yamagata, H., Terasawa, M., and Yukawa,H. (1994) A novel industrial process forL-aspartic acid production using anultrafiltrationmembrane. Catalysis Today,22, 621–628.

118 Jandel, A.-S., Hustedt, H., and Wandrey,C. (1982) Continuous production ofL-alanine from fumarate in a two-stagemembrane reactor. European Journal ofApplied Microbiology and Biotechnology, 15,59–63.

119 Asai, Y., Inui, M., Vertes, A., Kobayashi,M., and Yukawa, H. (1995) Cloning

and sequence determination of theaspartase-encoding gene fromBrevibacterium flavum MJ-233. Gene,158, 87–90.

120 Kisumi, M., Komatsubara, S., andTaniguchi, T. (1985) Method forproducing L-aspartic acid, US 4,692,409 toTanabe Seiyaku Co. Chemical Abstracts,102, 111473.

121 Nishimura, N., Taniguchi, T., andKomatsubara, S. (1989) Hyperproductionof aspartase by a catabolite repression-resistant mutant of Escherichia coli Bharboring multicopy aspA and parrecombinant plasmids. Journal ofFermentation and Bioengineering, 67,107–110.

122 Murase, S., Takagi, J.S., Higashi, Y.,Imaishi, H., Yumoto, N., and Tokushige,M. (1991) Activation of aspartase by site-directed mutagenesis. Biochemical andBiophysical Research Communications,177, 414–419.

123 Wang, L.-j., Kong, X.-d., Zhang, H.-y.,Wang, X.-p., and Zhang, J. (2000)Enhancement of the activity of L-aspartasefrom Escherichia coli W by directedevolution. Biochemical and BiophysicalResearch Communications, 276, 346–349.

124 Kawata, Y., Tamura, K., Yano, S.,Mizobata, T., Nagai, J., Esaki, N., Soda, K.,Tokushige, M., and Yumoto, N. (1999)Purification and characterization ofthermostable aspartase from Bacillus sp.YM55-1. Archives of Biochemistry andBiophysics, 366, 40–46.

125 Shi, W., Dunbar, J., Jayasekera, M.M.K.,Viola, R.E., and Farber, G.K. (1997) Thestructure of L-aspartate ammonia-lyasefrom Escherichia coli. Biochemistry, 36,9136–9144.

126 Fujii, T., Sakai,H., Kawata, Y., andHata, Y.(2003) Crystal structure of thermostableaspartase from Bacillus sp. YM55-1:structure-based exploration of functionalsites in the aspartase family. Journal ofMolecular Biology, 328, 635–654.

127 Goto, M., Nara, T., Tokumaru, I., Fugono,N., Uchida, Y., Terasawa,M., and Yukawa,


H. (1996) Method of producing fumaricacid, EP 0,693,557 to MitsubishiChemical Corporation. ChemicalAbstracts, 124, 143771.

128 Kobayashi, M., Terasawa, M., andYukawa, H. (1999) L-Aspartic acid, inEncyclopedia of Bioprocess Technology –Fermentation, Biocatalysis, andBioseparation (eds M.C. Flickinger andS.W. Drew), John Wiley & Sons, Inc.,New York, pp. 210–213.

129 Rozzell, J.D. (1999) Biocatalysis atcommercial scale. Myths and realities.Chimica Oggi – Chemistry Today, 17,42–47.

130 Kumagai, H. (2006) Amino acidproduction, in The Prokaryotes, 3rd edn,Vol. 1 (eds M. Dwerkin, S. Falkow, E.Rosenberg, K-.H. Schleifer, and E.Stackebrandt), Springer, Berlin,pp. 756–765.

131 Hashimoto, S.-i. andKatsumata, R. (1998)L-Alanine fermentation by an alanineracemase-deficient mutant of theDL-alanine hyperproducing bacteriumArthrobacter oxydans HAP-1. Journal ofFermentation and Bioengineering, 86,385–390.

132 Tate, S.S. and Meister, A. (1969)Regulation of the activity of L-aspartateb-decarboxylase by a novel allostericmechanism. Biochemistry, 8,1660–1668.

