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Journal of Biotechnology

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owards catalyst compartimentation in combined chemo- andiocatalytic processes: Immobilization of alcohol dehydrogenases forhe diastereoselective reduction of a �-hydroxy ketone obtained fromn organocatalytic aldol reaction

iuseppe Rulli a, Marcel Heidlindemannb, Albrecht Berkessel c,∗, Werner Hummeld,∗∗,arald Grögera,b,∗ ∗ ∗

Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, Henkestrasse 42, 91054 Erlangen, GermanyFaculty of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, GermanyDepartment of Chemistry, University of Cologne, Greinstraße 4, 50939 Cologne, GermanyInstitute of Molecular Enzyme Technology at the Heinrich-Heine-University of Düsseldorf, Research Centre Jülich, Stetternicher Forst, 52426 Jülich,ermany

r t i c l e i n f o

rticle history:eceived 2 May 2013eceived in revised form 27 August 2013ccepted 29 August 2013vailable online xxx

eywords:lcohol dehydrogenase

a b s t r a c t

The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turnedout to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized withtheir cofactors on a commercially available superabsorber based on a literature known protocol. The useof the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to highconversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the secondreaction cycle. A comparable result was obtained when no cofactor was added although a water richreaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastere-

lcoholiastereoselective synthesisnzyme catalysismmobilizationeduction

oselective reduction of organocatalytically prepared enantiomerically enriched aldol adducts, leading tohigh conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at alower catalyst and cofactor amount being still sufficient for biotransformations with “free” enzymes theimmobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strongdecrease of conversion was observed already in the second reaction cycle, thus indicating a significant

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leaching effect.

The combination of chemical reactions towards one-potrocesses represents an attractive opportunity to enable bothustainable and economically attractive production processesBroadwater et al., 2005). One of the particular challenges in

Please cite this article in press as: Rulli, G., et al., Towards catalyst compartlization of alcohol dehydrogenases for the diastereoselective reduction of

J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.031

his field is the combination of chemocatalytic and enzymaticeactions since these catalysts are often regarded not to be com-atible with each other. Examples of such combinations towards

Abbreviations: ADH, alcohol dehydrogenase; NADH, nicotinamide adenine dinu-leotide reduced form; NAD+, nicotinamide adenine dinucleotide oxidized form;ADPH, nicotinamide adenine dinucleotide phosphate reduced form; NADP+, nicoti-amide adenine dinucleotide phosphate oxidized form.∗ Corresponding author. Tel.: +49 221 470 3283.

∗∗ Corresponding author. ∗ ∗Corresponding author. Present address: Faculty of Chemistry, Bielefeld Univer-ity, Universitätsstrasse 25, 33615 Bielefeld, Germany. Tel.: +49 521 106 2057.

E-mail addresses: berkessel@uni-koeln.de (A. Berkessel),.hummel@fz-juelich.de (W. Hummel), harald.groeger@uni-bielefeld.de

H. Gröger).

168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jbiotec.2013.08.031

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© 2013 Elsevier B.V. All rights reserved.

chemoenzymatic one-pot processes are still rare, in particularwhen using aqueous reaction media (Bruggink et al., 2003;Pellissier, 2008; Hussain, 2012; Makkee et al., 1980; Allen andWilliams, 1996; Schoevaart and Kieboom, 2002; Simons et al., 2006;Burda et al., 2008; Prastaro et al., 2009; Baer et al., 2009; Rulli et al.,2011; Kinnell et al., 2012). Recently, we showed a proof of conceptfor the combination of an asymmetric organocatalytic reaction witha biotransformation, exemplified for an initial proline derivative-catalyzed aldol reaction with a subsequent reduction of the aldoladducts catalyzed by an alcohol dehydrogenase (ADH) according tothe concept shown in Scheme 1 (Baer et al., 2009; Rulli et al., 2011).

Notably, both processes running in water can be coupled witheach other without work-up of the aldol adduct according to thereactor concept shown in Fig. 1, part A (Rulli et al., 2011). A keyfeature is the addition of the ADH after the initial aldol reaction

imentation in combined chemo- and biocatalytic processes: Immobi-a �-hydroxy ketone obtained from an organocatalytic aldol reaction.

step. Besides such a one-pot process, compartimentation of thetwo types of catalyst represents an interesting alternative. In suchprocess technology and reactor concepts (according to Fig. 1, partB (semi-continuously operating process) or part C (continuously

