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Journal of Molecular Catalysis B: Enzymatic 103 (2014) 10–15

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

j ourna l ho me pa g e: www.elsev ier .com/ locate /molcatb

rtificial cofactor regeneration with an iron(III)porphyrin asADH-oxidase mimic in the enzymatic oxidation of l-glutamate to-ketoglutarate

ilko Greschnera, Carsten Lanzerathb, Tina Reßa, Katharina Tenbrinka,c, Sonja Borchertc,ndreas Mixa, Werner Hummelb,∗, Harald Grögera,c,∗∗

Faculty of Chemistry, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, GermanyInstitute of Molecular Enzyme Technology, Research Centre Jülich, Wilhelm-Johnen-Straße, 52426 Jülich, GermanyDepartment of Chemistry and Pharmacy, University of Erlangen-Nürnberg, Henkestr. 42, 91054 Erlangen, Germany

r t i c l e i n f o

rticle history:vailable online 28 December 2013

eywords:rtificial cofactor recyclingmino acid dehydrogenase

a b s t r a c t

In this contribution the use of an artificial in situ-cofactor regeneration with [Fe(III)TSPP]Cl for enzy-matic amino acid oxidation, exemplified for the l-glutamate dehydrogenase-catalyzed synthesis of�-ketoglutarate from sodium l-glutamate, is reported. In comparison of two l-glutamate dehydroge-nases, the one isolated from Clostridium difficile turned out to be the preferred enzyme. At a substrateconcentration of 15 mM of l-glutamate in situ-cofactor regeneration using [Fe(III)TSPP]Cl as an “artificial

iomimetic catalysis-Ketoglutaratexidation

NADH-oxidase” proceeded smoothly, leading to up to >99% overall conversion and 88% conversion relatedto the formation of �-ketoglutarate after 24 h. At an increased concentration of 50 mM of l-glutamate, asomewhat decreased conversion of 43% was observed (which, however, corresponds to a nearly doubledvolumetric productivity of 3.95 g/(L d) compared to the experiments at 15 mM). Thus, the iron complex[Fe(III)TSPP]Cl turned out to be capable to be used for cofactor regeneration of the cofactor NAD+ forenzymatic amino acid oxidation.

. Introduction

In the last decades an impressive number of enzymatic redoxrocesses has been established on industrial scale for the synthesisf a broad variety of commercial products [1,2]. In particular thiss true for reductive processes, and, e.g., today production routes ofhiral alcohols and in part l-amino acids required in the pharma-eutical industry are typically based on dehydrogenase-catalyzedeactions such as reduction and reductive amination, respectively.

key prerequisite for technical feasibility in all of such processes isfficiency of the in situ-cofactor regeneration. Due to the high pricef the cofactor its use in low (catalytic) amount is required.

Whereas for reductive processes a range of in situ-cofactoregenerations are available with several of them being scalable

s well [1–4], in the oxidative mode only a few strategies haveeen studied more in detail for synthetic purpose. The most promi-ent approach is based on the use of an NADH-oxidase [3–9].

∗ Corresponding author. Tel.: +49 2461 61 3790.∗∗ Corresponding author at: Faculty of Chemistry, Bielefeld University, Univer-itätsstraße 25, 33615 Bielefeld, Germany. Tel.: +49 521 106 2057.

E-mail addresses: w.hummel@fz-juelich.de (W. Hummel),arald.groeger@uni-bielefeld.de (H. Gröger).

381-1177/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.molcatb.2013.12.015

© 2014 Elsevier B.V. All rights reserved.

Attractive features are the use of molecular oxygen for oxidationof NADH (which is formed in the dehydrogenase-catalyzed oxida-tion process from NAD+) and the formation of water as by-productat least in case of some NADH-oxidases. On the other hand, thenumber of available NADH-oxidases is still limited and some ofthem form hydrogen peroxide as a by-product, which is disad-vantageous since H2O2 typically causes deactivation of enzymes.Alternatively, artificial in situ-cofactor regeneration has been stud-ied. An early breakthrough was achieved by the Steckhan group bymeans of a rhodium complex as a biomimetic NADH-oxidase [10].In this case hydrogen peroxide is again formed as a by-product,thus requiring a catalase as an additional enzyme component inthe reaction system for cleavage of hydrogen peroxide to molecularoxygen and water. Recently we demonstrated as a proof of con-cept the ability of an iron(III)porphyrin complex (3; [Fe(III)TSPP]Cl)for in situ-regeneration of the oxidized cofactors NAD+ and NADP+

