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DOI: 10.1002/adsc.201100256 Multi-Enzymatic Cascade Reactions: Overview and Perspectives Emanuele Ricca, a Birgit Brucher, b and Joerg H. Schrittwieser c, * a Laboratory of Transport Phenomenaand Biotechnology, Engineering Faculty, University of Calabria, via P. Bucci Cubo 42A, 87036 Arcavacata di Rende (CS), Italy b Institute of Process Engineering in Life Sciences, Section II: Technical Biology, arlsruhe Institute of Technology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany c Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria Fax: (+ 43)-316-380-9840; e-mail: [email protected] Received: April 5, 2011; Published online: August 25, 2011 Abstract: Multi-enzymatic cascade reactions, i.e., the combination of several enzymatic transformations in concurrent one-pot processes, offer considerable ad- vantages: the demand of time, costs and chemicals for product recovery may be reduced, reversible re- actions can be driven to completion and the concen- tration of harmful or unstable compounds can be kept to a minimum. This review summarizes the de- velopments in multi-enzymatic cascades employed for the asymmetric synthesis of chiral alcohols, amines and amino acids, as well as for C À C bond for- mation. In addition, a general classification of biocat- alytic cascade systems is provided and bioprocess en- gineering aspects associated with the topic are dis- cussed. 1 Introduction 2 Classification of Biocatalytic Cascade Reactions 3 Multi-Enzyme Cascade Processes 3.1 Multi-Enzyme Cascades for the Production of Enantiomerically Enriched Alcohols 3.2 Multi-Enzyme Cascades for the Production of Enantiomerically Enriched Amines and Amino Acids 3.3 Multi-Enzyme Cascades Employing Aldolases and Hydroxynitrile Lyases for C À C Bond Forma- tion 4 Bioprocess Engineering Aspects of Biocatalytic Cascades 4.1 The Gain of One-Pot in Biocatalytic Reactions 4.2 Enzyme-Enzyme and Chemo-Enzymatic Reac- tions 4.3 Industrial Applications and Future Develop- ments 5 Conclusions and Outlook Keywords: alcohols; amines; biocatalysis; cascade re- actions; C À C coupling 1 Introduction The development of cleaner and more efficient chem- ical processes has become one of the major goals of chemical research in the new millennium. Leading sci- entists in the field have come to the conclusion that organic synthesis – while having reached an impres- sive level of sophistication – still holds vast room for improvement, especially in ecological terms. [1–4] The replacement of stoichiometric with catalytic proce- dures, the use of enzymes or whole microorganisms in organic synthesis and the combination of several reac- tions in one pot without isolation of intermediates (cascade reactions) are considered important strat- egies for establishing environmentally benign and sus- tainable chemical processes. The integration of sever- al biocatalytic transformations in a multi-enzyme cas- cade system is particularly appealing since biocata- lysts are intrinsically “green” (biodegradable), highly selective and – most importantly – they are compati- ble with each other within certain ranges of operating conditions. Indeed, living organisms carry out a huge variety of enzymatic reactions in a common reaction medium – the cytosol. Consequently, numerous at- tempts have been made to reproduce such enzymatic cascade reactions in vitro and to apply them in organ- ic synthesis. Multi-enzyme systems of remarkable complexity have been developed. This review pro- vides an overview of the field as well as a classifica- tion of the most common approaches, covering the perspectives of chemistry and bioprocess engineering alike. Adv. Synth. Catal. 2011, 353, 2239 – 2262 # 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2239 REVIEWS

Multi-Enzymatic Cascade Reactions: Overview and Perspectives

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DOI: 10.1002/adsc.201100256

Multi-Enzymatic Cascade Reactions: Overview and Perspectives

Emanuele Ricca,a Birgit Brucher,b and Joerg H. Schrittwieserc,*a Laboratory of Transport Phenomena and Biotechnology, Engineering Faculty, University of Calabria, via P. Bucci Cubo

42A, 87036 Arcavacata di Rende (CS), Italyb Institute of Process Engineering in Life Sciences, Section II: Technical Biology, arlsruhe Institute of Technology,

Engler-Bunte-Ring 1, 76131 Karlsruhe, Germanyc Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria

Fax: (+43)-316-380-9840; e-mail: [email protected]

Received: April 5, 2011; Published online: August 25, 2011

Abstract: Multi-enzymatic cascade reactions, i.e. , thecombination of several enzymatic transformations inconcurrent one-pot processes, offer considerable ad-vantages: the demand of time, costs and chemicalsfor product recovery may be reduced, reversible re-actions can be driven to completion and the concen-tration of harmful or unstable compounds can bekept to a minimum. This review summarizes the de-velopments in multi-enzymatic cascades employedfor the asymmetric synthesis of chiral alcohols,amines and amino acids, as well as for C�C bond for-mation. In addition, a general classification of biocat-alytic cascade systems is provided and bioprocess en-gineering aspects associated with the topic are dis-cussed.

1 Introduction2 Classification of Biocatalytic Cascade Reactions3 Multi-Enzyme Cascade Processes

3.1 Multi-Enzyme Cascades for the Production ofEnantiomerically Enriched Alcohols

3.2 Multi-Enzyme Cascades for the Production ofEnantiomerically Enriched Amines and AminoAcids

3.3 Multi-Enzyme Cascades Employing Aldolasesand Hydroxynitrile Lyases for C�C Bond Forma-tion

4 Bioprocess Engineering Aspects of BiocatalyticCascades

4.1 The Gain of One-Pot in Biocatalytic Reactions4.2 Enzyme-Enzyme and Chemo-Enzymatic Reac-

tions4.3 Industrial Applications and Future Develop-

ments5 Conclusions and Outlook

Keywords: alcohols; amines; biocatalysis; cascade re-actions; C�C coupling

1 Introduction

The development of cleaner and more efficient chem-ical processes has become one of the major goals ofchemical research in the new millennium. Leading sci-entists in the field have come to the conclusion thatorganic synthesis – while having reached an impres-sive level of sophistication – still holds vast room forimprovement, especially in ecological terms.[1–4] Thereplacement of stoichiometric with catalytic proce-dures, the use of enzymes or whole microorganisms inorganic synthesis and the combination of several reac-tions in one pot without isolation of intermediates(cascade reactions) are considered important strat-egies for establishing environmentally benign and sus-tainable chemical processes. The integration of sever-al biocatalytic transformations in a multi-enzyme cas-cade system is particularly appealing since biocata-lysts are intrinsically “green” (biodegradable), highly

selective and – most importantly – they are compati-ble with each other within certain ranges of operatingconditions. Indeed, living organisms carry out a hugevariety of enzymatic reactions in a common reactionmedium – the cytosol. Consequently, numerous at-tempts have been made to reproduce such enzymaticcascade reactions in vitro and to apply them in organ-ic synthesis. Multi-enzyme systems of remarkablecomplexity have been developed. This review pro-vides an overview of the field as well as a classifica-tion of the most common approaches, covering theperspectives of chemistry and bioprocess engineeringalike.

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2 Classification of Biocatalytic CascadeReactions

It has been proposed to classify enzyme-catalyzedmulti-step reactions according to the type of inter-mediates that occur until the final product isformed.[5] If a first, enzyme-catalyzed step leads to theformation of an unstable intermediate, which sponta-neously undergoes further reactions before ultimatelyforming the stable product, the whole process wouldbe called a biocatalytic “cascade” or “domino” reac-tion. If, however, several enzyme-catalyzed transfor-mations (which in principle could also be carried outseparately) are coupled in a one-pot fashion, thiswould constitute a biocatalytic “tandem” reaction.However, in recent years there has been a tendencyto use the term “cascade” in a broader sense, includ-ing systems of the latter type as well.[3,4,6–8] According-ly, we would like to refer to a biocatalytic cascade asany reaction system where two or more transforma-

tions are carried out concurrently in the same reac-tion vessel, employing at least one biocatalyst. Thisincludes multi-enzymatic, chemo-enzymatic andenzyme-initiated spontaneous sequences. In thisreview we will focus exclusively on the first category.Biocatalytic cascade reactions in this sense can beclassified into one of four different “designs”.

(i) The most straightforward approach is constitutedby linear cascades, where a single substrate is convert-ed into a single product via one or more intermedi-ates in a multi-step one-pot fashion [Scheme 1 (a)].Cascade processes of this kind do not only help tosave time and reduce waste in multi-step synthesesthrough the elimination of downstream processingsteps, they also offer advantages when unstable ortoxic intermediates are involved, since these do notaccumulate but are transformed further into the finalproduct. This leads to safer processes, reduced side re-actions and better yields. In addition, linear cascadesmay help to drive reversible reactions to completionif the reaction product is further converted in a fol-

Emanuele Ricca, born 1981,graduated in chemical engi-neering at the University ofCalabria (UNICAL) in 2005with a thesis on artificialorgans in the group of Prof.G. Catapano. He then joinedProf. G. Iorio�s group for hisPhD on industrial biocatalysis.He was guest PhD student atthe University of Trieste withProf. L. Gardossi working onenzyme immobilization, andguest postdoc at Denmark Technical University(DTU) with Prof. J. Woodley on the evaluation ofindustrial bioprocesses. Currently he is a joint Re-searcher at UNICAL and the Italian NationalAgency for New Technologies (ENEA). His re-search interests include enzyme processes and kinet-ics, enzyme immobilization, development and opti-mization of bioprocesses for biofuels, transport phe-nomena in enzyme processes and natural systems.

