Biocatalytic stereoinversion of D-para-bromophenylalanine in a one-pot three-

enzyme reaction

Fahimeh Khorsand1, Cormac D. Murphy1, Andrew J. Whitehead2 and Paul C. Engel1*

1School of Biomolecular and Biomedical Science, Conway Institute, University

College Dublin, Belfield, Dublin 4, Ireland

and

2GlaxoSmithKline, Currabinny, Carrigaline, Co Cork, Ireland

* Corresponding author: Address as above E-mail: paul.engel@ucd.ie Phone: 00353 1 7166764

Key words: Phenylalanine dehydrogenase, D-amino acid transaminase, para-

bromophenylalanine, deracemisation

Abstract

Halogenated derivatives of phenylalanine can be used as cross-coupling reagents for

making drug-like molecules, and pure enantiomers of these precursors are therefore

highly desirable. In our exploration of enzymatic routes to simplify the

deracemisation process, the application of two enzymes, D-amino acid transaminase

and phenylalanine dehydrogenase, both from Lysinibacillus sphaericus, has given

promising results for the stereo-inversion of D-enantiomers of para-

bromophenylalanine as the model substrate and also p-chloro/fluorophenylalanine and

tyrosine. The addition of a coenzyme recycling system using ethanol and alcohol

dehydrogenase reduced the amount of coenzyme needed for the reaction catalysed by

phenylalanine dehydrogenase, reducing cost and permitting efficient and complete

conversion of the racemic amino acids to the L-enantiomer. Relative proportions of

the enzymes were optimized. The high purity of the L-enantiomer, with an e.e. over

99%, and the ease of the process make it an ideal alternative for deracemisation of the

studied compounds.

Introduction

Non-natural amino acids play an important role in the drug industry as core

components of many drug molecules or as essential precursors in chemical synthesis.

The synthesis, for example, of L-biarylalanines, found in many pharmaceuticals such

as inhibitors of viral 3C protease and endothelin-converting enzyme 1, 2, is dependent

on the reaction and incorporation of phenylalanine derivatives. The production of

highly enantiopure derivatives of L-phenylalanine is therefore one of the initial steps

in making L-biarylalanines and the drug molecules containing these compounds.

Since the synthesis of racemic amino acids is fairly well developed, effective methods

for obtaining one single enantiomer from a racemic mixture of amino acids are of

critical importance. This can be done through either dynamic kinetic resolution or

stereo-inversion 3. The application of the hydantoinase-carbamoylase system for the

synthesis of a broad range of D-amino acids as well as L-amino acids 4, 5, and also

protease- 6, lipase- 7 and acylase- 8 mediated reactions are examples of enzymatic

kinetic resolution. In stereo-inversion, one enantiomer is converted to the target

enantiomer or product while the other starting enantiomer remains unaffected. A well-

known example is the stereo-inversion of DL--amino acids by porcine kidney D-

amino acid oxidase (DAAO) and a hydride reducing agent (NaCNBH3) 9. However,

there are many approaches focusing on the application of two or more enzymes with

different enantioselectivities without any need for chemical reagents. The conversion

of DL-methionine to L-methionine by Nakajima et al 10 used four enzymes including

DAAO, catalase, leucine dehydrogenase and a formate dehydrogenase. The industrial

production of a number of D-amino acids such as D-phenylalanine, D-tyrosine, D-

glutamate and D-alanine has been successfully performed by using recombinant

bacteria expressing Lysinibacillus sphaericus D-amino acid transaminase and a

Proteus myxofaciens or a Proteus mirabilis L-amino acid deaminase 11. The use of

multi-enzyme deracemisation systems is an attractive approach applicable under both

in vitro and in vivo conditions and has been extensively studied over recent years.

The recent advances in protein engineering and metabolic engineering allow

modification of existing enzymes to take part in reactions with different substrates,

widening their industrial application12. The enzymatic deracemisation of the

halogenated para derivatives of phenylalanine has been investigated in this study.