through reduction of molecular oxygen and the coupling of thistype of cofactor regeneration with alcohol dehydrogenase- andglucose dehydrogenase-catalyzed oxidation reactions [10]. Thus,this iron(III)porphyrin-complex represents an “artificial NAD(P)H-

oxidase” for in situ-cofactor regeneration in enzymatic oxidationprocesses. Furthermore we did not observe hydrogen peroxideas a side product, which indicates that water is formed as a by-product. Since this initial study [10] with the iron porphyrin 3 has

W. Greschner et al. / Journal of Molecular Cat

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cheme 1. Concept for the biocatalytic synthesis of �-ketoglutarate with “artificial”n situ-cofactor regeneration using an ironporphyrin as an NADH-oxidase substitute.

ocused on oxidation of carbohydrates and an alcohol, we becamenterested to explore if this iron porphyrin also is compatible withther dehydrogenase-catalyzed oxidations. In particular the use ofFe(III)TSPP]Cl for artificial in situ-cofactor regeneration in gluta-

ate dehydrogenase (GluDH)-catalyzed oxidation of l-glutamateoward �-ketoglutarate found our interest due to the industrialelevance of �-ketoglutarate (2). In the following we report ouresults on the use of the artificial in situ-cofactor regeneration withFe(III)TSPP]Cl 3 in amino acid oxidation, exemplified for the l-lutamate dehydrogenase-catalyzed synthesis of �-ketoglutarate2) from l-glutamate (l-1) according to Scheme 1.

. Experimental

.1. Materials

The catalyst [Fe(III)TSPP]Cl (3; 5,10,15,20-tetrakis-(4-ulfonatophenyl)-porphyrin-iron(III)chloride) is commerciallyvailable and was purchased from TriPorTech GmbH (productumber: tpt00142617). The sodium l-glutamate monohydrate (1)as purchased from Tokyo Chemical Industry, the �-ketoglutarate

odium salt (2) was available from Applichem GmbH and bothere used as received.

.2. Preparation of the NAD+-dependent l-glutamateehydrogenases from Clostridium difficile and Fusobacteriumucleatum and determination of their enzyme activitiesaccording to Table 1 and Scheme 1)

.2.1. Microorganisms and culture conditionsE. coli BL21(DE3) and E. coli DH5 were grown in a 1 L-scale in LB

edium containing 100 �g/mL of ampicillin, 1.0% of tryptone, 1.0%f NaCl, and 0.5% of yeast extract as well as for non liquid medium.5% of agar. The pH was measured and adjusted to 7.5.

alysis B: Enzymatic 103 (2014) 10–15 11

2.2.2. Molecular cloning of the l-glutamate dehydrogenases fromC. difficile and F. nucleatum

Genomic DNA from C. difficile (DSM 12056) or F. nucleatumsubsp. nucleatum (DSM 15643) (purchased by the German Collec-tion of Microorganisms and Cell Cultures (DSMZ)) was used as atemplate for the amplification of the gludh genes. For cloning stepsfollowing primers were used:

GluDH from C. difficile forward:5′-TGCCGGTCTCGCATGTCAGGAAAAGATGTAAATGTCTTCGAG-

ATGGCGCAATC-3′, GluDH from C. difficile reverse:5′-CATGGAGCTCTTAGTACCATCCTCTTAATTTCATAGCTTCAGCA-

ACTTTCTTAATTGAA TGCATG-3′, GluDH from F. nucleatum forward:5′-TGCCGGTCTCGCATGAGTAAAGAAACTTTAAACCCATTAGAAA-