Birgit Brucher studied Geoe-cology at the University ofKarlsruhe and joined thegroup of Prof. Christoph Syl-datk in 2006 for her diplomathesis about hydantoinasesfrom extreme environments.After a short stay at the Uni-versity of Cape Town withProf. Stephanie Burton, sherejoined Prof. Syldatk�s group

for her PhD on the biocatalytic synthesis of unusualamino acids. Her research interests include thescreening and characterization of new biocatalysts,the optimization of enzymatic reactions and the de-velopment of associated analytical methods.

Joerg H. Schrittwieser, born1984, studied chemistry atthe University of Graz. In2007, he joined the researchgroup of Prof. WolfgangKroutil for work on his di-ploma thesis dealing withbiocatalytic cascades involv-ing alcohol dehydrogenases.After participating in twoshort-term research projectsat the Universidad Complu-tense in Madrid and the Re-search Centre of Applied Biocatalysis in Graz, hereturned to Prof. Kroutil�s labs to do his PhD. Hisresearch interests are the development and optimi-zation of multi-enzymatic cascade systems, as well asthe utilization of flavoenzymes for “green” oxidationchemistry.

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lowing irreversible transformation and therefore con-stantly drawn out of the equilibrium. A special caseof linear cascades are deracemization processes, inwhich one enantiomer of a racemic compound is con-verted into the enantiomer of opposite configurationvia a prochiral intermediate, leading to an opticallypure (or optically enriched) product (see the section“Deracemization and stereoinversion of alcohols usingalcohol dehydrogenase cascades” for recent exam-ples).

(ii) In orthogonal cascades, the (enzymatic) trans-formation of the substrate into the product is coupledwith further reactions in order to regenerate cofactorsor cosubstrates, or to remove by-products [Scheme 1(b)]. Cofactor regeneration for nicotinamide-depen-dent oxidoreductases by a second redox enzyme is aclassical example. However, more complex systemsmight be required – and have been developed – inspecial cases. For instance, in a biocatalytic cascadepublished by Soda and co-workers, keto acids aretransformed into d-amino acids by a d-amino acidtransaminase while three more enzymes (alanineracemase, alanine dehydrogenase and formate dehy-drogenase) are required for supplying the transami-nase�s cosubstrate d-alanine and for recycling of theinvolved NADH cofactor (see the section “Multi-en-zymatic syntheses of amines and amino acids employ-ing transaminases”).[9]ACHTUNGTRENNUNG(iii) Closely related to the orthogonal concept areparallel cascades, where two substrates are convertedinto two products by two distinct biocatalytic reac-tions, which are coupled via cofactors or cosubstrates[Scheme 1 (c)]. The difference to orthogonal cascadesmerely lies in the economic value of the substancesinvolved, since in parallel cascades both products areconsidered valuable compounds and are therefore iso-lated. In orthogonal cascades, on the other hand, theby-products are discarded. Detailed investigations onparallel cascades have recently been published byGotor and co-workers, who describe for instance the

combination of alcohol dehydrogenases and Baeyer–Villiger monooxygenases for the concurrent produc-tion of non-racemic alcohols and sulfoxides (see thesection “Alcohol dehydrogenases in linear and parallelmulti-enzyme cascade systems”).[10,11] The same au-thors have also coined the term PIKAT (parallel in-terconnected kinetic asymmetric transformations) forreaction systems of this kind.

(iv) Finally, in cyclic cascades one out of a mixtureof substrates is selectively converted into an inter-mediate which is then transformed back to the start-ing materials. Repetition of this cycle leads to the ac-cumulation of the compound which is left behind inthe first transformation [Scheme 1 (d)]. This concepthas been widely used for the chemo-enzymatic de-ACHTUNGTRENNUNGracemization of amino acids, hydroxy acids andamines. One enantiomer of the starting racemate isoxidized to a prochiral intermediate by an oxidase(e.g., d-amino acid oxidase). The intermediate is re-duced back to the racemic starting material by achemical reducing agent (e.g., NaBH4, amine-bor-anes) or by electrochemical means. Over severalcycles the non-oxidized enantiomer accumulates.Since all published systems using this approach arechemo-enzymatic rather than multi-enzymatic, we willnot give any detailed examples here. Instead we referto a number of excellent reviews covering thetopic.[12–16]

Of course, the presented classification is not a strictone, since cascade designs can be combined in manydifferent ways. One step of a linear cascade, for in-stance, might require additional orthogonal reactions(e.g., cofactor regeneration). Likewise, the degrada-tion of a reaction by-product in an orthogonal cascademay involve more than one step and may thereforeconstitute a linear cascade sequence of its own. In thefollowing sections we shall meet examples of multi-enzymatic processes which can be easily assigned toone single class as well as systems comprising ele-ments of different cascade designs.

3 Multi-Enzyme Cascade Processes

A vast number of biocatalytic cascade reactions hasbeen developed in the past decades, ranging from thesimple combination of oxidoreductases with a suitablecofactor regeneration system to highly complex andspecialized multi-enzymatic synthesis routes.[17] Themost important developments have been made – es-pecially in recent years – in the formation of opticallyactive alcohols, amines and amino acids by means ofbiocatalytic cascades, as well as in the integration ofC�C bond forming enzymes into cascade systems.These topics are covered by the following sections.[18]

Scheme 1. Four different “designs” of enzymatic cascadeprocesses: (a) linear cascade, (b) orthogonal cascade, (c)parallel cascade, and (d) cyclic cascade.

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3.1 Multi-Enzyme Cascades for the Production ofEnantiomerically Enriched Alcohols

For a long time, lipase-catalyzed kinetic resolutionand dynamic kinetic resolution have been the biocata-lytic methods of choice for the preparation of enan-tiomerically enriched alcohols. Recent years, however,have brought about impressive progress in the use ofalcohol dehydrogenases (ADHs) and ketoreductases(KREDs) for the asymmetric synthesis of alcohols bystereoselective reduction of the corresponding ke-tones. Combination of an ADH with a secondenzyme (e.g., formate dehydrogenase) for nicotin-ACHTUNGTRENNUNGamide cofactor regeneration represents a classical ex-ample of an orthogonal biocatalytic cascade reactionin this field. However, since NAD(P)H cofactor recy-cling has been the subject of several reviews,[13,19–25]

we will exclude the topic from this article. Instead weare going to focus on recent systems for the deracemi-zation of alcohols via stereoinversion as well as linearand parallel cascades involving ADHs.

3.1.1 Deracemization and Stereoinversion of AlcoholsUsing Alcohol Dehydrogenase Cascades

The specificity of oxidoreductases towards certainsubstrates and cofactors leads to the possibility ofcombining reduction and oxidation steps in a one-potprocess, something that is not feasible using classicalredox chemistry.[26] For instance, two stereocomple-mentary alcohol dehydrogenases can be combined to

achieve deracemization and stereoinversion of alco-hols via oxidation of one substrate enantiomer to thecorresponding ketone and stereoselective reduction ofthe latter to the alcohol enantiomer of opposite con-figuration.[12,16,27,28] It has been known for more than10 years that various microorganisms possessing ste-reocomplementary sec-ADHs are able to performthese transformations,[15] but only recently have theybeen achieved employing in vitro multi-enzyme sys-tems. Simple combination of two stereocomplemen-tary ADHs in one pot with cofactor cycling betweenthem does not suffice, since it allows equilibration tothe thermodynamically most stable product, which isthe racemic alcohol.[29] However, decoupling of thecofactor regeneration for the two enzymes providescontrol over the oxidation/reduction equilibria andhence over the stereochemical outcome of the reac-tion.

This concept has been put into practice by Kroutiland co-workers, who used whole cells of Alcaligenesfaecalis DSM 13975 or Rhodococcus erythropolisDSM 43066 for enantiospecific oxidation and differ-ent ADHs and cofactor recycling systems for stereo-selective reduction. Thus, a broad variety of secon-dary alcohols was deracemized without the formationof measurable amounts of ketone [Scheme 2 (a)].[30]

However, while excellent ee values were obtained inthis study using Prelog ADHs for the reduction step,the anti-Prelog system led to only moderate enantio-meric enrichment. This limitation was overcome bythe same authors in a second study using an evenmore complex multi-enzyme system:[31] Control over

Scheme 2. Deracemization of secondary alcohols via stereoinversion: (a) aerobic oxidation followed by ADH-catalyzed re-duction, (b) use of two ADHs with opposite enantioselectivity and cofactor specificity.

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the reaction equilibria was realized by using twoADHs with different cofactor specificity (NADH vs.NADPH) and two independent cofactor regenerationsystems – one for NADPH oxidation, another one forNAD+ reduction. Thus, two ADHs complementarywith respect to stereoselectivity and cofactor specifici-ty were combined with an NADPH-oxidase from Ba-cillus subtilis and an (NAD+-specific) formate dehy-drogenase in one pot without any compartmentaliza-tion [Scheme 2 (b)]. Applying this system, differentracemic sec-alcohols were transformed into the enan-tiomerically pure stereoisomers in quantitative yield.Recently, a similar system employing whole microbialcells has also been reported.[32] In this study, restingcells of Microbacterium oxydans ECU2010 possessingan NAD+-dependent (R)-selective alcohol dehydro-genase and Rhodotorula sp. AS2.2241 expressing anNADPH-dependent (S)-selective ketoreductase werecombined in a one-pot process. The reported yields(43–84%) are lower than those obtained by Vosset al. using isolated enzymes;[31] however, the enantio-meric purities were excellent (ee>99%) and the useof whole cells rendered external cofactor recyclingunnecessary.