These compounds are not normally considered as substrates for wild-type enzymes,

which react efficiently with natural amino acids. Nevertheless, some amino acid

metabolising enzymes do show activity with these non-natural amino acids owing to

the similarity of their structure with those of phenylalanine and tyrosine. The

incorporation/application of D and L-enantiomers of para-bromophenylalanine in

making D and L biarylalanines through chemoenzymatic reaction has been recently

studied by Ahmed et al 13. They used phenylalanine ammonia lyase and recombinant

D-amino acid dehydrogenase to prepare the pure enantiomers of L and D para-

bromophenylalanine. The preparation of the L-enantiomer of 4-chlorophenylalanine

from its DL mixture has also been performed by a two-enzyme system in E. coli cells

expressing D-amino acid dehydrogenase and branched-chain amino acid

aminotransferase 14.

In the current project, we have used a tandem reaction of two enzymes, D-amino acid

aminotransferase (DAAT) and L-phenylalanine dehydrogenase (PheDH), both from

Lysinibacillus sphaericus, to efficiently deracemise the para bromo, chloro and fluoro

derivatives of phenylalanine, as well as tyrosine, via stereo-inversion of the D-

enantiomers to L.

Since the predominant form of amino acids in Nature is L, D-amino acid-specific

enzymes are relatively rare. Nevertheless, some do exist. L. sphaericus D-amino acid

aminotransferase (DAAT) catalyses the transfer of an amino group from an amino

acid donor to a prochiral carbon in a keto acid 15 yielding an oxo-product and a new

amino acid. This enzyme is strictly specific for D-amino acids. In the present study,

the activity of this enzyme was checked with para-bromo-D-phenylalanine and good

activity was detected. With -ketoglutarate as amino acceptor, therefore, this enzyme

converts para-bromo-D-phenylalanine to para-bromophenylpyruvate according to

Equation 1.

p-bromo-D-phenylalanine + -ketoglutarate ↔ p-bromophenylpyruvate + D-

glutamate Equation 1

The second reaction is carried out by the L. sphaericus phenylalanine dehydrogenase,

an NAD+/NADH dependent oxidoreductase which catalyses the reversible reductive

amination of a keto acid to the corresponding L-amino acid. Although the wild-type

enzyme shows broad substrate specificity, the activity is not very high with some non-

natural amino acids that are of interest for chemical synthesis. This has encouraged

the use of mutagenesis to modify substrate specificity and, in our group, a number of

variants with altered specificity have been made both by site-directed mutagenesis

based on structural studies (homology modelling) 16, 17 and by random mutagenesis 18.

Among these variants, mutant N145A had already shown good activity with some

para derivatives of phenylalanine 19. Here we have screened the wild-type enzyme

and 19 mutant variants to find the most effective catalyst with para-bromo-L-

phenylalanine as the model substrate (Equation 2). The selected biocatalyst was used

to carry out the second reaction in our tandem process for stereoinversion (Equation

2).

p-bromophenylpyruvate + NADH + NH4+ ↔ p-bromo-L-phenylalanine + NAD+

Equation 2

The use of a dehydrogenase entails deployment of the costly nicotinamide cofactor

NADH, which is stoichiometrically consumed in Equation 2. This is not an

economically viable proposition and, as in all such biocatalytic processes, it is

necessary to find a way of recycling the cofactor, reconverting NAD+ to NADH for

reuse. This requires a third oxidoreductase, ideally one with a highly soluble and

cheap substrate. Alcohols like ethanol and 2-propanol, sugars such as glucose,

glucose-6-phosphate (G6P), and glucose-6-sulfate, formic acid, amino acids such as

glutamic acid and also dihydrogen have all been used as the hydrogen source in such

recycling reactions. Glutamate dehydrogenase and glutamate, for example, have been

remarkably useful in the reduction of NAD+ to NADH and were employed to

regenerate NADH for a multi-enzymatic system reducing carbon dioxide to methanol

20. Formate dehydrogenase has also found favour with a number of authors, offering

the advantage that the product, CO2, is lost to the atmosphere. In our own earlier

studies, using PheDH for chiral synthesis, we found the alcohol dehydrogenase

reaction very suitable. 5% ethanol not only drives the equilibrium to the right but also

helps to solubilize oxoacid substrates with limited aqueous solubility. From the

standpoint of equilibrium, propan-2-ol is a more favourable option as equilibrium for

the oxidation of a secondary alcohol lies further towards the carbonyl product.