GCGGACAAAAACAA GTTAAAAAAG-3′

GluDH from F. nucleatum reverse:5′-CATGGAGCTCTTAGTACCATCCTCTAATTTTCATAGCTTCAGCT-

ATTCTCTTAATAGAT TTCATATAAGTAGCTTGTCTA-3′

Forward primers contain a recognition site for the restric-tion enzyme BsaI, reverse primer for the restriction enzyme SacI.Primers were products of MWG Biotech. PCR was performed witha DNA Thermal Cycler from Eppendorf in a total volume of 0.05 mLcontaining 0.2 mM dNTPs, 30 pmol of each primer, 5 ng templateDNA, 0.010 mL of 5x reaction buffer (HV, Fermentas) and 2.5YUPhusion polymerase (Fermentas). After an initial denaturation for3 min at 98 ◦C PCR was run for 30 cycles with the following program:denaturation by incubation at 98 ◦C for 20Ys; annealing at 64 ◦C for20Ys; elongation at 72 ◦C for 1 min, finished by an final elongationfor 5 min. After amplification PCR fragments were digested withBsaI/SacI and purified by preparative agarose gel and gel extractionkit (AnalytikJena).

2.2.3. Construction of l-glutamate dehydrogenases GluDHexpression vectors

The GluDH genes from C. difficile and F. nucleatum, respectively,were cloned into the first multiple cloning site of the expressionvector pETDuet-1 (Novagen) under the control of the T7-promoteryielding pDGluDHCD and pDGluDHFN using T4 DNA ligase (Fer-mentas) according to the manufacturer’s instructions. Resultingvectors were transformed into competent E. coli DH5 cells andtransformants were selected on LB agar plates supplemented with100 �g ampicillin per mL. Correct amplification was confirmed byrestriction analysis and DNA sequencing. Sequencing was carriedout by MWG Germany. For protein production, vectors pDGluD-HCD and pDGluDHFN were transformed in the E.coli host strainBL21(DE3).

2.2.4. Production and purification of the recombinantl-glutamate dehydrogenases from C. difficile and F. nucleatum

The strains E. coli BL21(DE3)/pDGluDHCD and E. coliBL21(DE3)/pDGluDHFN were cultivated in 10 mL of LB mediumcontaining 100 �g/mL ampicillin overnight at 37 ◦C. These cultureswere used to inoculate 1YL of LB medium for expression containing100 �g/mL ampicillin at a final optical density 600 nm of 0.05.Culturing was done in shaking flasks (120 rpm) at 30 ◦C and theproduction of recombinant GluDH under the T7-promoter wasinduced by the addition of IPTG (isopropyl thio-�-d-galactoside) ina final concentration of 0.2 mM when the optical density at 595 nmreached 0.5, followed by further growing for enzyme productionfor 16 h. The following steps were done in the same way for theGluDHs from C. difficile and F. nucleatum. Cells were harvested bycentrifugation at 5000 × g for 30 min at 4 ◦C (centrifuge by Hettich),suspended for lysis (25% cell suspension) in 50 mM phosphate

(cooling periods for 3 min on ice between) (Sonopuls HD 60ultrasonic oscillator, power: 80%, cycles: 50; Bandelin, Germany).After sonification, separation of cell debris from the cell lysate

1 lar Catalysis B: Enzymatic 103 (2014) 10–15

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Table 1Enzyme activities of the prepared recombinant l-GluDHs from F. nucleatum and C.difficile

.

Entry Original strainof recombinantl-GluDH

Enzyme activity ofcrude extracta

[U/mg]

Enzyme activityafter heattreatment [U/mg]b

1 Fusobacterium nucleatum 6 16 (32)c

2 Clostridium difficile 8 20 (146)c

a For preparation of the crude extracts and conditions of spectrophotometricactivity determination, see Section 2.

2 W. Greschner et al. / Journal of Molecu

as done by centrifugation at 14,000 × g for 30 min at 4 ◦C (cen-rifuge by Hettich). After obtaining the clear supernatant a heatrecipitation was performed by incubating the enzyme solutiont a temperature of 59 ◦C (according to Anderson et al. [11] andharbia and Haroun [12] where the high thermal stability of bothluDHs are described). The residence time was adjusted to 60 min.eparation of denaturated proteins was done by centrifugation at4,000 rpm for 30 min at 4 ◦C and the supernatant was taken asnzyme solution for the subsequent experiments.