Another quite complex cascade system involving al-cohol-ketone interconversion was applied by Rivaand co-workers for the synthesis of 12-ketourso-deoxycholic acid from cholic acid (Scheme 3).[33] Inthis study, two alcohol oxidations and one ketone re-duction could be realized in one pot using differenthydroxysteroid dehydrogenases (HSDHs) and inde-pendent regeneration systems for NAD+ andNADPH. Overall, one secondary alcohol moiety wasoxidized while another one was stereoinverted. How-ever, insufficient cofactor specificity of the glucosedehydrogenase (GDH) used for NADPH recyclingled to undesired side reactions upon longer reactiontimes. To circumvent this problem, the oxidation andreduction steps had to be separated in time, whichwas realized via two approaches: (i) compartmentali-zation of the enzymes in a so-called “tea-bag” systemusing dialysis bags which were applied sequentiallyand (ii) compartmentalization in two distinct mem-brane reactors.

3.1.2 Alcohol Dehydrogenases in Linear and ParallelMulti-Enzyme Cascade Systems

Due to their high stereoselectivity, alcohol dehydro-genases are attractive biocatalysts for processes in-volving optically active intermediates that are furtherconverted in a linear cascade fashion. As an example,a-chloro ketones may be stereoselectively reduced tothe corresponding chloro alcohols by an ADH, fol-lowed by ring-closure to yield optically active epox-ides. Considerable effort has been made to combinethe chloro ketone reduction and epoxide formation ina one-pot process. In the simplest case, ring-closure iseffected by addition of base,[34,35] but it can also beachieved enzymatically using halohydrin dehalogenas-es.[36] Thus, enantiomerically enriched epoxides can beobtained from prochiral halo ketones in a two-enzyme cascade. The practicability of this conceptwas demonstrated by Seisser et al. using ADHs fromRhodococcus ruber DSM 44541 (Prelog selective) andLactobacillus brevis (anti-Prelog selective) as well asa non-stereoselective halohydrin dehalogenase fromMycobacterium sp. GP1 [Scheme 4 (a)].[6] Unfortu-nately, the unfavourable equilibrium of the ring-clo-sure reaction limited the yield in this biocatalytic cas-cade. This limitation could be overcome in a follow-up study by employing anionic exchange resins whichbind the chloride ion liberated in the epoxide formingstep and therefore drive the reaction to completion.[37]

Additionally, the authors could extend the cascade bya nucleophilic epoxide ring-opening step, thus produc-ing enantiopure b-hydroxy nitriles[38] and b-azido alco-hols from the corresponding chloro ketones in a“three-step, two-enzyme, one-pot process” [Scheme 4(b)].[39] Recently, Janssen and co-workers reported afurther extension of this concept:[40] The formation ofb-azido alcohols using designer cells expressing bothan ADH and a halohydrin dehalogenase was coupledwith Cu(I)-catalyzed “click” cycloaddition of theseazido alcohols to phenylacetylene in one pot[Scheme 4 (c)]. b-Hydroxytriazoles were obtained inmoderate yields (18–65%) and high enantiomericexcess (97–99%).

An alternative approach towards b-hydroxy nitrilesis direct reduction of the corresponding keto nitriles,

Scheme 3. Multi-enzyme synthesis of 12-ketoursodeoxycholic acid from cholic acid using sequential oxidation and reductionsteps.

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and this reaction can be coupled with nitrilase-cata-lyzed hydrolysis to give b-hydroxy acids as the finalproducts. Thus, employing a ketone reductase fromCandida magnoliae or an ADH from Saccharomycescerevisiae as well as nitrilases from Synechocystis sp.and Bradyrhizobium japonicum, both enantiomers of1-hydroxy-1-phenylpropanoic acid and some relatedcompounds have been synthesized in enantiopureform with yields up to 27% higher than those ob-tained in a sequential reaction (Scheme 5).[41]

The production of enantiomerically enriched alco-hols via ADH-catalyzed oxidative kinetic resolutionhas recently been coupled with Baeyer–Villigermono ACHTUNGTRENNUNGoxygenase (BVMO)-catalyzed oxidation reac-tions in a parallel cascade.[10,11] In this system, the al-cohol dehydrogenase enantioselectively oxidizes analcohol to the corresponding ketone consuming oneequivalent of NADP+ which is converted to NADPH.The BVMO, on the other hand, consumes NADPH,as it incorporates one oxygen atom from O2 into itssubstrate (e.g., a sulfide, yielding a sulfoxide) while

the other one is reduced to H2O by the nicotinamidecofactor. Thus, the two enzymatic reactions are inter-connected through the cycling of the redox cofactor(Scheme 6). Since both biotransformations yield enan-tiomerically enriched products, the authors havecoined the term “parallel interconnected kineticasymmetric transformations” (PIKAT) for reaction

Scheme 4. Combination of alcohol dehydrogenases (ADHs) and halohydrin dehalogenases for the asymmetric synthesis of(a) epoxides, (b) b-azido alcohols or b-hydroxy nitriles, and (c) b-hydroxytriazoles from the corresponding chloro ketones.

Scheme 5. Biocatalytic cascade synthesis of b-hydroxy acids from b-cyano ketones employing a ketoreductase, a nitrilase andglucose dehydrogenase (GDH) for cofactor regeneration.

Scheme 6. Concurrent kinetic resolution of alcohols andasymmetric synthesis of sulphoxides catalyzed by alcohol de-hydrogenases (ADHs) and Baeyer–Villiger monooxygenases(BVMOs) in a parallel cascade.

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systems of this kind. Chiral alcohols and sulfoxides aswell as chiral alcohols and esters have been concur-rently obtained by this method in high yields and ex-cellent enantiomeric excess using minimal amounts ofNADPH. Besides, the concurrent production of dif-ferent enantioenriched alcohols using a single ADHhas also been explored.[42]

3.2 Multi-Enzyme Cascades for the Production ofEnantiomerically Enriched Amines and Amino Acids

While the preparation of optically active alcoholsusing lipases or alcohol dehydrogenases can be con-sidered an established technology, the biocatalyticasymmetric synthesis of enantiomerically enrichedamines has long been problematic. Only recently, thedevelopment of efficient cascade systems involving w-transaminases has provided an efficient biocatalyticentry to a-chiral primary amines, many of which areimportant for the pharmaceutical industry. Conse-quently, w-transaminases have quickly entered indus-trial applications.[43] More traditionally, a-transami-nases and amino acid dehydrogenases (AADHs) areemployed for the production of optically active aminoacids. Besides, monoamine oxidases (MAOs) andamino acid oxidases (AAOs) are available for theenantioselective irreversible oxidation of amines andamino acids, respectively, and these enzymes havebecome widely used biocatalysts in chemo-enzymaticderacemization and stereoinversion systems based onthe cyclic oxidation/reduction approach (see “Classifi-cation of Biocatalytic Cascade Reactions”).[12–14,16] Inthis section we shall look at examples of how thementioned enzymes have been employed in cascadeprocesses for the production of optically enrichedamines and amino acids.

3.2.1 Multi-Enzymatic Synthesis of Amino AcidsEmploying Amino Acid Dehydrogenases

Amino acid dehydrogenases (AADHs) are NAD(P)+-dependent enzymes which convert amino acids to thecorresponding keto acids and ammonia. Since this re-action is reversible, they are also useful biocatalysts

for the asymmetric synthesis of amino acids via reduc-tive amination. Early studies employing for instancephenylalanine dehydrogenase[44,45] were limited by thenarrow substrate specificity of the enzyme. Leucinedehydrogenase[46–49] offers a broader substrate scopeand several aliphatic l-amino acids have been ob-tained in enantiomerically pure form using thisenzyme in combination with FDH for cofactor recy-cling.[9] As an alternative, internal cofactor regenera-tion may be established via enzymatic oxidation of ana-hydroxy acid, thus providing both the substrate andthe reduced nicotinamide cofactor for the AADH(Scheme 7).[47,50,51] Recently, this redox-neutral cas-cade has been extended by employing mandelateracemase in combination with d-mandelate dehydro-genase (d-MDH) and different AADHs for the con-version of racemic mandelic acid to l-phenyl glycinein a novel type of “dynamic kinetic asymmetric trans-formation” (DYKAT) system.[52]

In order to avoid substrate inhibition effects causedby high concentrations of phenyl pyruvate in reduc-tive amination processes using phenylalanine dehy-drogenase (PheDH), this reaction has been coupledwith in situ generation of the corresponding iminoacid from N-acetyl dehydrophenylalanine (acetamido-cinnamate) by action of acetamidocinnamate acylase(ACA-acylase) in a cascade process (Scheme 8).[53]

Complete conversion of acetamidocinnamate to l-phenylalanine was achieved within 75 h at a substrateconcentration of 300 mM. A process using immobi-lized whole cells of mutated Corynebacterium equistrains for production of l-phenylalanine by the samepathway has also been reported.[54]

The direct biocatalytic reductive amination of ketoacids represents a promising approach for the synthe-sis of d-amino acids as well. However, unlike their l-selective counterparts, d-AADHs are not ubiquitousin nature, but are only found in some bacterial strains,including several Pseudomonas species and E. coli.These enzymes are usually membrane-bound flavo-proteins coupled to the respiratory chain,[55] and re-quire cofactors like coenzyme Q, methylene blue or2,6-dichlorophenol-indophenol when used in solubi-lized form.[56,57] Owing to these difficulties, the poten-tial of natural d-amino acid dehydrogenases for appli-cation in preparative biotransformations is rather lim-

Scheme 7. Redox-neutral bi-enzymatic conversion of lactate to alanine employing alanine dehydrogenase (AlaDH) and lac-tate dehydrogenase (LDH).