133 Calton, G.J. (1992) The enzymaticpreparation of L-alanine, in BiocatalyticProduction of Amino Acids and Derivatives(eds J.D. Rozzell and F. Wagner), Hanser,Munich, pp. 59–74.

134 Furui, M. and Yamashita, K. (1983)Pressurized reaction method forcontinuous production of L-alanine byimmobilizedPseudomonas dacunhae cells.Journal of Fermentation Technology, 61,587–591.

135 Goto, M., Nara, T., Terasawa, M., andYukawa, H. (1991) Process for producingl-alanine, EP 0,386,476 to MitsubishiPetrochemical Co., Ltd. ChemicalAbstracts, 114, 80071.

136 Takamatsu, S., Umemura, I., Yamamoto,K., Sato, T., Tosa, T., and Chibata, I. (1982)Production of L-alanine from ammoniumfumarate using two immobilizedmicroorganisms. Elimination of sidereactions. European Journal of AppliedMicrobiology and Biotechnology, 15,147–152.

137 Tosa, T., Takamatsu, S., Furui, M., andChibata, I. (1984) Continuous productionof L-alanine: successive enzyme reactionswith two immobilized cells. Annals of theNew York Academy of Sciences, 434,450–453.

138 Hanson, K.R. and Havir, E.A. (1978)An introduction to the enzymologyof phenylpropanoid biosynthesis. RecentAdvances in Phytochemistry, 12, 91–137.

139 Nelson, R.P. (1976) Immobilizedmicrobial cells, US 3,957,580 to PfizerInc. Chemical Abstracts, 84, 2022.

140 Robers, F.F. Jr., Hamsher, J.J., andNelson, R.P. (1976) Production ofl-phenylalanine, GB 1,489,468 to PfizerInc. Chemical Abstracts, 84, 178203.

141 Fotheringham, I.G. (1999) Phenylalanine,in Encyclopedia of Bioprocess Technology –Fermentation, Biocatalysis, andBioseparation (eds M.C. Flickinger andS.W. Drew), John Wiley & Sons, Inc.,New York, pp. 1943–1954.

142 Kishore, G.M. (1985) Stabilization ofL-phenylalanine ammonia-lyase enzyme,EP 0,136,996 to Monsanto Company.Chemical Abstracts, 103, 21245.

143 Adachi, O.,Matsushita, K., Shinagawa, E.,and Ameyama, M. (1990) Crystallizationand properties of L-phenylalanineammonia-lyase from Rhodosporidiumtoruloides. Agricultural and BiologicalChemistry, 54, 2839–2843.

144 Rees, D.G. and Jones, D.H. (1996)Stability of L-phenylalanine ammonia-lyase in aqueous solution and as thesolid state in air and organic solvents.Enzyme and Microbial Technology, 19,282–288.

145 Abell, C.W. and Shen, R.S. (1987)Phenylalanine ammonia-lyase from the

References j111

yeast Rhodotorula glutinis. Methods inEnzymology, 142, 242–248.

146 Fritz, R.R.,Hodgins,D.S., andAbell, C.W.(1976) Phenylalanine ammonia-lyase.Induction and purification from yeast andclearance in mammals. The Journal ofBiological Chemistry, 251, 4646–4650.

147 Yamada, S., Nabe, K., Izuo, N.,Nakamichi, K., and Chibata, I. (1981)Production of L-phenylalanine from trans-cinnamic acid with Rhodotorula glutiniscontaining L-phenylalanine ammonia-lyase activity. Applied and EnvironmentalMicrobiology, 42, 773–778.

148 Takaç, S., Akay, B., and Özdamar, T.H.(1995) Bioconversion of trans-cinnamicacid to L-phenylalanine by L-phenylalanineammonia-lyase of Rhodotorula glutinis:parameters and kinetics. Enzyme andMicrobial Technology, 17, 445–452.