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mH

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Scheme 1. Combination of an organocatalytic aldol reaction with a diastereoselect

perating process)), both catalysts are immobilized and the reac-ion mixture is pumped subsequently through columns filled withuch immobilisates. In analogy to the “classic” batch-type one-ot process using “free”, non-immobilized catalysts no work-up isequired, thus offering the perspective to obtain economically andcologically favourable data. In addition, compartimentation of theatalysts offers further advantages such as, e.g., (i) an easy separa-ion and recycling of the catalysts, (ii) avoidance of compatibilityroblems of the two catalyst systems due to a possible negative

nteraction with each other (via inhibition, deactivation effects)nd (iii) the option to operate at different reaction conditionse.g., reaction temperature). Accordingly, we became interested inhe development of suitable immobilisates of both the organo-nd biocatalyst. After a suitable formulation of the immobilizedrganocatalyst for the enantioselective aldol reaction of acetoneith a substituted benzaldehyde was identified (Kristensen et al.,

009; Rulli et al., 2013) we then focused on immobilization of thelcohol dehydrogenases which turned out to be suitable as biocat-lysts for the diastereoselective reduction of the �-hydroxy ketonebtained by means of the initial organocatalytic aldol reaction.

In the following we report our results on the extension ofhe superabsorber-based co-immobilization method for ADHs andofactors developed by Jeromin (2009, 2011) towards the ADHsrom L. kefir and Rhodococcus sp. and the application of suchmmobilized enzymes for reductions in a water-rich reaction

edium as preferred reaction embodiment for such biotransfor-ation steps. Besides acetophenone as a model substrate ourajor interest was on the use of these immobilized biocatalysts

or the diastereoselective reduction of an organocatalytically pre-ared aldol adduct (�-hydroxy ketone). A further focus was on thevaluation of the efficiency of the immobilized ADHs in terms of re-se as well as catalyst stability and leaching under those reaction

Please cite this article in press as: Rulli, G., et al., Towards catalyst compartlization of alcohol dehydrogenases for the diastereoselective reduction of

J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.031

onditions.The initial step consisted in the choice of an immobilization

ethod for the ADHs from Lactobacillus kefir (Weckbecker andummel, 2006) and Rhodococcus sp. (commercially available from

Fig. 1. Comparison of different reactor concepts for the combination of organo-

zymatic reduction of the “in situ”-formed �-hydroxy ketone in a one-pot process.

evocatal GmbH, Germany; product number 1.1.030), which turnedout to catalyze successfully the diastereoselective reduction of aldoladducts (�-hydroxy ketones, see Scheme 1) when being used in“free” (non-immobilized) form (Baer et al., 2009; Rulli et al., 2011).Among available immobilization methods (Pierre, 2004; Liese et al.,2006) we chose a protocol developed by Jeromin (2009, 2011) forimmobilization of ADHs from Lactobacillus brevis and Thermoanaer-obicum sp. on a commercial superabsorber in slightly modifiedform. As a binding mechanism Jeromin postulated an absorptionor entrapment of the ADHs (and cofactors) on or in the superab-sorber, which consists of polymerized acrylic acid (Jeromin, 2011).This method attracted us in particular due to the economical attrac-tiveness and readily availability of superabsorbers as well as theopportunity to include and retain the enzyme and cofactor in theimmobilization matrix (which, however, has been demonstrated inthe presence of isopropanol with a low water content as reactionmedium), thus suppressing cofactor leaching during the process(Jeromin, 2009).

To start with the immobilization of the ADH from L. kefir (whichis structurally closely related to the ADH from L. brevis used byJeromin, 2009), immobilization conducted in analogy to the proto-col of Jeromin with slight modification of the work-up proceededsmoothly leading to an enzyme immobilisate with an activityof 4.8 U/mg (components for immobilization of the ADH from L.kefir: 10 mL pH 7 buffer, 50 mM; 50 mg MgCl2; 79 mg NADP+;2.5 mL isopropanol; 1 mL ADH from L. kefir, 2400 U/mL relativeto acetophenone; 1 g superabsorber Favour SXM 9155® (EvonikIndustries AG); after 10 min of stirring of the mixture 5 mL of waterwere added; work-up consist of evaporation of volatile materialsin vacuo for 18 h; amount of water in the immobilisate: 34%(m/m)after 24 h drying at 107 ◦C). When conducting an initial reduc-tion using acetophenone (1) as a model substrate (which also

imentation in combined chemo- and biocatalytic processes: Immobi-a �-hydroxy ketone obtained from an organocatalytic aldol reaction.

has been used in previous studies, see Weckbecker and Hummel,2006) and added cofactor NADP+, we found a high conversion of>95% under non-optimized conditions by means of this immobi-lized ADH from L. kefir (Fig. 2). When recycling the immobilisate

and biocatalysis towards a process without work up of the intermediate.