However, with the readily available and cheap alcohol dehydrogenase from yeast, the

rate of oxidation of propan-2-ol is very slow compared to that for ethanol.

Accordingly in this study, the first two enzymes, DAAT and PheDH, have been

combined with yeast alcohol dehydrogenase, using ethanol to recycle the

nicotinamide coenzyme and providing us with a one-pot, three-enzyme process

offering easy and cost-effective deracemisation of our target compound. Following

initial proof of principle with para-bromophenylalanine, the process has been tried

out also with the corresponding fluoro, chloro and hydroxy derivatives.

Experimental

Chemicals

Analytical grade reagents were used in most cases. -ketoglutarate and D/L-amino

acids were purchased from Sigma Chemical Co. (St Louis, MO, USA). Fluka (Buchs,

Switzerland) supplied pyridoxal phosphate (PalP). NAD+ and NADH were from

Apollo (Manchester, UK). Yeast alcohol dehydrogenase (ADH) and rabbit muscle

lactate dehydrogenase (LDH) were purchased from Sigma.

Bacteria and plasmids

Recombinant E. coli TG1 cells harbouring the plasmid ptac85, containing the gene

encoding L. sphaericus PheDH were used to express the phenylalanine

dehydrogenases. The D-amino acid transaminase (DAAT) gene inserted in plasmid

ING 2024 and cloned in E. coli BW25113 dad A was a kind gift from Ingenza Ltd.

Expression and purification of phenylalanine dehydrogenases

PheDH and mutant variants were prepared by a simplified version of the procedure

described by Seah et al.16,17 Recombinant E. coli TG1 cells containing the ptac85

plasmid encoding either the wild-type L. sphaericus PheDH gene or a mutated variant

were cultivated in LB medium containing ampicillin (100 μg/mL) at 37°C with

shaking. Enzyme expression was induced by adding 1M isopropyl -D-

thiogalactopyranoside (IPTG) after the OD600 of the culture reached 0.6. The cells

were cultivated for a further 6 hours, harvested (11,000 × g, 40 min) and suspended

in 10 mL 10 mM potassium phosphate (pH 7.8), followed by ultrasonication for

10 min at 4°C and centrifugation at 40,000 × g for 20 min to remove cell debris. The

supernatant, brought to 30% saturation with (NH4)2SO4, was centrifuged at 20,000 × g

for 20 min. The supernatant was adjusted to 60% (NH4)2SO4 saturation. After

centrifugation, the new precipitate was dissolved in buffer A (0.01 M potassium

phosphate buffer (pH 7.8) containing 1 mM EDTA and 5 mM 2-mercaptoethanol) and

dialysed against the same buffer at 4°C to remove (NH4)2SO4. The enzyme solution

was loaded on a Procion Red-P3BN Sepharose CL-6B column (6 x 2 cm), unbound

materials were washed off with buffer A and the enzyme was eluted with 0.5 M KCl

in the same buffer. Enzyme purity was checked by SDS-PAGE (12%) at each step.

Purified PheDH was stored at 4°C. and its concentration was calculated using the

absorbance at 280 nm and an extinction coefficient of 1.17 x 10-3 g-1 mL cm-1 17.

Expression and purification of D-amino acid transaminase

The recombinant DAAT was overexpressed in E. coli BW25113 cells cultivated and

harvested as described above for E. coli TG1 but using LB medium containing 50

mg/mL kanamycin. Harvested cells were suspended in 10 mL 20 mM Tris-HCl

(pH 8), followed by ultrasonication and centrifugation as above. The precipitate from

35% ammonium sulfate was resuspended in 5 mL 20 mM Tris-HCl buffer pH 8 and

dialysed against the same buffer to remove the salt. The enzyme solution was loaded

on a Q Sepharose column equilibrated with 20 mM Tris-HCl buffer pH 8 and eluted

with a 0 to 0.5 M NaCl gradient. The concentration of purified DAAT was determined

using the Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and the purity

was checked by SDS-PAGE.