149 Vollmer, P.J., Montgomery, J.P.,Schruber, J.J., and Yang, H.-H. (1985)Method for stabilizing the enzymicactivity of phenylalanine ammonia lyaseduring l-phenylalanine production, EP0,143,560 toGenexCorporation.ChemicalAbstracts, 103, 69802.

150 Evans, C.T., Conrad, D., Hanna, K.,Peterson, W., Choma, C., andMisawa, M.(1987) Novel stabilization ofphenylalanine ammonia-lyase catalystduring bioconversion of trans-cinnamicacid to L-phenylalanine. AppliedMicrobiology and Biotechnology, 25,399–405.

151 El-Batal, A.I. (2002) Optimization ofreaction conditions and stabilization ofphenylalanine ammonia lyase-containingRhodotorula glutinis cells duringbioconversion of trans-cinnamic acid toL-phenylalanine. Acta MicrobiologicaPolonica, 51, 139–152.

152 Vollmer, P.J. and Schruben, J.J. (1986)Stabilization of phenylalanine ammonia-lyase inabioreactorusing reducingagents,US 4,574,117 to Genex Corporation.Chemical Abstracts, 104, 166923.

153 Evans, C.T.,Hanna, K., Payne, C., Conrad,D., and Misawa, M. (1987)

Biotransformation of trans-cinnamic acidto L-phenylalanine: optimization ofreaction conditions using whole yeastcells. Enzyme and Microbial Technology, 9,417–421.

154 Orndorff, S.A., Costantino, N., Stewart,D., and Durham, D.R. (1988) Strainimprovement of Rhodotorula graminis forproduction of a novel L-phenylalanineammonia-lyase. Applied andEnvironmental Microbiology, 54, 996–1002.

155 Orndorff, S.A. and Durham, D.R. (1989)Phenylalanine ammonia lyase-producingstrains, US, 4,757,015 to GenexCorporation. Chemical Abstracts, 110,73881.

156 Evans, C.T., Hanna, K., Conrad, D.,Peterson, W., and Misawa, M. (1987)Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation ofyeast strains suitable for commercialproduction of L-phenylalanine. AppliedMicrobiology and Biotechnology, 25,406–414.

157 Baedeker, M. and Schulz, G.E. (1999)Overexpression of a designed 2.2 kb geneof eukaryotic phenylalanine ammonia-lyase in Escherichia coli. FEBS Letters, 457,57–60.

158 Xiang, L. and Moore, B.S. (2005)Biochemical characterization of aprokaryotic phenylalanine ammonialyase. Journal of Bacteriology, 187,4286–4289.

159 Xiang, L. and Moore, B.S. (2006)Biochemical characterization of aprokaryotic phenylalanine ammonia lyase[Correction]. Journal of Bacteriology, 188,5331.

160 Weiner, D., Varvak, A., Richardson, T.,Podar,M., Burke, E., andHealey, S. (2006)Lyase enzymes, nucleic acids encodingthem and methods for making and usingthem, WO 2006/099207 to DiversaCorporation. Chemical Abstracts, 145,309308.

161 Yanaka, M., Ura, D., Takahashi, A.,and Fukuhara, N. (1994) Productionof beta-substituted alanine derivative,


JP 6,113,870 to Mitsui Toatsu ChemicalsInc. Chemical Abstracts, 121, 155941.

162 Liu,W. (1991) Synthesis of optically activephenylalanine analogs using Rhodotorulagraminis, US 5,981,239 to Great LakesChemical Corp. Chemical Abstracts, 131,321632.

163 de Vries, J.G., de Lange, B., de Vries,A.H.M., Mink, D., van Assema, F.B.J.,Maas, P.J.D., and Hyett, D.J. (2006)Process for the preparation ofenantiomerically enriched indoline-2-carboxylic acid, EP 1,676,838 to DSM IPAssets BV. Chemical Abstracts, 145,124453.

164 Gloge, A., Zoñ, J., K€ov�ari, Á., Poppe, L.,and R�etey, J. (2000) Phenylalanineammonia-lyase: the use of its broadsubstrate specificity for mechanisticinvestigations and biocatalysis – synthesisof L-arylalanines. Chemistry – A EuropeanJournal, 6, 3386–3390.