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ited. Recently, however, mutagenesis was used tocreate a nicotinamide cofactor-dependent, highly ste-reoselective d-AADH out of meso-2,6-d-diaminopi-melic acid dehydrogenase (DAPDH).[58] A combina-tion of rational and random mutagenesis afforded afive-point mutant which was used for the reductiveamination of various keto acids to yield the corre-sponding d-amino acids in high optical purity. Regen-eration of NADPH consumed in the reaction wasachieved with glucose/GDH.

Apart from asymmetric synthesis, amino acid dehy-drogenases have also been applied for the kinetic res-olution of amino acids. For instance, d-tert-leucinewas obtained in enantiomerically pure form (ee>99%) through l-selective oxidation of the racemicmixture using leucine dehydrogenase in combinationwith NADH oxidase from Lactobacillus brevis(Scheme 9).[59]

Alanine dehydrogenase (AlaDH) has been coupledwith lactate dehydrogenase (LDH) for the kinetic res-olution of 3-fluoroalanine to the d-enantiomer and l-3-fluorolactic acid (Scheme 10).[60] This approach rep-resents the inverse counterpart to the LDH/AlaDH-cascade for the asymmetric synthesis of alanine fromlactate as described above. Just like in the reversesystem, internal cofactor regeneration is realized, re-sulting in a redox-neutral process. d-3-Fluoroalanineand l-3-fluorolactic acid were obtained in 60% (ee =88%) and 80% (ee>99%) yield, respectively. An in-teresting aspect of this work is that the equilibrium ofthe whole system – with both steps being reversible –lies on the side of the hydroxy acid, while calculationsshowed that for simple amino acid/hydroxy acid pairslike alanine and pyruvate the amino acid is the ther-modynamically more stable compound.[52] Apparently,the additional fluorine atom leads to a stabilization ofthe hydroxy acid, possibly because of a strong intra-molecular H-bond.

In a very elegant approach, deracemization ofamino acids was achieved by combining d-amino acidoxidase (d-AAO) for enantioselective oxidation andleucine dehydrogenase (LeuDH) for asymmetric re-ductive amination.[61] Cofactor regeneration for thelatter enzyme was supplied by formate dehydrogenase(FDH). Furthermore, catalase was added for hydro-gen peroxide degradation in order to prevent oxida-tive enzyme inactivation (Scheme 11). Various a-amino acids were deracemized using this process, af-fording the l-enantiomers in yields higher than 95%and 99% ee.

Scheme 8. Coupling of asymmetric synthesis of l-phenylalanine catalyzed by phenylalanine dehydrogenase (PheDH) with insitu formation of phenyl pyruvate via deacylation of acetamidocinnamate catalyzed by acetamidocinnamate acylase (ACA-acylase).

Scheme 9. Kinetic resolution of tert-leucine employing l-leu-cine dehydrogenase (LeuDH) for enantioselective oxidationand NADH oxidase (NOX) for cofactor regeneration.

Scheme 10. Kinetic resolution of 3-fluoroalanine in a redox-neutral cascade process.

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3.2.2 Multi-Enzymatic Synthesis of Amines andAmino Acids Employing Transaminases

Transaminases are often classified into two groups ac-cording to their substrate scope: (i) a-transaminases,which act on the a-amino group in amino acids, and(ii) w-transaminases, which can convert amines thatlack a vicinal carboxylic acid functionality. A difficul-ty in the application of both classes lies in the factthat the amino group transfer is usually reversible, re-sulting in incomplete conversion. Therefore, transami-nases have been a target for the development of cas-cade processes which allow for shifting the equilibri-um to the product side.

a-Transaminases have been used extensively in theproduction of amino acids through kinetic resolutionand asymmetric synthesis. While many studies rely onthe use of an excess of cosubstrate to drive the reac-tion to completion, some multi-enzymatic approacheshave been developed as well. For instance, an orthog-onal cascade process for the synthesis of the a-aminoacid herbicide l-phosphinothricin from the corre-sponding keto acid precursor has been reported.[62] Toachieve irreversibility, the authors make use of thespontaneous decarboxylation of oxaloacetic acid,which is formed from aspartate by transamination.However, aspartate is not accepted as amino donorby the l-phosphinothricin-forming enzyme (4-amino-butyrate:2-ketoglutarate transaminase from E. coli),so glutamate is used instead as a primary aminodonor and recycled by glutamate:oxalacetate trans-ACHTUNGTRENNUNGaminase (GOT) from Bacillus stearothermophilus con-suming aspartate as a secondary amino donor(Scheme 12). Up to 85% conversion and excellent ste-reoselectivity (ee>99%) were achieved within 24 h ata substrate concentration of 500 mM.

Aspartate has also been used as an amino donor ina multi-enzymatic synthesis of l-2-aminobutyratefrom l-threonine (Scheme 13).[63] The rather complexmulti-step sequence starts with in situ formation of 2-ketobutyrate from l-threonine catalyzed by threoninedeaminase from E. coli. A tyrosine transaminase(TyrAT) from E. coli converts 2-ketobutyrate and as-partate to l-2-aminobutyrate and oxaloacetate, whichspontaneously decarboxylates to give pyruvate. Since

the latter compound can be accepted as a substrateby the transaminase, which would result in incompleteconversion, a third enzyme is employed for pyruvateremoval. Acetolactate synthase from Bacillus subtilisconsumes two molecules of pyruvate to form aceto-lactate, which again spontaneously decarboxylates togive acetoin as the final by-product. All three en-zymes were expressed separately in E. coli and usedin the form of fresh whole cells, allowing simple ad-justment of the appropriate relative activities via thecell mass. Full conversion was achieved within 24 h ata 500-mM scale; however, the recovery of l-2-amino-butyrate was only 54% because of metabolic con-sumption by the living microorganisms.

a-Transaminases have also been coupled withamino acid dehydrogenases (AADHs) to achieve anindirect biocatalytic reductive amination. Most nota-bly, they have been used for the asymmetric synthesisof d-amino acids from keto acids, which for a longtime could not be achieved in a direct manner, sinceno appropriate d-selective AADHs were known (see

Scheme 11. Deracemization of amino acids in a four-enzymecascade process.

Scheme 12. Asymmetric synthesis of the herbicide l-phos-phinothricin applying two a-transaminases.

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above). These systems comprise four enzymes:[9,64–66]

In a first step, an amino acid dehydrogenase forms anl-amino acid which is stereoinverted by a suitableamino acid racemase to give the d-enantiomer. Thelatter finally serves as amino donor in the transforma-tion of a keto acid to a d-amino acid catalyzed by ad-amino acid transaminase (d-AAT). Cofactor regen-eration for the AADH is usually supplied by formatedehydrogenase [Scheme 14 (a)]. Thus, several naturalas well as non-natural d-amino acids have been ob-tained in good yields and high ee-values. Since aminoacid transaminases are usually less substrate-specificthan AADHs, an analogous methodology has beenapplied for the preparation of l-amino acids which

could not be produced by direct enzymatic reductiveamination, e.g., b- and g-branched glutamic acid de-rivatives. In this case the amino acid racemase is ofcourse omitted [Scheme 14 (b)].[67–69]

Interestingly, amino acid dehydrogenases and a-transaminases have also been coupled in a linear way,realizing a system for deracemization of aminoacids.[70] Branched-chain amino acid transaminase(BCAAT) from Sinorhizobium meliloti ATCC 51124was cloned and overexpressed in E. coli. Additionally,d-amino acid dehydrogenase DadA present in thehost organism was induced by adding l-alanine to thegrowth medium, thus giving a whole-cell biocatalystcapable of deracemizing a-amino acids by an oxida-

Scheme 13. Asymmetric synthesis of l-2-aminobutyrate by a three-enzyme multi-step cascade.

Scheme 14. Indirect biocatalytic reductive amination of a-keto acids to give a-amino acids: (a) system used for d-aminoacids, (b) system used for l-amino acids. The amino functionality formed by an amino acid dehydrogenase is transferred byan a-transaminase to give the final product.

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tion/reduction sequence [Scheme 15 (a)]. The use ofresting whole cells in this study assured a supply ofthe needed cofactors and cosubstrates for the reac-tions. l-4-Chlorophenylalanine was obtained from itsracemate in enantiomerically pure form and quantita-tive yield within 48 h at a 0.5 g L�1 scale. A very simi-lar concept has been applied for the deracemizationof naphthylalanine to the l-enantiomer.[71] In thisstudy, the isolated enzymes d-amino acid oxidasefrom Rhodotorula gracilis and l-aspartate transami-nase from E. coli were used. Cysteinesulphinic acidserved as the amino donor for the transaminationstep, assuring irreversibility through spontaneous de-composition of the b-keto sulphinic acid by-product[Scheme 15 (b)].