Activity assays

Phenylalanine dehydrogenase activity

PheDH activity was assayed using a Cary 50 ultraviolet–visible spectrophotometer

(Agilent Technologies, Cork, Ireland) at 25 °C in 1-cm light path cuvettes. The

increase in A340 caused by reduction of NAD+ to NADH (: extinction coefficient is

6220 M−1 cm−1) was monitored after adding enzyme to a solution containing 10 mM

para-bromo-L-phenylalanine, 2 mM NAD+ and 100 mM KCl in 50 mM Gly-KOH

buffer, pH 10.4.

Coupled assay of D-amino acid transaminase

The standard assay for DAAT 21 measures the production of pyruvate from D-

alanine. Reaction mixtures (1 mL in 1 cm light-path cuvettes at 37 °C) contained

0.15 mM NADH, 15 mM -ketoglutarate, 5 mM D-alanine in 100 mM Tris/HCl (pH

8.5) and 25 Unit lactate dehydrogenase as the coupling enzyme.

The activity of the enzyme towards para-bromo-D-phenylalanine was measured with

a similar coupled assay using phenylalanine dehydrogenase (10 U, approx. 80 µg) in

place of lactate dehydrogenase.

One-pot stereo-inversion of para-bromo-D-phenylalanine

The relative amounts of DAAT and PheDH were optimised by varying the

concentrations of both. The reaction solution (1.5 mL) contained 6.5 mM para-

bromo-D-phenylalanine, 15 mM -ketoglutarate, 10 mM NADH, 20 M PalP, 100

mM KCl and 400 mM NH4Cl in 0.1 M Tris-HCl, pH 8.5. In the absence of recycling,

the NADH was supplied at a sufficiently high concentration to avoid coenzyme

concentration becoming the limiting factor. Reaction at 37oC in 15 mL Falcon tubes

was initiated by adding both enzymes. To stop the reaction for HPLC analysis, 700

L of the mixture was transferred to a microtube and incubated at 95oC for 10 min to

denature the enzymes. Denatured protein was precipitated by 10 min centrifugation at

20000 x g. The supernatant was adjusted to a pH between 4 and 7, suitable for the

Astec CHIROBIOTIC T column. The sample was diluted five-fold with the mobile

phase and filtered for HPLC analysis. Percentage conversion was calculated from

integrated peak areas for the two enantiomers.

With an optimal ratio of DAAT and PheDH established, a recycling system was

introduced to explore how far the coenzyme concentration could be reduced. The

initial reaction solution contained 6.5 mM para-bromo-D-phenylalanine and 15 mM

-ketoglutarate, 20 M PalP, 0.2 mg DAAT, 4 g PheDH (~0.5 U), 5 L ethanol, 1

mg ADH (300 U). Starting at 2 mM, the concentration of NADH was progressively

reduced to find the lowest required amount. Reactions were stopped after two hours

and the product was checked by chiral HPLC.

Enzyme recycling and scale-up

The Amicon Ultra-15 centrifugal filter unit (30K) was used to separate the reaction

solution from the enzyme molecules, which are not able to pass through the 30K filter

in the tube. The 6 mL reaction solution contained 6.5 mM DL-para-

bromophenylalanine, 15 mM -ketoglutarate, 20 M PalP, 20 L ethanol, 0.2 mM

NADH, 88 g PheDH, 2.3 mg DAAT and 14 mg ADH. After 2 hours, the tube was

centrifuged at 2000 × g to separate the reaction solution from the enzymes and the

production of L-enantiomer was checked by HPLC. The tube containing the enzymes

trapped on the filter was used for more than 10 reaction cycles over 10 days. The tube

was stored on ice when not in use.

Results and Discussion

Activity of PheDH with para-bromo-L-phenylalanine

An enzymatic approach to racemic resolution inevitably depends on the activity and

selectivity of the chosen enzymes towards the desired substrate(s). Additionally, the

efficiency and stability of the enzymes over repeated reaction cycles must be

considered. Initially, the idea of using PheDH as the sole enzyme was investigated by

testing different mutants. Theoretically, a suitable PheDH could convert the L half in

a racemic mixture of e.g. para-bromophenylalanine to the keto acid. After separation

from the untouched para-bromo-D-phenylalanine, reductive amination of the keto

acid using the same enzyme could yield the pure L-enantiomer, thus effecting a

quantitative resolution. The maximum yield would be 50% of each enantiomer, but

the expected high purity of the resulting products made this route worth exploring.