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Fig. 2. Experiments towards characterization of the reaction performance of immobilized ADH from L. kefir in a water/isopropanol reaction medium (75:25) exemplifiedfor the reduction of acetophenone. (Experimental protocol: 1 g of immobilized ADH from L. kefir (4.8 U/mg) was added to a mixture of buffer (7.5 mL), isopropanol (2.5 mL)a , the mp was aw

awc

msJptHvTtltafhsalnbmltbtcrir

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nd 58 �L acetophenone (1, 0.5 mmol). After optional addition of 20 mg of cofactorhase-separation filter and evaporation of the solvent gave a crude-product whichas washed in 20 ml isopropanol prior to reuse for the second cycle.)

nd re-using it in a further biotransformation a conversion of 92%as observed, which indicates a slight loss of activity under these

onditions.Since the cofactor is co-immobilized in the superabsorber

atrix, we evaluated next if the biotransformation also proceedsuccessfully in the absence of added cofactor. In the earlier work oferomin such a synthetic set-up has been reported to be suitable inarticular when using a “dry” immobilisate (14% (m/m) water con-ent) and a water/isopropanol mixture of 10:90 (Jeromin, 2009).owever, in our biotransformation we applied preferably a sol-ent mixture of water/isopropanol in a volumetric ratio of 75:25.hus, the reaction medium contains a significantly increased por-ion of water, which then might led to an undesired high degree ofeaching of the cofactor from the immobilization matrix and (dueo the resulting dilution of the cofactor) a decreased reaction ratend conversion. For the biotransformation with immobilized ADHrom L. kefir in the absence of added cofactor we observed still aigh conversion of >95%. A re-use of this immobilisate led to alightly decreased conversion of 91% (no additional cofactor wasdded for the second reaction cycle). This indicates that a severeeaching of the cofactor bound in the superabsorber matrix doesot occur in the first reaction cycle when using this type of immo-ilisate even at a high amount of water (75% (v/v)) in the reactionedium. At the same time, however, it has to be considered that a

arge amount of cofactor was used in the immobilization step andhat a more detailed study with lower cofactor amount still had toe done at this stage in order to get a more detailed insight intohe leaching issue (see later section and Fig. 3). Notably, a signifi-

Please cite this article in press as: Rulli, G., et al., Towards catalyst compartlization of alcohol dehydrogenases for the diastereoselective reduction of

J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.031

antly lower conversion of 55% was obtained when replacing theeaction medium aqueous buffer/isopropanol (75/25 (v/v)) by puresopropanol. [This experiment was carried out in analogy to the firsteaction cycle described in Fig. 2 (without further added cofactor).]

ixture was stirred for 18 h and diluted with methylene chloride. Filtration over analyzed via 1H NMR. For recycling of the immobilisates the filtered superabsorber

Next we focused on the immobilization of the ADH fromRhodococcus sp. on the superabsorbent polymer Favor SXM 9155®

(Evonik Industries AG) according to the protocol of Jeromin (2009)in slightly modified form as described above. Once again, anactive immobilized ADH was obtained, however, with a somewhatdecreased activity of 0.5 U/mg compared to the immobilized ADHfrom L. kefir. When using this immobilized ADH from Rhodococ-cus sp. (components for immobilization of the (S)-ADH fromRhodococcus sp.: 10 mL pH 7 buffer, 50 mM; 79 mg NAD+; 2.5 mLisopropanol; 1 mL (S)-ADH from Rhodococcus sp., 71 U/mL rela-tive to 4′-chloroacetophenone; 1 g superabsorber Favor SXM 9155®

(Evonik Industries AG); after 10 min of stirring of the mixture 5 mLof water were added; work-up consist of evaporation of volatilematerials in vacuo for 18 h; amount of water in the immobilisate:72%(m/m) after 24 h drying at 107 ◦C) for the reduction of acetophe-none (1) in a synthetic experiment with water/isopropanol (75:25)as a reaction medium, a high conversion of >95% was obtainedunder non-optimized reaction conditions in the presence of addedcofactor NAD+ (Scheme 2).