In the last years, w-transaminases have emerged asversatile and powerful biocatalysts for the asymmetricsynthesis of a broad variety of amines with applica-tions in both academia and industry.[72,73] Severalmulti-enzymatic systems have been developed forshifting the equilibrium of the transamination to thedesired side. This holds true predominantly for appli-cation of transaminases in asymmetric synthesismode, since thermodynamics strongly favour ketone/amino acid over amine/keto acid. Since alanine is usu-ally employed as the amino donor, removal of thecorresponding by-product pyruvate has become thestrategy of choice.[74] Various commercial transaminas-es have been employed for the asymmetric synthesisof a broad range of a-chiral primary amines from thecorresponding ketones in a three-enzyme one-pot

system, where pyruvate was removed via reduction tolactate using a lactate dehydrogenase (LDH).[75–78] Co-factor regeneration for the latter enzyme was suppliedby glucose dehydrogenase (GDH), rendering thesystem irreversible [Scheme 16 (a)]. A high-through-put transaminase screening system based on this cas-cade process has been developed,[77,79] and the meth-odology has been employed for the preparation ofchiral selenium-amine ligands,[80] the pharmaceuticalintermediate (R)-4-phenylpyrrolidin-2-one[81] and theanti-Alzheimer drug (S)-rivastigmine.[82] Alternatively,pyruvate decarboxylases (PDCs) can be used for theremoval of pyruvate in transamination reactions, of-fering some considerable advantages: Pyruvate is de-graded to highly volatile acetaldehyde and CO2, assur-ing irreversibility, and no cofactor is needed in thistransformation [Scheme 16 (b)]. High optical puritiesand reasonable yields have been obtained employingthis system.[83]

Finally, pyruvate may also be recycled using anamino acid dehydrogenase (AADH), ammonia and asuitable cofactor regeneration system, as describedabove for the multi-enzymatic synthesis of aminoacids via indirect reductive amination(Scheme 17).[77,84,85] However, while such a system inprinciple only needs sub-stoichiometric amounts ofthe amino donor alanine, an excess was used toachieve a higher reaction rate for the rate-limitingtransamination step.

Multi-enzymatic approaches are less common whenw-transaminases are applied for the kinetic resolution

Scheme 15. Deracemization of a-amino acids using (a) a d-selective amino acid dehydrogenase and an l-selective a-trans-ACHTUNGTRENNUNGaminase, or (b) a d-selective amino acid oxidase and an l-selective a-transaminase.

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of amines using keto acids (usually pyruvate) asamino acceptors, since this reaction is thermodynami-cally favoured and addition of one equivalent of co-substrate is usually sufficient. Still, a more elegant ap-proach is to use catalytic amounts of pyruvate and toregenerate it in the process. This can be achieved withthe help of amino acid oxidases, thus using molecularoxygen as the stoichiometric oxidant (Scheme 18).Employing (S)- or (R)-selective w-transaminases, l-or d-amino acid oxidases and 8 mol% of pyruvate,different aromatic amines were resolved to opticalpurity (ee>99%) within 3 h.[76] By coupling the reac-tion system with a peroxidase-based assay on hydro-gen peroxide, the kinetic resolution can be used forthe colorimetric screening of transaminase activityand selectivity.[86]

Kroutil and co-workers recently demonstrated thatkinetic resolution and asymmetric synthesis using w-

transaminases of opposite stereopreference can becombined in order to establish a biocatalytic deracem-ization system for chiral amines (Scheme 19).[87] How-ever, interference of the two stereocomplementarytransaminases was observed when the reaction wascarried out in a one-pot one-step fashion, leading todecreased enantiomeric excess. This problem wasovercome by deactivating the first transaminase viaheat treatment after the kinetic resolution was com-plete and carrying out the asymmetric synthesis after-wards. Alternatively, the use of immobilized enzymesallows separation by filtration.[88] This concept hasbeen applied to a variety of a-chiral primary amines,including the pharmaceutically relevant mexiletine.[89]

Very interestingly, a- and w-transaminases havebeen coupled in a parallel cascade system for the con-current production of (S)-amino acids and (R)-amines.[90] In this approach, amination of a keto acidwith a suitable amino donor affords the correspondingamino acid and a keto acid by-product, which servesas the amino acceptor in the transaminase-catalyzedkinetic resolution of an amine (Scheme 20). As an ex-ample, (S)-a-aminobutyrate and (R)-a-methylbenzyl-amine could be obtained concurrently by this strategy

Scheme 17. Formal biocatalytic reductive amination of ke-tones to give amines. Alanine is employed as the aminodonor for the w-transaminase reaction and regeneratedusing an amino acid dehydrogenase and FDH for cofactorrecycling.

Scheme 16. Two cascade strategies for the asymmetric synthesis of a-chiral primary amines from ketones employing w-trans-aminases: (a) removal of pyruvate using lactate dehydrogenase and GDH/glucose, (b) removal of pyruvate by pyruvate de-carboxylase (PDC).

Scheme 18. Kinetic resolution of a-chiral primary aminesemploying w-transaminase and amino acid oxidase.

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reaching conversions of 90% (ee>99%) and 48%(ee =95%), respectively. Alanine serves as an aminogroup “shuttle” in this reaction system. However,since equimolar amounts of the substrates – keto acidand racemic amine – were used in this study, the cas-cade is not catalytic in alanine, and indeed one equiv-alent has been added.

A final example provides the transition to the nextsection, which covers biocatalytic cascades involvingC�C bond forming reactions: A transketolase from E.coli and a transaminase from Pseudomonas aerugino-sa PAO2 overexpressed in a single host were used forthe biocatalytic synthesis of 2-amino-1,3,4-butanetriolfrom hydroxypyruvate, glycolaldehyde and 1-phenyle-

thanamine (a-methylbenzylamine) as an amino donor(Scheme 21).[91] Thus, two stereogenic centers wereformed via consecutive C�C bond formation andtransamination in a one-pot process giving exclusivelythe syn-diastereomer. While the absolute stereochem-istry of the final product was not determined, the(2S,3R)-configuration was assumed according to theknown stereoselectivity of the transketolase.

3.3 Multi-Enzyme Cascades Employing Aldolases andHydroxynitrile Lyases for C�C Bond Formation

The concept of coupling the synthetic power ofcarbon-carbon bond forming enzymes with other en-zymes in a single reactor truly imitates nature in gen-erating complex compounds from relatively simplebuilding blocks. Aldolases and hydroxynitrile lyasesplay the most important role for C�C bond formationin biotransformations and frequently appear in cas-cade processes due to the following reasons: (i) toavoid expensive starting materials, (ii) to broaden therange of possible products, (iii) to shift unfavourablereaction equilibria and (iv) to achieve more cost-effi-cient products by better space-time yields.

Aldolases are the most widely applied C�C bondforming enzymes in organic synthesis. They catalyzean aldol-type condensation reaction of a donor com-pound (nucleophile) to an acceptor compound (elec-trophile) leading to the formation of one or two new

Scheme 19. Deracemization of a-chiral primary amines employing a combination of w-transaminase (w-TA) and amino acidoxidase (AAO) for the oxidative step as well as a combination of w-TA, lactate dehydrogenase (LDH) and glucose dehydro-genase (GDH) for the reductive step.

Scheme 20. Concurrent kinetic resolution of amines andasymmetric synthesis of amino acids using both w- and a-transaminases.

Scheme 21. Bi-enzymatic formation of 2-amino-1,3,4-butanetriol from glycolaldehyde and hydroxypyruvate.

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stereogenic centers. They present an attractive alter-native to standard chemical methods as they requireno protecting group chemistry. Most aldolases showhigh specificity towards a certain donor substrate(usually a ketone) but tolerate a broad range of ac-ceptor substrates (an aldehyde). Thus they can beclassified according to their donor substrate. The mostimportant natural donors are dihydroxyacetone phos-phate (DHAP), pyruvate or phosphoenol pyruvate,acetaldehyde and glycine.[92] Mechanistically, aldolasescan be differentiated in two major classes. Type I al-dolases catalyze the reaction by forming a Schiff baseintermediate between the donor substrate and ahighly conserved lysine residue in the active site. TypeII aldolases are dependent on a metal cation (mostlyZn2+) as a cofactor which acts as a Lewis acid in theactivation of the donor substrate.

3.3.1 In-situ Synthesis of DihydroxyacetonePhosphate (DHAP)

Many aldolases, including the well known fructose1,6-diphosphate aldolase and l-rhamnulose 1-phos-phate aldolase, utilize dihydroxyacetone phosphate asthe donor substrate. The need for the expensiveDHAP donor is a major drawback in the applicationof these enzymes. Therefore several ways for the in-situ formation of DHAP have been developed, includ-

ing several (chemo-)enzymatic routes which allowDHAP formation to be connected to the followingcatalytic steps in a one-pot synthesis. Generally threedifferent enzymatic routes for the in-situ formation ofDHAP can be distinguished:[93] glycerol phosphateoxidase (GPO)-catalyzed oxidation of glycerol 3-phosphate, phosphorylation of dihydroxyacetone(DHA), and formation of DHAP from fructose 1,6-diphosphate by a coupled aldolase/triosephosphateisomerase reaction, mimicking DHAP formation inthe glycolytic pathway.