165 Renard, G., Guilleux, J.C., Bore, C.,Malta-Valette, V., and Lerner, D.A. (1992)Synthesis of L-phenylalanine analogs byRhodotorula glutinis. Bioconversion ofcinnamic acids derivatives. BiotechnologyLetters, 14, 673–678.

166 Thorne, C.B., Gómez, C.G., andHousewright, R.D. (1955)Transamination of D-amino acids byBacillus subtilis. Journal of Bacteriology, 69,357–362.

167 Thorne, C.B. and Molnar, D.M. (1955)D-amino acid transamination in Bacillusanthracis. Journal of Bacteriology, 70,420–426.

168 Yonaha, K., Misono, H., Yamamoto, T.,and Soda, K. (1975) D-amino acidaminotransferase of Bacillus sphaericus.Enzymologic and spectrometricproperties. The Journal of BiologicalChemistry, 250, 6983–6989.

169 Rozzell, J.D. (1987) Immobilizedaminotransferases for amino acidproduction. Methods in Enzymology, 136,479–497.

170 Crump, S.P. and Rozzell, J.D. (1992)Biocatalytic production of amino acids by

transamination, in Biocatalytic Productionof Amino Acids and Derivatives (eds J.D.Rozzell and F. Wagner), Hanser, Munich,pp. 43–58.

171 Fotheringham, I.G., Pantaleone, D.P.,and Taylor, P.P. (1997) Biocatalyticproduction of unnatural amino acids,mono esters, and N-protected derivatives.Chimica Oggi – Chemistry Today, 15,33–37.

172 Rozzell, J.D. and Bommarius, A.S.(2002) Transaminations, in EnzymeCatalysis in Organic Synthesis (eds K.Drauz and H. Waldman), Wiley-VCHVerlag GmbH, Weinheim, pp. 873–893.

173 Tewari, Y.B., Goldberg, R.N., and Rozzell,J.D. (2000) Thermodynamics of reactionscatalysed by branched-chain-amino-acidtransaminase. Journal of ChemicalThermodynamics, 32, 1381–1398.

174 Schulz, A., Taggeselle, P., Tripier, D., andBartsch, K. (1990) Stereospecificproduction of the herbicidephosphinothricin (glufosinate) bytransamination: isolation andcharacterization of a phosphinothricin-specific transaminase from Escherichiacoli. Applied and EnvironmentalMicrobiology, 56, 1–6.

175 Cho, B.-K., Seo, J.-H., Kang, T.-W., andKim, B.-G. (2003) Asymmetric synthesisof L-homophenylalanine by equilibrium-shift using recombinant aromaticL-amino acid transaminase. Biotechnologyand Bioengineering, 83, 226–234.

176 Hanzawa, S., Oe, S., Tokuhisa, K.,Kawano, K., Kobayashi, T., Kudo, T., andKakidani, H. (2001) Chemo-enzymaticsynthesis of 3-(2-naphthyl)-L-alanine by anaminotransferase from the extremethermophile, Thermococcus profundus.Biotechnology Letters, 23, 589–591.

177 Bessman, S.P. and Layne, E.C. Jr. (1950)Stimulation of the non-enzymaticdecarboxylation of oxalacetic acid byamino acids. Archives of Biochemistry, 26,25–32.

178 Walter, J.F. and Sherwin, M.B. (1986)Improved transamination process for

References j113

producing L-amino acids, GB 2,161,159to W R Grace & Co. Chemical Abstracts,104, 205538.

179 Rozzell, J.D. (1985) Production of L-aminoacids by transamination, US 4,518,692 toGenetics Institute Inc.Chemical Abstracts,102, 219635.

180 Rozzell, J.D. (1987) Production of aminoacids using coupled aminotransferases,US 4,826,766 to Genetics Institute Inc.Chemical Abstracts, 107, 57455.