To find an enzyme sufficiently active with para-bromo-L-phenylalanine, twenty

variants of PheDH were screened. Some were not active enough to be studied further.

Fig. 1 shows those with fairly good activity towards para-bromophenylalanine and

also lists their mutations. Three mutants, nos. 3, 10 and 11, showed higher activity

than wild-type PheDH with para-bromo-L-phenylalanine, and one of these, No. 3, is

much more active with para-bromo-L-phenylalanine than with unsubstituted

phenylalanine.

An earlier study showed that inactivity with the D-enantiomer need not imply that

this isomer does not bind the active site. 18 The activity of the PheDH variants was

therefore checked with para-bromo-L-phenylalanine as substrate in the absence and

presence of equimolar para-bromo-D-phenylalanine in order to assess the risk of

inhibition by the inactive enantiomer (Fig. 1). Although enzyme no. 3, with the

mutation N145A, has the highest activity towards para-bromo-L-phenylalanine, the

activity drops remarkably in the racemic mixture, reaching only 20% of the activity

with the L-enantiomer alone. The inhibition is similarly more or less present for other

variants. Although, with enantiopure starting substrate, the activity of enzyme no. 3

with para-bromo-L-phenylalanine is quite high compared to other mutants, it does not

show the same superiority with a racemic substrate. Inhibition of 70 to 80% is already

seen with equimolar enantiomers, and, considering that the ratio of D to L in solution

increases as the reaction progresses, the rate would constantly decrease, making it

difficult to achieve full conversion. Using a coenzyme recycling system with

diaphorase and thiazolyl blue tetrazolium bromide (MTT), the full conversion of

para-bromo-L-phenylalanine to the keto acid was not achieved after a 24-hour

incubation of enzyme no. 3 with the racemic mixture of substrate (data not shown).

This brings into question the appropriateness of the PheDH mutants as an efficient

tool for the racemic resolution of para-bromophenylalanine by oxidative deamination.

Interestingly, however, the inhibition effect of the D-enantiomer is almost negligible

for the reductive amination reaction (data not shown). This would be of great merit if

we could couple the reaction with a D-amino acid-specific enzyme.

Oxidative deamination of para-bromo-D-phenylalanine by DAAT

The D-amino acid transaminase of L. sphaericus (DAAT) is known to be active with

a broad range of substrates. If DAAT is sufficiently active with the D-enantiomer in a

racemic mixture, PheDH could transform the resulting keto acid to the L-enantiomer

resulting in 100% conversion to the L-isomer (Fig. 2).

For DAAT purified in two steps (see above and Fig 3) the standard coupled activity

assay with D-alanine showed a specific activity of 110 U/mg, consistent with the

previous report.19 Replacing D-alanine with para-bromo-D-phenylalanine as substrate

necessitated a change from LDH to PheDH as the coupling enzyme. The reaction was

easily monitored by detecting the absorbance change. The specific activity of DAAT

measured with 2 mM para-bromo-D-phenylalanine was 2 U/mg. It should be noted

that much higher activity could be achieved were it not for the low water solubility of

this substrate. The reaction rate with a racemic mixture of para-bromophenylalanine

was similar to the rate of reaction with D-enantiomer as the only starting material.

Therefore, this activity is not inhibited by the inactive enantiomer.

The bienzymatic reaction for conversion of para-bromo-D-phenylalanine to para-

bromo-L-phenylalanine

Since the conditions required for transamination by DAAT and reductive amination

by PheDH are similar, it was possible to use the same buffer (Tris-HCl, pH 8.5) and

temperature (37oC) in a single-pot reaction, avoiding troublesome extraction of the

intermediate to start the second reaction in a new environment.

Using combinations of different concentrations of both enzymes, the lowest amounts

of enzymes giving almost 100% product yield over a two-hour reaction course were

investigated (Table 1). Under our conditions, in a 1.5 mL incubation, 0.2 mg DAAT

and 4 g PheDH no. 3 were able to convert all the D-enantiomer to the L-form as

shown by the HPLC results (Fig. 4). Owing to the very high activity of PheDH with

para-bromophenylpyruvate, only a small amount of this enzyme is required. Starting

with equal concentrations of both enantiomers, all the D was consumed leading to a

doubled concentration of L-enantiomer. The chromatograms clearly show the

efficiency of the studied reaction for deracemisation of para-bromophenylalanine.