.2.5. Determination of the enzyme activity of l-glutamateehydrogenase

Enzyme activity was determined for the oxidative deaminationf l-glutamate. The standard reaction mixture contained 50 mM l-lutamate, 2.5 mM of NAD+ in 100 mM Tris–HCl buffer (pH 9.0), andnzyme in a final volume of 1 mL. For product inhibition studies,0 mM �-ketoglutarate was added. The substrate was replaced byater in a blank experiment. Enzyme solution (0.01 mL) was added

nd the change in absorbance at 340 nm was monitored for 1 min at0 ◦C. One unit of the GluDH is defined as the amount of enzyme thatatalyzes the formation of 1 �mol of NADH per min in the oxidativeeamination of l-glutamate under the conditions mentioned aboveith a molar absorption coefficient of 6.22 mM−1 cm−1 for NADH.

he specific activity is expressed as units per mg of protein.

.2.6. NADH oxidase from Lactobacillus brevisPreparation of NADH oxidase from L. brevis as a recombinant

nzyme in E. coli BL21(DE3) and the determination of its activitysing 0.1 mM NADH at pH 7.0 were carried out as described byeueke et al. [5].

.2.7. Determination of the amount of protein in enzyme crudextracts

The determinations of the amount of protein in the crude andartially purified extracts were done by means of the methodf Bradford [13] using bovine serum albumin as a standard. Thexpression levels were monitored by means of a polyacrylamideel electrophoresis using commercial Bis/tris-gels (4–12%) andhe NuPAGE-System from Invitrogen. Prestained SeeBlue Proteintandard from Invitrogen was used as molecular weight standard.

.3. Determination of time-dependent stability of–ketoglutarate in buffered aqueous solution

To a solution of sodium l-glutamate monohydrate (l-1; 22.5 mg;.12 mmol) and sodium �–ketoglutarate (2; 5.0 mg; 0.03 mmol) inhosphate buffer (3.0 mL; pH 8; 100 mM) or in carbonate buffer3.0 mL; pH 9.6; 100 mM) the GluDH from C. difficile or F. nuclea-um (15YU referring to the activity for l-glutamate oxidation) wasdded. The mixture was stirred with 550 rpm at room temperatureor 48 h. Samples of 0.3 mL were periodically taken and the pro-ein was removed through ultrafiltration-centrifugation (10 kDa

WCO; 12.000 rpm). To each sample 0.05 mL of a freshly prepared0 mM pivalic acid solution and 0.250 mL deuterium oxide weredded and analyzed by 1H-NMR spectroscopy using pivalic acidtrimethylacetic acid) as an internal standard in combination with

presaturation technique for suppression of the water signal.

.4. Typical procedure for the synthesis of ˛-ketoglutarate (2)according to Tables 2 and 3)

To a solution, consisting of sodium l-glutamate monohy-

rate (l-1; 14.0 mg; 75 �mol respectively 46.8 mg; 250 �mol) andFe(III)TSPP]Cl (3; 5.1 mg; 5 �mol) or NADH-oxidase from L. bre-is (22.5YU referring to the activity for NADH) in phosphate (orarbonate/bicarbonate) buffer (5.0 mL; 100 mM), the GluDH from

b Heat treatment for 1 h at 60 ◦C; for details, see Section 2.c In parentheses literature values for the specific activities of the purified proteins

are given (in U/mg).

C. difficile or from F. nucleatum (15YU referring to the activ-ity for l-glutamate oxidation) was added. With the addition ofNAD+ (6.6 mg; 10 �mol) the reaction was started and stirred with550 rpm at room temperature. Workup and determination of theconversion were done as described in Section 2.3.