With these immobilized forms of the two required ADHs from L.kefir and Rhodococccus sp. in hand, we then applied these heteroge-neous biocatalysts for the desired diastereoselective reduction ofthe organocatalytically prepared chiral aldol adduct (R)-3 (whichwas prepared by means of an immobilized organocatalyst in asemi-continuous process according to Rulli et al., 2013 and showed89% ee). When using the immobilized (R)-enantioselective ADHfrom L. kefir as a biocatalyst, the reduction proceeded successfullyunder formation of the desired (R, R)-diastereomer with 92%

imentation in combined chemo- and biocatalytic processes: Immobi-a �-hydroxy ketone obtained from an organocatalytic aldol reaction.

conversion, a high diastereomeric ratio of >20:1 (syn/anti) andexcellent >99% ee (Scheme 3). The (R, S)-stereoisomer was formedin the presence of the immobilized ADH from Rhodococcus sp. withalso >95% conversion, a diastereomeric ratio of 1:16 (syn/anti) and

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Fig. 3. Diastereoselective reduction of the organocatalytically prepared chiral aldol adduct (R)-3 (95% ee) using an immobilized ADH from Rhodococcus sp. at decreasedcatalytic amount. (Experimental protocol: 80 mg of immobilized (S)-ADH from Rhodococcus sp. (0.2 U/mg, approx. 60% water content) was added to a mixture of phosphatebuffer (100 mM, pH 7, 7.5 mL), isopropanol (2.5 mL) and 109–111 mg �-hydroxyketone (R)-3 (corresponding to 0.55–0.56 mmol). After stirring the mixture for 24 h, thesuperabsorbed ADH was filtered and washed with 256 �L isopropanol. The filtrate was extracted three times with methylene chloride and filtrated over a phase-separationfi MR s1 e in act on ste

a1i

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SeiF

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lter. Evaporation of the solvent gave a crude product which was analyzed via 1H N.0 mL/min, 30 ◦C, 220 nm). The analytical data obtained for compound (R,S)-4 werhe filtered superabsorber was re-used without any further treatments or purificati

n enantioselectivity of >99% ee. Thus, both stereoisomers of the,3-diol products, namely (R, R)-4 and (R, S)-4, are now accessible

n the presence of the immobilized ADHs biocatalysts.Based on this proof of concept we next became interested to

valuate this method in detail in terms of efficiency and enzymend co-enzyme leaching during recycling. For this purpose, the

Please cite this article in press as: Rulli, G., et al., Towards catalyst compartlization of alcohol dehydrogenases for the diastereoselective reduction of

J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.031

eaction conditions had to be changed since the initially usednzyme activities (as described above in the “standard protocol”ith 1042 U/mmol of substrate (R)-3, Scheme 3) are significantly

cheme 2. Experiments towards a characterization of the reaction performance of immoxemplified for the reduction of acetophenone. (Experimental protocol: 1 g of immobilizedsopropanol (2.5 mL) and 58 �L acetophenone (1, 0.5 mmol). After optional addition of 20 miltration over a phase-separation filter and evaporation of the solvent gave a crude-prod

pectroscopy and chiral HPLC (Chiralpak® column OJ-H, CO2:isopropanol 97:3, flowcordance with the data given in Rulli et al. (2011). For subsequent reaction cyclesps in the following cycle according to the above mentioned protocol.)

higher than the enzyme activities used in our earlier syntheticreactions with the “free” enzymes (10 U/mmol of substrate 4′-chloroacetophenone, see Rulli et al., 2011) as well as the enzymeactivities used by Jeromin (250 U/mmol of ketone, see Jeromin,2009). Accordingly, we studied if the immobilized ADHs can also beused and re-cycled when operating at a much lower initial enzyme

imentation in combined chemo- and biocatalytic processes: Immobi-a �-hydroxy ketone obtained from an organocatalytic aldol reaction.

activities being in the range of those of the biotransformationswith “free” enzymes. Thus, the components applied for immobiliza-tion of the ADH from Rhodococcus sp. according to the protocol by

bilized ADH from Rhodococcus sp. in a water/isopropanol reaction medium (75:25) (S)-ADH from Rhodococcus sp. (0.5 U/mg) was added to a mixture of buffer (7.5 mL),g of cofactor, the mixture was stirred for 18 h and diluted with methylene chloride.

uct which was analyzed via 1H NMR.)