The fact that PheDH catalyses the reverse reaction (reductive amination) at pH values

less basic than needed for the forward reaction (oxidative deamination) is absolutely

helpful for stereo-inversion of para-bromo-D-phenylalanine to the L-enantiomer. The

presence of both enzymes in the same solution is the main reason for rapid completion

of the reaction. Since the product of the first reaction is simultaneously removed from

the medium by the second reaction, transamination goes quickly to completion in

comparison with the situation with DAAT as the sole enzyme in the reaction.

The application of ADH for recycling the coenzyme

As mentioned earlier, oxidised nicotinamide coenzymes can be reconverted to the

reduced form by enzymatic methods thus avoiding the need for stoichiometric

addition, and in the present study yeast alcohol dehydrogenase and ethanol were

selected for this purpose. Ethanol, however, like other organic solvents, can inhibit

enzymatic reactions. To ensure that the presence of ethanol does not negatively affect

the progress of reaction, activities of the two enzymes were separately checked with

different concentrations of ethanol. The presence of low concentrations of ethanol did

not inhibit the studied reactions and in fact had a slight positive effect in some cases

(Fig. 5). The relevant reaction conditions required only a low ethanol concentration

(0.33%), and so no inhibition was expected. The full stereo-inversion of 6.5 mM

para-bromo-D-phenylalanine in the presence of various concentrations of coenzyme

was analysed by HPLC and the reaction yields were determined (Table 2). The

reaction proceeded to completion with NADH concentrations as low as 0.2 mM and

probably would still have done so with even less. Using even 0.02 mM NADH, a

325:1 molar ratio of substrate to coenzyme, 95% of substrate was transformed to the

product over two hours and full conversion was achieved after 5 hours incubation.

Considering 0.2 mM NADH as the appropriate amount of coenzyme for the two-hour

reaction, the application of ADH/ethanol reduced the required amount of NADH

nearly 30-fold, thus substantially moderating costs. The results unambiguously

confirm the efficiency of this recycling reaction.

Scale-up of the reaction using a centrifugal filter unit

In the present study, the reaction was scaled up with two purposes; firstly, to show the

possibility of reusing enzymes and secondly, to get sufficient product for further

studies. The centrifugal filter unit offered the advantage of easy separation of product

solution from enzymes (Fig. 6). The enzymes remained untouched above the filter

barrier and the small molecules like amino acids and coenzyme passed through the

membrane by centrifugation. The same tube was used for 12 rounds of reaction on

different days and the reaction was monitored to reach completion in each round. The

functionality of the enzymes gradually decreased over the period and the reaction time

became longer so that ultimately complete conversion was not attained within two

hours. The first cycle was complete in under 90 min; in the 10 th round four days later

with the same tube, only 90% of D-enantiomer was transformed to the L within 2

hours. The conversion yield decreased to 84% after 2 hours reaction of the 11th round

on the 11th day. More than 60 mL solution was ultimately obtained, theoretically

containing 6.5 mM pure para-bromo-L-phenylalanine, which was extracted from the

other components of reaction with 1-butanol. The solvent was removed by a rotary

evaporator and the final product was obtained as a white solid.

A gradual loss in activity after recycling the enzymes was not unexpected, and no

special methods for stabilising enzymes were employed. However, the enzymes

remained remarkably stable and functional considering the storage time and the

number of reaction cycles. An obvious next stage to explore in the context of

industrial application would be either encapsulation or immobilization of the enzymes

on a solid support.

Authentication of the product.

Reactions were monitored throughout by chiral HPLC and, in view of the known

specificity of the enzymes used, the nature of the product was not in serious doubt.

However, the collected material from the multi-cycle scale-up was subjected to NMR

analysis. The product was obtained as a white solid after butanol extraction of 20 mL

of reaction solution (29.9 mg, 88% purity). This material was characterised by 1H

NMR (400 MHz, DMSO) δ 2.9 (1H, dd, -CβH), 3.06 (1H, dd, -CβH), 3.68 (1H, dd, -

CαH) , 7.14-7.2 (2H, d, 2Ar-H), 7.42-7.48 (2H, d, 2Ar-H) and also 13C NMR (101

MHz, DMSO) δCO), 136.5, 131.5, 132.5, 121.4 (CBr), 53.5 (Cα), 35.5 (Cβ).