181 Bartsch, K., Schneider, R., and Schulz, A.(1996) Stereospecific production of theherbicide phosphinothricin (glufosinate):purification of aspartate transaminasefrom Bacillus stearothermophilus,cloning of the corresponding gene,aspC, and application in a coupledtransaminase process. Applied andEnvironmental Microbiology, 62,3794–3799.

182 Fotheringham, I. (2000) Engineeringbiosynthetic pathways: new routes tochiral amino acids. Current Opinion inChemical Biology, 4, 120–124.

183 Ager, D.J., Fotheringham, I.G., Li, T.,Pantaleone, D.P., and Senkpeil, R.F.(2000) The large scale synthesis of�unnatural� amino acids. Enantiomer, 5,235–243.

184 Fotheringham, I. and Taylor, P.P. (2006)Microbial pathway engineering foramino acid manufacture, in Handbookof Chiral Chemicals, 2nd edn (ed. D.J.Ager), CRC Press, Boca Raton, FL,pp. 31–45.

185 Fotheringham, I.G., Li, T., Senkpeil, R.F.,and Ager, D. (2000) Transaminasebiotransformation process employingglutamic acid, WO 2000/23609 to NSCTechnologies LLC. Chemical Abstracts,132, 292811.

186 Li, T., Kootstra, A.B., and Fotheringham,I.G. (2002) Nonproteinogenic a-aminoacid preparation using equilibriumshifted transamination. Organic ProcessResearch & Development, 6, 533–538.

187 Soda, K., Yonaha, K., Misono, H., andOsugi, M. (1974) Purification and

crystallization of D-amino acidaminotransferase of Bacillus sphaericus.FEBS Letters, 46, 359–363.

188 Lee, S.-G., Hong, S.-P., Song, J.J.,Kim, S.-J., Kwak, M.-S., and Sung, M.-H.(2006) Functional and structuralcharacterization of thermostable D-aminoacid aminotransferases from Geobacillusspp. Applied and EnvironmentalMicrobiology, 72, 1588–1594.

189 Tanizawa, K., Masu, Y., Asano, S., Tanaka,H., and Soda, K. (1989) Thermostable D-amino acid aminotransferase from athermophilic Bacillus species.Purification, characterization, and activesite sequence determination.The Journal of Biological Chemistry, 264,2445–2449.

190 Tanizawa, K., Asano, S., Masu, Y.,Kuramitsu, S., Kagamiyama, H., Tanaka,H., and Soda, K. (1989) The primarystructure of thermostable D-amino acidaminotransferase from a thermophilicBacillus species and its correlation with L-amino acid aminotransferases. TheJournal of Biological Chemistry, 264,2450–2454.

191 Peisach,D., Chipman,D.M., VanOphem,P.W.,Manning, J.M., andRinge,D. (1998)Crystallographic study of steps along thereaction pathway of D-amino acidaminotransferase. Biochemistry, 37,4958–4967.

192 Sugio, S., Petsko, G.A., Manning, J.M.,Soda, K., and Ringe, D. (1995) Crystalstructure of a D-amino acidaminotransferase: how the proteincontrols stereoselectivity.Biochemistry, 34,9661–9669.

193 Okada, K., Hirotsu, K., Sato, M., Hayashi,H., and Kagamiyama, H. (1997) Three-dimensional structure of Escherichia colibranched-chain amino acidaminotransferase at 2.5 Â resolution.Journal of Biochemistry, 121, 637–641.

194 Fotheringham, I.G., Bledig, S.A., andTaylor, P.P. (1998) Characterization of thegenes encoding D-amino acidtransaminase and glutamate racemase,


two D-glutamate biosynthetic enzymes ofBacillus sphaericusATCC 10208. Journal ofBacteriology, 180, 4319–4323.

195 Yohda, M., Okada, H., and Kumagai, H.(1991) Molecular cloning and nucleotidesequencing of the aspartate racemasegene from lactic acid bacteriaStreptococcus thermophilus. Biochimica etBiophysica Acta, 1089, 234–240.

196 Taylor, P.P., Pantaleone, D.P., Senkpeil,R.F., andFotheringham, I.G. (1998)Novelbiosynthetic approaches to the productionof unnatural amino acids usingtransaminases.Trends in Biotechnology, 16,412–418.