3. Results and discussion

First, we searched for an l-glutamate dehydrogenase (l-GluDH)synthetically suitable for oxidative deamination of sodium l-glutamate (l-GluDH, l-1) since our own preliminary experimentsand literature data [8,14] revealed strong product inhibition ofcommercially available l-glutamate dehydrogenases for this pur-pose. For example, GluDH from C. symbiosum shows a severeproduct inhibition (leading to a reduction of activity of 70%) causedby only 3 mM of �-ketoglutarate (2) when converting 5 mM ofl-1 [8]. The resulting productivities with 1.07 g/(L d) at the high-est [8] limit preparative applicability of this enzymatic approachbased on glutamate dehydrogenase and NADH-oxidase. Thus, theestablished technical process is based not on biocatalysis but onoxidation of l-1 in air in the presence of a copper complex [8].Since in our initial literature search for alternative l-GluDHs werecognized that data with respect to product inhibition of l-GluDHsare very rare, we focused on l-GluDHs with a catabolic function inmicrobial metabolisms, since the natural function of such enzymesis the conversion (degradation) of l-Glu (l-1) to �-ketoglutarate(2). Among about 30 catabolic l-GluDHs in literature we prior-ized the enzymes of F. nucleatum and C. difficile. These GluDHsare reported to have a high specific activity of about 32YU/mg(GluDH from F. nucleatum) [12] and 146YU/mg (GluDH from C. dif-ficile) [11] in comparison to GluDHs from other sources like suchas Peptostreptococcus asaccharolyticus (7.8YU/mg) [15], Bacteroidesfragilis (7.3YU/mg) [16], Pyrobacterium islandicum (3.5YU/mg) [17],and Janthinobacterium lividum (3.1YU/mg) [18].

Recombinant expression and production of sufficient amountsof both biocatalyst were conducted applying an E. coli expressionsystem as mentioned above. Both enzymes, namely the GluDHsfrom C. difficile and F. nucleatum, were successfully expressed inE. coli as a soluble and fully active protein. The specific enzymeactivities in the crude extracts of 6 and 8YU/mg of protein forthe GluDHs from F. nucleatum and C. difficile, respectively, couldbe increased nearly threefold up to 16 and 20YU/mg after heatprecipitation for the recombinant l-GluDHs as summarized in

Table 1. From 4 g of cells (wet weight) we obtained 1.580YU of therecombinant l-GluDH from F. nucleatum and 1.390YU of the recom-binant l-GluDH from C. difficile (corresponding to yields of 76% and88%, respectively referred to activities of the crude extracts). The

W. Greschner et al. / Journal of Molecular Catalysis B: Enzymatic 103 (2014) 10–15 13

Table 2Biotransformation of sodium glutamate (l-1, 15 mM) into 2 with a l-GluDH and [Fe(III)TSPP]Cl (3, 1 mM) or NADH-oxidase from Lactobacillus brevis at 20 ◦C and pH 8

.

Entry l-GluDH [U/mL] NOX[U/mL] Conv.a 2 h [%] Conv.a 4 h [%] Conv.a 8 h [%] Conv.a 24 h [%]

1 1.5 – 51(50) 65(64) 76(78) 86(66)2 5.0 – 49(51) 77(60) 91(89) >99(88)

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Scheme 1. A first proof of concept for the compatibility of glutamatedehydrogenases with the iron(III)porphyrin complex 3 has beendemonstrated when conducting the process at pH 8.0 and witha substrate concentration of 15 mM (Table 2). High conversions

3 1.5 4.5 36(37)

a The conversion is defined as the amount of consumed substrate 1 related to the

o the original amount of substrate 1 (in %) is given).

iochemical data in terms of enzyme activities of the two recom-inant l-GluDHs are listed in Table 1.

In addition, our expectation that such recombinant catabolicluDHs show product inhibition to a less extend than aboveentioned commercially l-GluDHs has been fulfilled by the two

rioritized enzymes. Product inhibition caused by 50 mM of 2 wasn the range of 30% for the l-GluDH from C. difficile and around 55%or the l-GluDH from F. nucleatum at pH 9.0, thus leading to resid-al enzyme activities of 70% and around 45%, respectively (Fig. 1).he lowered activities of both enzymes are caused by the additionf �-ketoglutarate, not by an instability of the enzymes themselvesecause stability studies showed that no deactivation at all coulde observed after a period of 24 h. In comparison to the publishedata obtained by l-GluDH from C. symbiosus the results clearly indi-ate a significantly lowered product inhibition level, even at higheroncentrations in the range of 50 mM.

With the two recombinant l-GluDHs in hand, we next focusedn the desired preparative enzymatic synthesis of �-ketoglutarate2). Initial work addressed the set up of a robust and reliable ana-ytical method for determination of the conversion. Due to thenown potential of �-keto acids to decompose, determination ofonversion (related to the formation of 2) through the absolutemount of �-ketoglutarate (2) via comparison with an internaltandard appeared to us more attractive compared to calculationf conversion based on a relative comparison of substrate/product

atio. As an analytical method we chose proton NMR spectroscopysing pivalic acid (trimethylacetic acid) as an internal standard

n combination with a presaturation technique (to improve the

ig. 1. Product inhibition of the l-GluDHs from C. difficile (GluDH CD) and F. nuclea-um (GluDH FN) by �-ketoglutarate (2) (assay: 50 mM l-1; pH 9.0; 50 mM 2; 2.5 mMAD+).