Application of the method to other para-derivatives of phenylalanine

As well as para-bromophenylalanine other para-derivatives of phenylalanine can be

utilised as intermediates in synthesis. So, it is useful to explore the discussed method

of deracemisation with these other compounds. PheDH no. 3 has already been shown

to be active with the para-chloro and –fluoro derivatives of phenylalanine 18. The

activity of wild-type PheDH and variants mutated at N145 was also fairly high with

para derivatives of phenylpyruvate 24. This could be of particular importance as

PheDH catalyses the reductive deamination of the -keto acids in our proposed

deracemisation method. Wild-type PheDH is strongly active towards L-tyrosine, the

hydroxylated derivative of phenylalanine 16, 25. Therefore the second, PheDH reaction

in the deracemisation procedure was expected to proceed smoothly with the

mentioned derivatives of phenylalanine, but the viability of the transamination was

under question. The activity of DAAT with the D-enantiomers of the three amino

acids, para-chlorophenylalanine, para-fluorophenylalanine and tyrosine, was

explored using the assay method based on PheDH as the coupling enzyme. Following

the detection of good activities, the three-enzymatic reaction was used for these three

substrates and resulted in full stereo-inversion of their D-enantiomers to the L (data

not shown). In the case of tyrosine the reaction required a longer time (only 97%

conversion after 4 hours).

Conclusion

Owing to different requirements for reaction conditions in chemocatalysis, one-pot

multistep reactions are more challenging compared to enzymatic catalysis. Since the

optimum conditions for reactions catalysed by different enzymes are often similar,

biocatalytic cascades are much more readily applied. These series of reactions may be

performed either in sequential mode, with the next catalyst added only after the

previous step is completed, or in simultaneous mode, with all reactants and enzymes

present from the outset. Performing the reactions of a multistep process in a “one-pot”

fashion, without the isolation of the intermediates, has many benefits since isolation

and purification steps are typically time-consuming, yield-reducing and require large

amounts of chemicals/solvents. Avoiding these isolation steps therefore reduces

operation time, cost and environmental impact 26.

Considering the objective of this study, which was the efficient deracemisation of a

racemic mixture of amino acids, different methods have been developed.

Stereoselective deamination of an unwanted enantiomer by an amino acid oxidase

followed by amination of the keto acid back to an amino acid of desired chirality by

asymmetric biocatalysis or to a racemic mixture by non-stereoselective chemical

reduction are well-known processes. However, the production of H2O2, with a

consequent requirement for addition of catalase, is a substantial obstacle. The

application of two transaminases with opposite stereoselectivities was another

strategy to obtain pure enantiomers of amino acids through one-pot deracemisation 27.

Although the transaminases are not considered specific to a narrow range of

substrates, it is not always easy to find two enzymes targeting different enantiomers of

a substrate efficiently. Here, we have developed a novel method for the

deracemisation of a racemic mixture of para-bromo-L-phenylalanine and some

similar compounds by using a combination of a transaminase and an amino acid

dehydrogenase.

The mutant variants derived from the wild-type L. sphaericus phenylalanine

dehydrogenase have demonstrated striking potential for biocatalysis. The possibility

of changing the substrate specificity of enzyme in favour of a new substrate has

helped us to assemble the tools not immediately available in Nature. The high activity

of PheDH N145A with para-bromo-L-phenylalanine was a promising start, but

uexpectedly potent inhibition by the D-enantiomer proved problematic. In principle it

should be possible in the future to eliminate this problem through molecular

modelling and further mutagenesis. Even so, the use of a single enzyme would

inevitably involve two reaction steps under different conditions. The addition of

DAAT to the reaction offered a viable alternative route for conversion of the D-

enantiomer to the ketoacid, avoiding the inhibition. Crucially the subsequent

conversion to L-para-bromophenylalanine by PheDH, increasing the potential yield to

100%, can be carried out at the same time in the same reaction vessel.