197 Fotheringham, I.G., Taylor, P.P., andTon, J.L. (1998) Preparation ofD-amino acids by direct fermentativemeans, US 5,728,555 toMonsanto Company. ChemicalAbstracts, 128, 216437.

198 Galkin, A., Kulakova, L., Yoshimura, T.,Soda, K., and Esaki, N. (1997) Synthesis ofoptically active amino acids from a-ketoacids with Escherichia coli cells expressingheterologous genes. Applied andEnvironmental Microbiology, 63,4651–4656.

199 Galkin, A., Kulakova, L., Yamamoto, H.,Tanizawa, K., Tanaka, H., Esaki, N., andSoda, K. (1997)Conversion ofa-keto acidsto D-amino acids by coupling of fourenzyme reactions. Journal of Fermentationand Bioengineering, 83, 299–300.

200 Mart�ınez del Pozo, A., Merola, M., Ueno,H., Manning, J.M., Tanizawa, K.,Nishimura, K., Soda, K., and Ringe, D.(1989) Stereospecificity of reactionscatalyzed by bacterial D-amino acidtransaminase. The Journal of BiologicalChemistry, 264, 17784–17789.

201 Van den Tweel, W.J.J., Smits, J.P., and deBont, J.A. (1986)Microbial metabolism ofD- and L-phenylglycine by Pseudomonasputida LW-4. Archives of Microbiology, 144,169–174.

202 Van den Tweel, W.J.J., Ogg, R.L.H.P., andde Bont, J.A.M. (1988) Process for thepreparation of a D-a-amino acid from the

corresponding a-keto acid, EP 0,315,786to Stamicarbon BV. Chemical Abstracts,108, 166114.

203 Van den Tweel, W.J.J., Smits, J.P., Ogg,R.L.H.P., and de Bont, J.A.M. (1988) Theinvolvement of an enantioselectivetransaminase in the metabolism ofD-3- and D-4-hydroxyphenylglycine inPseudomonas putida LW-4. AppliedMicrobiology and Biotechnology, 29,224–230.

204 M€uller, U., van Assema, F., Gunsior, M.,Orf, S., Kremer, S., Schipper, D.,Wagemans, A., Townsend, C.A., Sonke,T., Bovenberg, R., and Wubbolts, M.G.(2006) Metabolic engineering of theE. coli L-phenylalanine pathway forthe production of D-phenylglycine(D-Phg). Metabolic Engineering, 8,196–208.

205 Wiyakrutta, S. and Meevootisom, V.(1997) A stereo-inverting D-phenylglycineaminotransferase from Pseudomonasstutzeri ST-201: purification,characterization and application forD-phenylglycine synthesis. Journal ofBiotechnology, 55, 193–203.

206 Kongsaeree, P., Samanchart, C.,Laowanapiban, P., Wiyakrutta, S., andMeevootisom, V. (2003) Crystallizationand preliminary X-ray crystallographicanalysis of D-phenylglycineaminotransferase from Pseudomonasstutzeri ST201. Acta Crystallographica.Section D, Biological Crystallography, 59,953–954.

207 Brunhuber, N.M.W. and Blanchard, J.S.(1994) The biochemistry and enzymologyof amino acid dehydrogenases. CriticalReviews in Biochemistry and MolecularBiology, 29, 415–467.

208 Brunhuber, N.M.W., Thoden, J.B.,Blanchard, J.S., and Vanhooke, J.L. (2000)RhodococcusL-phenylalaninedehydrogenase: kinetics, mechanism,and structural basis for catalytic specifity.Biochemistry, 39, 9174–9187.

209 Sanwal, B.D. and Zink, M.W. (1961)L-Leucine dehydrogenase of Bacillus

References j115

cereus. Archives of Biochemistry andBiophysics, 94, 430–435.

210 Bommarius, A.S. (2002) Reduction ofC¼ N bonds, in Enzyme Catalysis inOrganic Synthesis (eds K. Drauz and H.Waldman), Wiley-VCH Verlag GmbH,Weinheim, pp. 1047–1063.