Wong and Whitesides have first reported the in-situpreparation of DHAP from DHA catalyzed by a glyc-erol kinase coupled to cofactor regeneration with pyr-uvate kinase [Scheme 22 (a)].[94] They isolated DHAPin good yield (80–90%). A similar approach was de-scribed by Itoh et al.[95] and Sanchez-Moreno et al.,[96]

who cloned and overexpressed a dihydroxyacetonekinase (DHA-kinase) from Schizosaccharomycespombe IFO 0354 and Citrobacter freundii, respective-ly [Scheme 22 (b)]. Acetate kinase served for ATP re-generation. The in-situ DHAP generation by this ap-proach can be utilized for the synthesis of unusualsugars with a variety of different aldehydes;[97] howev-er, the authors report that ethyl 3-methyl-4-oxocroto-nate inhibited DHA-kinase activity and therefore re-quired a time separation of the DHAP formation andthe following aldolase catalyzed condensation reac-tion. Recently, Iturrate et al. designed a fusion protein

Scheme 22. In-situ formation of DHAP: (a–c) from DHA, (d) from fructose 1,6-diphosphate, and (e) from glycerol 3-phos-phate; PK =pyruvate kinase, AK=acetate kinase, PPi =pyrophosphate, Pi =phosphate.

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that possesses both aldolase and kinase activities.[98,99]

The fusion protein consists of a monomeric fructose-1,6-diphosphate aldolase from Staphylococcus carno-sus and a homodimeric dihydroxyacetone kinase fromC. freundii linked by five amino acids. With this ap-proach they accomplished a 20-fold increase in the re-action rate which they attribute to substrate channel-ing effects.

An alternative to these ATP-dependent enzymes isthe phosphorylation of DHA by an acid phosphatasefrom Shigella flexneri [Scheme 22 (c)] which usescheap pyrophosphate as a phosphorus source.[100] Con-comitant aldolase condensation increases the yielddue to the two-way action of the phosphatase. It first-ly phosphorylates DHA and secondly dephosphory-lates the aldol adduct which leads to phosphate cy-cling. A drawback is the low DHA affinity of thetested phosphatase. Therefore, a non-specific phos-phatase from Salmonella enterica ser. typhimuriumLT2 was improved by directed evolution which led toa six-fold increase in activity.[101]

Another approach directly emulates DHAP forma-tion in glycolysis: First fructose 1,6-diphosphate isconverted to DHAP and glyceraldehyde 3-phosphateby an aldolase. Triose phosphate isomerase in turnconverts glyceraldehyde 3-phosphate into DHAP[Scheme 22 (d)].[102] Fessner et al. implemented thisapproach in a seven-enzyme one-pot procedure whichuses sucrose as a starting material.[103] By using readilyavailable dialdehydes and DHAP derived from fruc-tose 1,6-diphosphate, Eyrisch et al. were able to syn-thesize various disaccharides by tandem aldol addi-tions.[104]

Fessner et al. first reported the in-situ synthesis ofDHAP by a glycerol phosphate oxidase [Scheme 22(e)].[105] The flavin-dependent enzyme can be rapidlyreoxidized by elemental oxygen with liberation of hy-drogen peroxide. Addition of catalase suppressesenzyme inactivation by H2O2. Combination of DHAPformation with an aldolase proved to be advantageousas the rapid consumption of DHAP prevented prod-uct inhibition of the oxidase. This approach was com-bined with in-situ sugar precursor formation byEyrisch et al.[106] They describe the formation of boththe aldol donor and acceptor components by air oxi-dation using microbial oxidases. The sugar precursorwas oxidized by a d-galactose oxidase from Ductyliumdendroides. The hydrogen peroxide resulting fromDHAP and sugar generation were both decomposedby catalase. The following condensation of the sugarand DHAP was performed by an l-rhamnulose 1-phosphate aldolase to give methyl b-l-threo-d-galac-to-8-nonosulo-1,5-pyranoside 9-phosphate. However,due to aldolase inhibition by the d-galactose oxidasecofactor Cu2+ only low overall yields (14%) could beachieved in a one-pot approach. The in-situ formationof glycerol 3-phosphate from cheap prochiral glycerol

in the presence of phytase was realized by Schoevaartet al. in an elegant sequential one-pot reaction giving5-deoxy-5-ethyl-d-xylulose in 57% overall yield.[107]

The key of their process lies in the phytase on/off-switch by pH change. Charmantray et al. synthesizedglycerol 3-phosphate chemically by controlled ring-opening of rac-glycidol in the presence ofNa2(K2)HPO4 or Na3PO4 at a pH above 10.[108] Fol-lowing enzymatic reactions were performed after pHadjustment to 6.8.

3.3.2 Aldolase Catalyzed Synthesis of Sialic AcidDerivatives

N-Acetylneuraminic acid aldolases (NeuAc aldolases)use pyruvate as the donor substrate and catalyze thealdol reaction between pyruvate and mannose ormannose derivatives. They have gained a lot of inter-est as the produced sialic acids find use in cancer ther-apy and as anti-infectives. The reaction is reversiblewith an equilibrium constant close to one. The equi-librium can be pulled towards aldol formation byadding an excess of pyruvate, but the latter seriouslyinterferes with product isolation. A possible solutionis the application of multi-enzyme one-pot reactions.

Ichikawa et al. demonstrated the synthesis of asialyl trisaccharide in a nine-enzyme one-pot proce-dure starting from N-acetylmannosamine.[109] The re-action starts with the NeuAc aldolase-mediated con-version of N-acetylmannosamine to N-acetylneura-minic acid in the presence of pyruvic acid [Scheme 23(a)]. The produced N-acetylneuraminic acid thenreacts to cytidine 5’-monophospho-N-acetyl-neura-minic acid with cytidine 5’-monophosphosialic acidsynthetase (CMP-sialic acid synthetase), thus imped-ing the aldolase back reaction. Further enzymaticsteps lead to the formation of the sialyl trisaccharar-ide N-acetylneuraminic acid-a-(2,6)-galactose-b-(1,4)-N-acetylglucosamine in an overall yield of 22%. Yuet al. tested three different CMP-sialic acid synthetas-es with different substrates and could synthesize vari-ous CMP-sialic acid derivatives.[110] In a similar ap-proach, Cao et al. used C-5-hydroxy-substituted man-nose derivatives to produce a variety of C-5-substitut-ed sialosides employing a CMP-sialic acid synthetaseand a sialyl-transferase.[111] .

Taking advantage of the two-way reaction ofNeuAc aldolases, Miyazaki et al. developed an ele-gant [3-13C]-labelling method for N-acetylneuraminicacid analogues [Scheme 23 (b)].[112] In a first step N-acetylneuraminic acid analogues are degraded to N-acetylmannosamine and pyruvate by NeuAc aldolase.The reaction equilibrium is shifted towards degrada-tion by addition of lactate dehydrogenase which re-moves pyruvic acid from the reaction mixture. Afterthe reaction is completed, the cofactor for lactate de-

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hydrogenase is degraded by the addition of nucleotidepyrophosphatase. Then [3-13C]-pyruvic acid is addedand incorporated into N-acetylneuraminic acid by theNeuAc aldolase. This method allows [3-13C]-labellingin satisfactory yield and with an excellent degree of13C-enrichment.

N-Acetylmannosamine, the substrate for NeuAc al-dolases, is rather expensive and difficult to prepare ona large scale. A possibility to circumvent this is the in-situ formation of N-acetylmannosamine from N-ace-tylglucosamine by N-acetylglucosamine 2-epimerase[Scheme 23 (c)]. Kragl et al. realized an enzymaticmembrane reactor containing free N-acetylglucos-ACHTUNGTRENNUNGamine 2-epimerase and NeuAc aldolase.[113] After aninitiation phase, N-acetylneuraminic acid could beproduced continuously over several hours with aspace-time yield of 109 g L�1 d�1. Recently, N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic

acid aldolase were overexpressed as double-taggedgene fusions, thus greatly facilitating enzyme isola-tion.[114]

3.3.3 Multi-Enzyme Cascades Involving2-Deoxy-d-ribose 5-Phosphate Aldolase (DERA)

2-Deoxy-d-ribose 5-phosphate aldolases (DERAs)belong to the class of acetaldehyde-dependent aldo-lases. In contrast to DHAP aldolases, the donor sub-strate specificity is not as strict, allowing for chainelongation by two or three carbon atoms.[115,116]

DERA aldolases have been successfully applied tothe production of nucleosides. Horinouchi et al. de-scribe the production of 2’-deoxyribonucleosides fromglucose, acetaldehyde and a nucleobase in a multi-en-zymatic sequential reaction (Scheme 24).[117] They use

Scheme 23. One-pot reactions with NeuAc aldolase (N-acetylneuraminic acid aldolase); CMP=cytidine monophosphate,CTP=cytidine 5’-triphosphate, LDH = lactate dehydrogenase, ADH=alcohol dehydrogenase.