60(60) 77(76) 82(77)

al amount of substrate 1 (in parentheses the amount of generated product 2 related

quality of the baseline of the spectra due to suppression of thelarge signal resulting from the solvent water) [19]. Next we stud-ied the stability of �-ketoglutarate (2) under reaction conditionsstarting from an �-ketoglutarate (2, 10 mM)/l-glutamate mixture(l-1, 40 mM) which simulates a 20% conversion of an enzyme-catalyzed oxidation of 50 mM L-glutamate. After optimization ofsampling and work-up for analytical determination (showing adeviation in the range of ca. 3%), we found high recovery ratesof 97% or more for �-ketoglutarate (2) within a time range of48 h (Fig. 2). Notably, recovery rates were high not only for the�-ketoglutarate/l-glutamate mixture but also in the presence of acrude extract of the recombinant l-GluDH from C. difficile. How-ever, when using a crude extract of the l-GluDH from F. nucleatumwe observed a significantly higher loss of 2 probably caused by an�-ketoglutarate decomposing enzyme present in the partially puri-fied sample of the L-GluDH from F. nucleatum. In general stability ofthe samples appeared to be high at pH 8.0 as well as at 9.6. Selectedexamples of this stability study are given in Fig. 2.

Next we conducted biotransformations with in situ-cofactorregeneration based on the use of the iron(III)porphyrin[Fe(III)TSPP]Cl (3) as an artificial NADH-oxidase according to

Fig. 2. Time-dependent stability of �-ketoglutarate (2) determined out of a mixtureof �-ketoglutarate (2, 10 mM) and l-glutamate (1, 40 mM) (aqueous solution) at pH8.0 or 9.6. Additionally, crude extracts of GluDH from C. difficile (C.d.) or from F.nucleatum (F.n.) were added to the �-ketoglutarate/glutamate mixture.

14 W. Greschner et al. / Journal of Molecular Catalysis B: Enzymatic 103 (2014) 10–15

Table 3Biotransformation of l-glutamate (l-1, 50 mM) into �-ketoglutarate (2) with l-GluDH and Fe(III)TSPP (3, 1 mM) at pH 8 (according to Scheme 1).

Entry l-GluDH l-GluDH [U/mL] Temp. [◦C] Conv.a 1 h [%] Conv.a 4 h [%] Conv.a 24 h [%]

1 C. difficile 5 20 22(22) 33(33) 47(43)b

2 F. nucleatum 5 20 5(5) 9(8) 16(5)3 F. nucleatum 5 12 8(8) 11(11) 28(27)c

a The conversion is defined as the amount of consumed substrate 1 related to the original amount of substrate 1 (in parentheses the amount of generated product 2 relatedt

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ere obtained in particular in the presence of the l-GluDH from. difficile (entries 1 and 2). For example, after optimization and aeaction time of 8 h a conversion of 91% (based on consumed sub-trate l-1) as well as a product-related conversion of 89% (based onhe amount of formed 2, entry 2) were observed, whereas using aeduced amount of the enzyme resulted in a somewhat decreasedonversion of 76% (entry 1). The total turnover numbers for theofactor NAD+ calculated from entries 1 and 2 (Table 2) are withinhe range of 6.5–7.4.

Furthermore, we carried out experiments with NADH-oxidaseNOX) from Lactobacillus brevis in order to compare the “artifi-ial NADH oxidase” (iron(III)porphyrin [Fe(III)TSPP]Cl (3)) with thenzyme NADH oxidase (Table 2; entry 3). Unfortunately, the activ-ty of the enzyme NADH oxidase is relatively low at higher pHalues, therefore an excess of activity was added to obtain completeegeneration of NAD+.

Table 2 shows that, in terms of efficiency, the conversion ratesf the biomimetic [Fe(III)TSPP]Cl (3) are in the same order of mag-itude as the enzyme NADH oxidase.