211 Hummel, W. and Kula, M.R. (1989)Dehydrogenases for the synthesis ofchiral compounds. European Journal ofBiochemistry, 184, 1–13.

212 Ohshima, T. and Soda, K. (2000)Stereoselective biocatalysis: amino aciddehydrogenases and their applications, inStereoselective Biocatalysis (ed. R.N. Patel),Marcel Dekker, New York, pp. 877–902.

213 Ohshima, T., Nishida, N.,Bakthavatsalam, S., Kataoka, K.,Takada, H., Yoshimura, T., Esaki, N.,and Soda, K. (1994) The purification,characterization, cloning and sequencingof the gene for a halostable andthermostable leucine dehydrogenasefrom Thermoactinomyces intermedius.FEBS Journal, 222, 305–312.

214 Bommarius, A.S., Drauz, K., Hummel,W., Kula, M.-R., and Wandrey, C. (1994)Some new developments in reductiveamination with cofactor regeneration.Biocatalysis, 10, 37–47.

215 Krix, G., Bommarius, A.S., Drauz, K.,Kottenhahn, M., Schwarm, M., and Kula,M.-R. (1997) Enzymatic reduction ofa-keto acids leading to L-amino acids, D- orL-hydroxy acids. Journal of Biotechnology,53, 29–39.

216 Hummel, W., Weiß, N., and Kula, M.R.(1984) Isolation and characterization of abacterium possessing L-phenylalaninedehydrogenase activity. Archives ofMicrobiology, 137, 47–52.

217 Hummel, W., Sch€utte, H., Schmidt, E.,Wandrey, C., and Kula, M.R. (1987)Isolation of L-phenylalaninedehydrogenase from Rhodococcus sp.M4 and its application for theproduction of L-phenylalanine.Applied Microbiology and Biotechnology,26, 409–416.

218 Ohshima, T., Takada, H., Yoshimura, T.,Esaki, N., and Soda, K. (1991)Distribution, purification, andcharacterization of thermostablephenylalanine dehydrogenase fromthermophilic actinomycetes. Journal ofBacteriology, 173, 3943–3948.

219 Hanson, R.L., Schwinden, M.D.,Banerjee, A., Brzozowski, D.B., Chen,B.-C., Patel, B.P., McNamee, C.G.,Kodersha, G.A., Kronenthal, D.R., Patel,R.N., and Szarka, L.J. (1999) Enzymaticsynthesis of L-6-hydroxynorleucine.Bioorganic and Medicinal Chemistry, 7,2247–2252.

220 Sch€utte, H., Flossdorf, J., Sahm, H., andKula, M.R. (1976) Purification andproperties of formaldehyde dehydrogenaseand formate dehydrogenase from Candidaboidinii.European Journal of Biochemistry, 62,151–160.

221 Kula, M.R. and Wandrey, C. (1987)Continuous enzymatic transformation inan enzyme-membrane reactor withsimultaneous NADH regeneration.Methods in Enzymology, 136, 9–21.

222 Shaked, Z. and Whitesides, G.M. (1980)Enzyme-catalyzed organic synthesis:NADH regeneration by using formatedehydrogenase. Journal of the AmericanChemical Society, 102, 7104–7105.

223 Slusarczyk,H., Felber, S., Kula,M.-R., andPohl, M. (2000) Stabilization of NAD-dependent formate dehydrogenase fromCandida boidinii by site-directedmutagenesis of cysteine residues.European Journal of Biochemistry, 267,1280–1289.

224 Weuster-Botz, D., Paschold, H., Striegel,B., Gieren, H., Kula, M.-R., and Wandrey,C. (1994) Continuous computercontrolled production of formatedehydrogenase (FDH) and isolation on apilot scale. Chemical Engineering &Technology, 17, 131–137.

225 W€oltinger, J., Drauz, K., and Bommarius,A.S. (2001) The membrane reactor in thefine chemicals industry. Applied CatalysisA – General, 221, 171–185.