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permeabilized yeast cells in a first step to produce d-glyceraldehyde 3-phosphate from glucose through theglycolytic pathway. In a second step, condensation ofd-glyceraldehyde 3-phosphate and acetaldehyde cata-lyzed by a recombinant DERA aldolase from Kleb-siella pneumoniae leads to the formation of 2-deoxyri-bose 5-phosphate. In a third step, a recombinant phos-phopentomutase catalyzes the isomerization to 2-de-oxyribose 1-phosphate. In a last step, a commercialnucleoside phosphorylase catalyzes the nucleobasetransfer yielding 2’-deoxynucleosides. The overallyield of this reaction sequence was low due to inhibi-tion effects of phosphate and the phosphorylated gly-colysis intermediates. Coupling the different reactionsteps in a one-pot cascade allowed a 3-fold increaseof 2’-deoxyribonucleoside production.[118]

3.3.4 Multi-Enzyme Cascades Involving l-ThreonineAldolases

l-Threonine aldolases catalyze the reversible cleavageof l-threonine to glycine and acetaldehyde. Theybelong to the glycine-dependent aldolases and requirepyridoxal 5’-phosphate. The reaction equilibrium canbe pulled towards aldol formation by adding a secondirreversible reaction. This was realized for the produc-tion of (R)-2-amino-1-phenylethanol from benzalde-hyde and glycine by a novel DYKAT combining l-threonine aldolase with an l-tyrosine decarboxylase(Scheme 25), taking advantage of the L/D and syn/anti selectivity of the respective enzymes.[119] As aconsequence, the aldol equilibrium is shifted to thealdol product, thus resulting in high conversions. It

could be shown that this scheme is applicable to awide variety of substituted benzaldehydes allowingthe production of several enantioenriched aromatic1,2-amino alcohols.[120]

3.3.5 Multi-Enzyme Cascades InvolvingHydroxynitrile Lyases

Hydroxynitrile lyases (also known as oxynitrilases)are powerful tools for the synthesis of enantiopure cy-anohydrins which constitute versatile building blocksin organic synthesis. Hydroxynitrile lyases appear in awide variety of higher plants and are classified intonon-FAD and FAD containing enzymes.[121]

Mateo et al. utilized hydroxynitrile lyases in combi-nation with an unspecific nitrilase immobilized as across-linked enzyme aggregate (CLEA) for the pro-duction of enantiopure (S)-mandelic acid [Scheme 26(a)].[122] The process is based on the enantioselectivehydrocyanation of benzaldehyde to mandelonitrile byhydroxynitrile lyase followed by enzymatic hydrolysisto mandelic acid in the presence of a non-selective ni-trilase. The hydroxynitrile lyase determines the con-figuration of the stereogenic center in the molecule.The main challenge lies in the different reactionoptima of the used enzymes: While hydroxynitrilelyases have a pH optimum of approximately 5, nitri-lases are usually most effective at close to neutral pHvalues. Additionally, reactions should be performed atlow pHs and in the presence of organic solvents inorder to suppress non-enzymatic hydrocyanation.Therefore a recombinant nitrilase from Pseudomonasfluorescens EBC 191 was employed which still pos-

Scheme 24. Production of 2’-deoxyribonucleosides by DERA (2-deoxy-d-ribose 5-phosphate aldolase).

Scheme 25. Application of l-threonine aldolase for the production of 1,2-amino alcohols.

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sesses significant activity at pH 5.5. Under optimizedconditions (S)-mandelic acid could be produced withhigh ee (98%). In a similar process, a hydroxynitrilelyase and a novel nitrile hydratase from Nitriliruptoralkaliphilus, both immobilized as CLEAs, were cou-pled for the synthesis of enantiopure aliphatic a-hy-droxycarboxylic amides [Scheme 26 (b)].[123] The ap-plication of CLEAs improved the stability of the ni-trile hydratase in cell-free preparations.

4 Bioprocess Engineering Aspects ofBiocatalytic Cascades

Multi-enzyme and chemo-enzymatic reactions offer awide range of opportunities and new paths to synthe-sis. Many achievements in this field have been ob-tained – and can be further gained – by means of ge-netics, screening and evolution engineering,[124,125]

which have been mainly focusing on improvement ofenzyme activity and stability.[126–128]

However, the development of effective processesrelies not only on the biocatalyst performance in se,but also on how it is implemented into the reactorand the entire process. Bioprocess engineering con-sists in the application of the engineering approach toachieve effective development and implementation ofbioprocesses on scale.[129] In the sphere of the reactionstep of a bioprocess, this requires the evaluation ofmany possible choices related to reaction media, typeof biocatalyst (whole cell, soluble enzyme, immobi-lized enzyme, a combination of biocatalysts), reactorconfiguration (well-mixed tank, fixed bed, fluid bed,membrane, etc.) mode of operation (batch, continu-ous, fed-batch, with or without recycle, step-wise feedof substrates, many reactors connected in series or inparallel or in mixed configurations, etc.). In addition,all the choices about the reaction step do not standalone, but they must be integrated into the process,taking into account up- and down-stream steps in

order to design flow-sheets for feasible and cost-effi-cient operations.

In this section, multi-enzyme and chemo-enzymaticreactions are discussed from the point of view of bio-process engineering in order to put in evidence theirstrong and weak points and to give a basis for rationaldecision making in biocatalytic synthesis.

4.1 The Gain of One-Pot in Biocatalytic Reactions

Operating “one-pot” reactions, as compared to non-integrated processes, has indubitable advantages thatcan deeply influence the efficiency of a process. Theycan be summarized as follows: (i) shift of reversiblereaction equilibria, (ii) higher yields, (iii) increase ofreaction rate, (iv) overcoming of inhibition effects,and (v) overcoming of solubility problems. All thesehave a direct effect on the process flow-sheet, thus onprocess feasibility, ease of operation and costs.

Especially in the case of compounds with similarstructures, conversion and selectivity >99% are de-sired in industry because product separation can heav-ily affect overall costs.[130] Shifting equilibrium in one-pot reactions, without introducing further steps andunit operations, is an effective way to reach this aim,as it was successfully reported.[119,131,132] When one ormore reversible reactions are in series with an irrever-sible one controlling the kinetics, very high yield andselectivity are reached;[103] this is of paramount impor-tance in dynamic processes.[133] Thus, higher yields area consequence of equilibrium shifts and they result ina reduction of waste and no need for further separa-tion; from the process point of view this means lowequipment and operational costs and also a reductionof raw material costs because of the complete conver-sion of substrates into desired products.

In case of substrate or product inhibition, reactionsare characterized by low reaction rates and conver-sions.[134] In these situations, a one-pot reactor canyield better performance than a series of reactors,providing in situ substrate supply and product with-drawal.[135] The reason for this is that, if the compoundresponsible for inhibition is continuously released/re-moved by means of the reaction, it cannot accumulatewithin the reaction volume in considerable amounts.This is especially true in the case of cascade reactions,where intermediates have a very short life time. Theresulting high reaction rates and conversions directlyturn into lower costs due to reduced reaction timesand reactor sizes as well as no necessity for recycling.A good example of coupling enzymatic reaction andfermentation in one pot is the SSF (simultaneous sac-charification and fermentation) process for bioethanolproduction from lignocellulosic biomass. In this pro-cess, the enzyme is inhibited by simple sugars comingfrom the enzymatic hydrolysis of cellulose (product

Scheme 26. One-pot reactions with hydroxy nitrile lyases.The opposite enantiomer (omitted in the scheme) can alsobe accessed, since enzymes with (R)- and (S)-selectivity areavailable.

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inhibition). When the process is run in SSF mode,sugars are released gradually and simultaneously con-verted into ethanol by fermentation.[136] From thepoint of view of compound solubility, one-pot reac-tions can be advantageous, as sparingly soluble com-pounds are gradually released and removed from thesystem.

Drawbacks in the implementation of one-pot reac-tions also exist. First of all, when two separate reac-tions are run in a process, optimal operating condi-tions can be chosen for each of them. Conversely,when multiple reactions are run in the same pot, opti-mal conditions for each reaction can often not be sat-isfied and this means that in practice none of the re-actions runs at its optimum in terms of reaction rateand stability. Mathematical modelling could be auseful means to find optimal conditions for the entireone-pot system, as discussed below (see section“Enzyme-Enzyme and Chemo-Enzymatic Reac-tions”). Other limitations of one-pot processes couldarise from the presence of side-products such as lac-tate and gluconolactone (see Scheme 3), SO2

(Scheme 15) and acetaldehyde [Scheme 16 (b)]: someof these compounds can lead to enzyme inhibitionphenomena that would not occur if the biocatalytictransformations were run in a step-wise fashion.

4.2 Enzyme-Enzyme and Chemo-EnzymaticReactions

One-pot multi-step reactions can be catalyzed bymulti-enzyme systems, chemical catalysts or by a com-bination of them. In this section relevant aspects ofmulti-enzyme and chemo-enzymatic reactions aretreated. Multi-enzyme reactions can be catalyzed bythe following combinations of biocatalysts: (i) wholecells, (ii) soluble enzymes, (iii) a combination of solu-ble enzymes and whole cells, (iv) immobilized en-zymes, and (v) a combination of soluble and immobi-lized enzymes.