Subsequently, we increased the substrate concentration of l-1o 50 mM and operated at a pH-value of 8 which turned out to behe preferred pH in terms of product stability. When using the l-luDH from C. difficile we gained after optimization a conversion of7% at room temperature (Table 3, entry 1). In the case of l-GluDHrom F. nucleatum we faced the problem of an immense loss of 2 andow conversions (entry 2). In order to suppress chemical degrada-ion of �-ketoglutarate (2), experiments were done at a somewhatecreased temperature of 12 ◦C. When using the l-GluDH from. nucleatum as a biocatalyst at this reaction temperature, aroduct-related conversion of �-ketoglutarate (2) (often defineds so-called “reaction yield”) of 27% was found after 24 h (Table 3,ntry 3).

Further experiments under different conditions like varying theH value, more intensive cooling during the reaction or stepwiseddition of the substrate l-1 did not show higher conversionshan those presented in Table 3. Moreover the cooling was thenly way to prevent the chemical degradation of �-ketoglutaraten prolonged reaction times, especially when the GluDH from F.ucleatum was used.

. Conclusion

In conclusion, we reported the use of an artificial in situ-cofactoregeneration with [Fe(III)TSPP]Cl (3) for enzymatic amino acid oxi-ation, exemplified for the l-glutamate dehydrogenase-catalyzedynthesis of �-ketoglutarate (2) from l-glutamate (l-1). In com-arison of the two investigated dehydrogenases, the l-glutamateehydrogenase from C. difficile turned out to be the preferrednzyme. At a substrate concentration of 15 mM of l-glutamate (l-1)n situ-cofactor regeneration using [Fe(III)TSPP]Cl (3) as an “arti-cial NADH-oxidase” proceeded smoothly, leading to up to >99%

verall conversion and 88% conversion related to the formationf desired product 2 after 24 h. At an increased concentration of0 mM of substrate l-1, a somewhat decreased conversion of 43%as observed (which, however, corresponds to a nearly doubled

[

volumetric productivity of 3.95 g/(L d) compared to the experi-ments at 15 mM). In addition, this productivity is almost four timeshigher as the one reported previously in literature when using anNADH-oxidase [8]. Thus, the iron complex [Fe(III)TSPP]Cl turnedout to be capable to be used for cofactor regeneration of the cofactorNAD+ for enzymatic amino acid oxidation.

The regeneration of NAD+ is a prerequisite if NAD+-dependentdehydrogenases are designated for oxidation reactions, for exam-ple, converting hydroxy acids, amino acids or alcohols into thecorresponding keto compounds. Only a few enzymatic methodsare described in the literature to achieve this aim, but all of thempresent also disadvantages: the use of glutamate dehydrogenase[20,21], lactate dehydrogenase [22], or horse liver alcohol dehy-drogenase [23] always leads to the formation of by-products, thuscomplicating the isolation of the desired product. The reactions cat-alyzed by these enzymes are reversible therefore it is difficult toreach a complete conversion regarding the primary desired oxidiz-ing reaction. Peroxide forming NADH oxidases require the additionof catalase. Furthermore, all enzymatic systems depend typically onone coenzyme species, either NADH or NADPH, which hampers thedevelopment of a generalizable reaction platform. Complementingenzymes as catalysts to regenerate the oxidized coenzyme, the ironcomplex [Fe(III)TSPP]Cl can be used successfully for this purpose.In addition, it can be stored over a long period of time and it acceptsNADH as well as NADPH. Furthermore, peroxide is not formed bythe reaction catalyzed by this complex.

Currently, further extension of this type of “artificial cofactorregeneration system” by means of the [Fe(III)TSPP]Cl (3) is inprogress as well as studies with respect to the clarification of thereaction mechanism.

Acknowledgements

The authors thank the German Federal Environmental Founda-tion (Deutsche Bundesstiftung Umwelt, DBU) for generous supportwithin the DBU-network “ChemBioTec” of the funding priority“Biotechnology” (Project AZ 13234-32). We further thank. Dr.Philipp Böhm for helpful discussions and technical support.

References

[1] K. Drauz, H. Gröger, O. May (Eds.), Enzyme Catalysis in Organic Synthesis, vols.1–3, 3rd ed., Wiley-VCH, Weinheim, 2012.

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