One obvious criticism of this reaction system is that substrate, and therefore product,

concentrations are low. This is a consequence of the low solubility of the substrate in

an aqueous buffer. Our attempts to remedy this by addition of miscible organic

solvents offered little or no improvement (results not shown). A possibility yet to be

explored is the use of a biphasic system. In a previous study of chiral synthesis of

amino acids from ketoacids with poor aqueous solubility 28, we observed efficient

conversion in systems where the enzyme and coenzyme resided in the aqueous phase

whilst the substrate resided mainly in the organic phase, which served effectively as a

high-concentration reservoir. A similar approach might well overcome the solubility

problem here also.

Finally, further work is also now needed to explore the immobilization of these

enzymes, which should improve stability and allow many cycles of use 29,30,31, and to

investigate a wider range of substrates. Nevertheless but current results already

indicate that this combination of biocatalysts offers a versatile and robust tool for

deracemisation of substituted aromatic amino acids.

Acknowledgement

This work was supported by an Irish Research Council Enterprise Partnership Scheme

scholarship with GlaxoSmithKline as the Enterprise Partner. We thank Dr. Ian

Fotheringham of Ingenza Ltd. for the generous gift of the E. coli clone containing the

DAAT gene.

References

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Legends

Fig. 1 The specific activity of PheDH variants with 10 mM L-phenylalanine (blue

bars) and with 10 mM para-bromo-L-phenylalanine in the absence (red bars) and

presence (green bars) of 10mM para-bromo-D-phenylalanine. The table shows the

mutations in each variant.

Fig. 2 Enzymatic deracemisation using the possibility of targeting both enantiomers of

bromophenylalanine. PheDH specifically interconverts the L-enantiomer and the keto

acid. An enzyme able to do the same for the D-enantiomer would allow the complete

deracemisation.

Fig. 3 The purification steps of DAAT analysed by SDS-PAGE. From the left side,

lanes show the protein marker, crude extract, 35% ammonium sulfate precipitate and

Q Sepharose eluate.

Fig. 4 HPLC chromatograms from reactions with 6.5mM para-bromo-DL-

phenylalanine, 15mM -ketoglutarate, 10mM NADH, 20M PalP, 0.2mg DAAT and

4g PheDH. (a) contains all components other than enzymes; therefore no reaction

occurs and the presence of both untouched amino acid enantiomers can be seen as two

peaks with almost equal peak areas. (b) adding enzymes ends up in stereo-inversion of

all the D to the L-enantiomer. The reaction was incubated at 37oC for two hours.

HPLC conditions: isocratic flow of 90% methanol on Astec CHIROBIOTIC chiral

column, flow rate of 0.3 mL/min and detection at 225 nm

Fig. 5 The effect of ethanol concentration on the activity of PheDH N145A (red bars)

and DAAT (black bars).

Fig. 6 Scale-up and recycling of enzymes using an Amicon centrifugal filter unit. The

combination of three reacting enzymes converts the D-enantiomer of the racemic

amino acid substrate quantitatively to the L even with amino acid to coenzyme ratios

in excess of 100. Centrifugation separates the small molecules such as substrates and

products from the enzymes. The enzymes remaining on the filter layer catalyse the

next round of reaction with a new solution of substrates.

Table 1

Deracemisation of 6.5 mM DL-para-bromophenylalanine in a reaction mixture

containing 15 mM α-ketoglutarate, 10 mM NADH, 20 µM PalP, 100 mM KCl and

400 mM NH4Cl in 0.1 M Tris-HCl, pH 8.5. In a 1.5 mL incubation the total additions

of PheDH and DAAT were varied in order to discover minimal amounts required to

achieve production of 99% L-amino acid within 2h.

DAAT mass (mg) PheDH mass (g) L-bromophenylalanine%

0.05 8 85.7

0.05 16 90.1

0.1 16 96.5

0.1 8 97.5

0.15 8 98.4

0.2 8 99

0.2 4 99

Table 2

Effect of NADH concentration on the product yield after 2 hours reaction

NADH Conc. (mM) Yield % in the presence of NADH recycling system

Yield % in the absence of NADH recycling system

0.02 95 2.6

0.2 99 4.3

1 99 22

2 99 39

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6