226 Kragl, U., Vasic-Racki, D., and Wandrey,C. (1992) Continuous processes withsoluble enzymes. Chemie IngenieurTechnik, 64, 499–509.

227 Kragl, U., Kruse, W., Hummel, W., andWandrey, C. (1996) Enzyme engineeringaspects of biocatalysis: cofactorregeneration as example. Biotechnologyand Bioengineering, 52, 309–319.

228 Wichmann, R., Wandrey, C., B€uckmann,A.F., and Kula, M.R. (1981) Continuousenzymatic transformation in an enzymemembrane reactor with simultaneousNAD(H) regeneration. Biotechnology andBioengineering, 23, 2789–2802.

229 Bommarius, A.S., Schwarm, M., andDrauz, K. (1998) Biocatalysis to aminoacid-based chiral pharmaceuticals –examples and perspectives. Journal ofMolecular Catalysis B – Enzymatic,5, 1–11.

230 Asano, Y., Yamada, A., Kato, Y.,Yamaguchi, K., Hibino, Y., Hirai, K., andKondo, K. (1990) Enantioselectivesynthesis of (S)-amino acids byphenylalanine dehydrogenase fromBacillus sphaericus: use of natural andrecombinant enzymes. The Journal ofOrganic Chemistry, 55, 5567–5571.

231 Hanson, R.L., Howell, J.M., LaPorte, T.L.,Donovan, M.J., Cazzulino, D.L.,Zannella, V., Montana, M.A.,Nanduri, V.B., Schwarz, S.R., Eiring, R.F.,Durand, S.C., Wasylyk, J.M., Parker, W.L.,Liu, M.S., Okuniewicz, F.J., Chen, B.-C.,Harris, J.C., Natalie, K.J., Ramig, K.,Swaminathan, S., Rosso, V.W., Pack, S.K.,Lotz, B.T., Bernot, P.J., Rusowicz, A., Lust,D.A., Tse, K.S., Venit, J.J., Szarka, L.J., andPatel, R.N. (2000) Synthesis of allysineethylene acetal using phenylalaninedehydrogenase from Thermoactinomyces

intermedius. Enzyme and MicrobialTechnology, 26, 348–358.

232 Galkin, A., Kulakova, L., Tishkov, V.,Esaki, N., and Soda, K. (1995) Cloning offormate dehydrogenase gene from amethanol-utilizing bacteriumMycobacterium vaccae N10. AppliedMicrobiology and Biotechnology, 44,479–483.

233 Takada, H., Yoshimura, T., Ohshima, T.,Esaki, N., and Soda, K. (1991)Thermostable phenylalaninedehydrogenase of Thermoactinomycesintermedius: cloning, expression, andsequencing of its gene. Journal ofBiochemistry, 109, 371–376.

234 Gr€oger, H., Werner, H., Altenbuchner, J.,Menzel, A., and Hummel, W. (2005)Process for preparing optically activeamino acids using a whole-cell catalyst,WO 2005/093081 to Degussa AG.Chemical Abstracts, 143, 365753.

235 Menzel, A., Werner, H., Altenbuchner, J.,and Gr€oger, H. (2004) From enzymes to�designer bugs� in reductive amination:a new process for the synthesis ofL-tert-leucine using a whole cell-catalyst.Engineering in Life Sciences, 4, 573–576.

236 Gr€oger, H., May, O., Werner, H., Menzel,A., andAltenbuchner, J. (2006)A �second-generation process� for the synthesis ofL-neopentylglycine: asymmetric reductiveaminationusing a recombinantwhole cellcatalyst. Organic Process Research &Development, 10, 666–669.

237 Vedha-Peters, K., Gunawardana, M.,Rozzell, J.D., and Novick, S.J. (2006)Creation of a broad-range and highlystereoselective D-amino aciddehydrogenase for the one-step synthesisof D-amino acids. Journal of the AmericanChemical Society, 128, 10923–10929.

References j117

amino acids, peptides and proteins in organic chemistry (origins and synthesis of amino acids) ||...

Documents