The catalyst type for a new biocatalytic process isone of the most important choices in processdesign.[137] The choice of one of the above possibilitiesdepends on many factors and must be made uponconsideration of the specific case under study. In gen-eral, passing from whole cells to soluble enzymes andthen to immobilized enzymes must be justified by spe-cific needs of the process, because it implies growingcosts for enzyme separation, purification and eventu-ally immobilization. However, whole cells catalyzedreactions can give side products and this requiresproduct purification; the compromise in this case maybe between the impact of catalyst costs and of separa-tion costs. The synthesis of amino-alcohols by transke-tolase and b-alanine-pyruvate transaminase representsan example for the use of whole cells in a bi-enzym-

ACHTUNGTRENNUNGatic one-pot reaction. Both enzymes were over-ACHTUNGTRENNUNGexpressed in E. coli in two separate plasmids. Howev-er, the overall product yield was lower than that ob-tained with purified enzymes because of product deg-radation by the host strain.[91] Soluble enzymes andwhole cells have been successfully combined for theproduction of enantiopure (S)-3-carboxymethyl and(S)-3-carb ACHTUNGTRENNUNGoxyethyl-g-butyrolactones as well as severalenantiopure dialkyl (S)-2-hydroxyglutarates. The firststep of the reaction consisted of the esterification of2-oxoglutaric acid to the corresponding 2-oxoglutaratedialkyl esters by a commercial lipase. In a second stepthe alkyl esters were reduced by whole cells of thefungus Mucus rouxii yielding the corresponding g-bu-tyrolactones or dialkyl (S)-2-hydroxyglutarates de-pending on the length of the alkyl chain in theester.[138]

Biocatalyst immobilization offers many advantagesin terms of biocatalyst reuse and feasibility of contin-uous processes. The best way of immobilizationshould be found in order to get stable processes with-out enzyme leakage. This is often reached through co-valent immobilization of enzymes, but other immobi-lization techniques, generally simple but not alwaysefficient, are available, such as enzyme adsorption.[137]

The option of immobilizing an enzyme instead ofusing it in soluble form should be adopted when thecatalyst is very costly or when an in-situ separation ofsome of the reacting mixture components is required.But of course other issues, such as the advantage ofrunning a continuous process, could be determinant.Interestingly, a combination of oxynitrilase and nitri-lase was immobilized in the form of a cross-linkedenzyme aggregate (CLEA),[139] resulting in better per-formance than the use of two separate CLEAs of oxy-nitrilases and nitrilases.[122]

The combination of enzymatic and chemical synthe-sis offers further possibilities. The challenge in thiscontext is to find suitable reaction conditions. Obvi-ously, separate reaction steps are possible in order torun each step at its optimal conditions, but the idea ofusing them simultaneously in a one-pot reaction isvery attractive because of capital costs reduction, im-provement of equilibrium and in situ substrate supplyand product removal. Matching between chemicaland enzymatic conditions must be made both ways:chemical substrates should not poison enzymes andbiocatalysis conditions should allow feasible chemis-try. In this balance, the medium (aqueous or organic)plays an important role, with water being very attrac-tive because of its low cost and easy control of pH.On the other hand, organic molecules are oftenscarcely soluble in water and chemical catalysts andintermediates can be water-sensitive.[135] Nevertheless,successful examples of chemo-enzymatic integrationhave been reported in both aqueous[140,141] and organic

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media[142,143] and with mixed homogeneous/heteroge-neous catalysis.[144,145]

Further improvements in multi-step biocatalysis canderive from reactor engineering. Reactor configura-tion, for example, can not only determine differentconversions, but it should be chosen according to thespecific needs of a reaction. If enzyme deactivation/inhibition is determined by one of the substrates, acontinuous stirred tank reactor (CSTR) can be thebest choice, because it works at low substrate concen-tration values when conversion is high. However, ifproduct inhibition exists, then CSTR is not a goodoption and other configurations, such as plug flow re-actors should be preferred. In case of both limitations,cascades of reactors, with distributed addition of sub-strates if necessary, could be adopted.[146] In the caseof product inhibition, in-situ product removal bymeans of membrane reactors[134,147] or evaporation ofthe product when very volatile[130] could also be suc-cessfully adopted. Other, more complex combinationscould be required in case of multiphasic systems.[148]

A field where applications of reactor engineeringare particularly useful is coenzyme regeneration. A(native enzyme)/(native coenzyme)/(batch reactor)system was used for chemo-enzymatic synthesis withNADPH recycling;[149,150] while an (immobilizedenzyme)/(native coenzyme)/(batch reactor) systemwas used for 12-ketochenodeoxycholic acid synthe-sis.[151] Membrane reactors were also used in differentways, such as (native enzyme)/(macromolecular coen-zyme)/(uncharged membrane reactor)[152] and (nativeenzyme)/(native coenzyme)/(charged membrane reac-tor).[153]

Many combinations of enzyme forms and reactorconfigurations are feasible, but their efficient use re-quires appropriate choice of operating conditions and,possibly, optimization. Two main patterns are possiblein this context: heuristics and fundamental modeling.Heuristic rules have proven very useful to determineoperating windows in the case of d-xylulose 5-phos-phate synthesis by triose phosphate isomerase andtransketolase: within the domain of operating varia-bles (substrate concentration, pH, enzymes loadingratio), the operating window has been determined byelimination of conditions implying low catalyst activi-ty, unfavourable yields and substrate instability andinhibition.[154] Modelling of biocatalytic reactions isdefinitely a more precise and versatile tool for the de-termination of feasible operating conditions and foroptimization, but it requires a superior experimentaleffort for the determination of kinetic parameters;much research is still required in this direction. How-ever, once kinetics are known, they can be used forperformance prediction and design of reactors withdifferent configurations. A general approach for theimplementation of mathematical models of multi-enzyme systems is reported by Santacoloma et al.[155]

A comprehensive model for transketolase-mediatedcarbon-carbon bond formation has been implementedtaking into account not only substrate and product in-hibition, but also toxicity of one of the substrates; itsuse for reaction performance prediction and optimiza-tion was shown by means of contour and 3D plots.[156]

An interesting approach for bi-enzymatic catalysis op-timization has been proposed based on constructionof isoproductivity and isoefficiency charts by meansof projection of 3D plots on the XY domain of en-zymes loading;[157,158] this is definitely a powerful toolfor optimization of multi-enzyme reactions. A furthervariable that should be subjected to optimization istemperature, but currently, this has not been exten-sively done. In enzymatic reactions, the determinationof reaction and deactivation kinetics has proven cru-cial to determine optimal reaction temperatures fordifferent enzyme loadings, and reaction performancescould largely benefit from it.[159,160]

Finally, multi-step reactions must always be imple-mented into the entire bioprocess in order to rapidlydevelop promising processes and determine their fea-sibility. This requires a big effort, especially in consid-eration of the fact that specific rules for decision onprocess flow-sheet do not exist and each new casemust be separately analyzed considering its specificpeculiarities. Process synthesis offers a valuable toolfor decision-making based on heuristics. For example,if some possible flow-sheets have been determinedand a choice has to be done, an interesting approachto process screening has been proposed that allowsfor preliminary exclusion of unfeasible options, by as-signing scores to kinetics, yield, bioreactor and (up-and/or) down-stream. With this approach, 5 out of 20process options have been selected in the bioprocessfor the synthesis of d-xylulose 5-phosphate.[129] Manyof the bioprocess engineering aspects relevant to one-pot reactions have been reported in the presentpaper; however, their use should be considered fromcase to case. More comprehensive references on pro-cess synthesis in general exist in the literature.[148]

As a conclusion, after procedures aimed at exclud-ing unfeasible options have been applied and the pro-cess has reached an advanced level of scientific devel-opment, economic evaluation of alternatives remainsthe instrument to push a process to implementationon scale.

4.3 Industrial Applications and Future Developments

Industrial examples of multi-enzyme catalysis are thetwo dynamic kinetic resolution (DKR)-based process-es by the Degussa group:[16] the first process consistsof the DKR of 5-substituted hydantoins catalyzed bywhole cells expressing l-carbamoylase, hydantoinracemase and hydantoinase; the second process con-

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sists of the DKR of N-acetylamino acids, catalyzed byacylase and N-acetylamino acid racemase.[161] A greatpotential of multi-step synthesis is in the process forN-acetyl-d-neuraminic acid (Neu5Ac), a starting ma-terial for anti-influenza virus agents that is of greatcommercial value. Neu5Ac can be obtained in a one-pot reaction catalyzed by epimerases and aldolaseseven with different techniques for enzyme immobili-zation which makes the multi-enzyme process very at-tractive for large-scale production.[113,114,162,163]

From the viewpoint of future developments, contri-butions to the success of enzymatic and chemo-enzy-matic reactions in organic media can derive from sol-vent and enzyme engineering. Solvent engineeringhas already allowed for successful biocatalysis in or-ganic solvents, ionic liquids[164] and supercritical/sub-critical fluids.[142] Enzyme engineering is based on ra-tional redesign and random mutagenesis methods andscreening aiming at improved substrate specificity,catalyst stability, enantioselectivity and reactionrate.[133,165] Another possible application of domino re-actions could be in labs-on-a-chip, i.e., in the use ofcascade reactions in micro-bioreactors; this could givea way to gain access to all the intermediates.[166]

5 Conclusions and Outlook

Multi-enzyme cascade reactions have emerged as animportant research area within the field of biocataly-sis. They have been employed for regeneration of co-factors, shifting reaction equilibria and overcoming in-hibition effects, or simply for reducing the time,energy and material demands of a given process.One-pot procedures for deracemization and stereoin-version have become possible, and parallel cascadesallow the concurrent production of several opticallyactive compounds from racemic starting materials. Allthese developments are important steps towards a“greener” chemical future. However, in order to findapplication in industry, biocatalytic cascade systemsmust be robust and scalable. As our understanding ofenzymes and our means for fine-tuning their activityprogress, ever more sophisticated multi-enzyme pro-cesses will become possible. Protein engineering mayhelp to extend the operating window of biocatalyststo make them more compatible with each other. Theuse of co-immobilization or fusion proteins couldallow for exploitation of substrate channeling effectsand for simpler down-stream processing. Finally, thegrowing number of readily available enzymes leads tovast possibilities for their creative combination. Thefuture may see biocatalytic cascade processes of un-precedented complexity, efficiency and elegance.

Acknowledgements

Dr. Mari P�iviç, Prof. Wolfgang Kroutil, Prof. Christoph Syl-datk and Prof. John Woodley are thanked for fruitful discus-sions. A thankful acknowledgement is due to the OrganizingCommittee of CASCAT Training School (Siena 2009, COSTAction CM0701), where this work was conceived and initiat-ed.

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