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Scheme 3. Diastereoselective reduction of the organocatalytically prepared chiral aldol adduct (R)-3 (89% ee) using an immobilized ADH from L. kefir or Rhodococcussp. (Experimental protocol: 1 g of immobilized (R)-ADH from L. kefir (4.8 U/mg) or (S)-ADH from Rhodococcus sp. (0.5 U/mg) was added to a mixture of buffer (7.5 mL),isopropanol (2.5 mL) and 100 �L �-hydroxyketone (R)-3 (103.9 mg, 95% purity, corresponding to 0.48 mmol). After stirring the mixture for a reaction time of 24 h and dilutionw of th( analyte

Jopt6A5wflwmbtpichauewithetrranioc

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ith methylene chloride, filtration over a phase-separation filter and evaporationChiralpak® column OJ-H, hexane:isopropanol 99:1, flow 1.0 mL/min, 220 nm). The

t al. (2011).)

eromin (2009) were then changed as follows: a mixture consistingf 178 �L phosphate buffer (pH 7, 100 mM), 15 mg NAD+, 13 �L iso-ropanol and 190 �L ADH from Rhodococcus sp. (265 U/mL relativeo 4′-chloroacetophenone) was stirred for 10 min prior to adding3 mg of superabsorber Favor SXM 9155® (Evonik Industries AG);.fter an additional 10 min of stirring the mixture solidified and10 �L isopropanol were added. Subsequent filtration and washingith 255 �L isopropanol, followed by 15 min drying under an airow then gave 160 mg (60% (m/m) of water) of superabsorbed ADHith an activity of 0.2 U/mg (which was determined via a photo-etric assay according to Jeromin, 2009). With this heterogeneous

iocatalyst derived from Rhodococcus sp. ADH we again performedhe desired diastereoselective reduction of the organocatalyticallyrepared chiral aldol adduct (R)-3 (0.5 mmol; synthesized accord-

ng to Rulli et al., 2011) at room temperature and with 0.5 mol%atalyst and 24 h reaction time, leading to 95% ee; Fig. 3). In this case,owever, now a significantly lowered activity of ADH (29 U/mmol)nd coenzyme amount (4.5 mol% for the immobilization step) wassed for the immobilisate, which corresponds to the range of thenzyme activities applied when operating earlier (Rulli et al., 2011)ith “free” enzymes. In the initial biotransformation with this ADH

mmobilisate, the reduction proceeded successfully under forma-ion of the desired (R, S)-diastereomer with >95% conversion, aigh diastereomeric ratio of >20:1 (syn/anti) and excellent >99%e (Fig. 3). After recovery of the heterogeneous components con-aining the superabsorber-immobilized enzyme, a second and thirdeaction cycle using the recycled immobilized biocatalyst was car-ied out. However, as shown in Fig. 3, the conversion drops to 40%fter the second cycle (while enantioselectivity is not affected), ando conversion was observed after cycle number three. These results

ndicate that significant leaching of enzyme and/or coenzymeccurs under the used reaction conditions during each reactionycle.

In summary, we reported an extension of the superabsorber-ased co-immobilization method for ADHs and cofactors developedy Jeromin towards the ADHs from L. kefir and Rhodococcus sp.,

Please cite this article in press as: Rulli, G., et al., Towards catalyst compartlization of alcohol dehydrogenases for the diastereoselective reduction of

J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.031

hich earlier turned out to be suitable for a chemoenzymatic one-ot synthesis with organocatalysts. Exemplified for the ADH from. kefir, the use of this immobilized enzyme in the reduction of ace-ophenone as a model substrate led to high conversion (>95%) in the

e solvent gave a crude product which was analyzed via 1H NMR and chiral HPLCical data obtained for compounds 4 were in accordance with the data given in Rulli

first reaction cycle, followed by a slight decrease of conversion inthe second reaction cycle. As a reaction medium a mixture of aque-ous buffer/isopropanol (75/25 (v/v)) was used. The immobilizedADHs also turned out to be suitable catalysts for the diastereose-lective reduction of organocatalytically prepared enantiomericallyenriched aldol adducts, leading to high conversion, diastereomericratio and enantioselectivity for the resulting 1,3-diols of type 4.However, when applying a lower amount of the immobilized bio-catalyst (which is still sufficient for a quantitative conversion after24 h reaction time) subsequent recycling revealed a significantleaching of enzyme and/or co-factor. Thus, although these ADH andcofactor immobilisates are suitable for lab-scale applications in awater-rich reaction medium, process improvement for their effi-cient use in chemoenzymatic synthesis under compartimentationof organo- and biocatalysts in fixed-bed reactors is still a challengeand subject of current research.

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft (DFG) forgenerous support within the priority programme SPP1179“Organokatalyse” (BE 998/11-1, GR 3461/2-1). Membership in theEuropean Cooperation in Science and Technology (COST) Action no.CM0905 Organocatalysis (ORCA) is gratefully acknowledged. Wealso thank Mr. Martin Aschenbrenner for technical assistance.

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