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Biocatalysis — key to sustainable industrial chemistryRoland Wohlgemuth

The ongoing trends to process improvements, cost reductions

and increasing quality, safety, health and environment

requirements of industrial chemical transformations have

strengthened the translation of global biocatalysis research

work into industrial applications. One focus has been on

biocatalytic single-step reactions with one or two substrates,

the identification of bottlenecks and molecular as well as

engineering approaches to overcome these bottlenecks.

Robust industrial procedures have been established along

classes of biocatalytic single-step reactions. Multi-step

reactions and multi-component reactions (MCRs) enable a

bottom-up approach with biocatalytic reactions working

together in one compartment and recations hindering each

other within different compartments or steps. The

understanding of the catalytic functions of known and new

enzymes is key for the development of new sustainable

chemical transformations.

Address

Sigma–Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland

Corresponding author: Wohlgemuth, Roland

([email protected])

Current Opinion in Biotechnology 2010, 21:713–724

This review comes from a themed issue on

Chemical biotechnology

Edited by Phil Holliger and Karl Erich Jaeger

Available online 26th October 2010

0958-1669/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.copbio.2010.09.016

IntroductionThe creation of value-added products by chemical trans-

formations has contributed significantly to the quality of

life over the centuries and has reached a high level, but it

has been suggested that many of the stoichiometric

reactions in current use should be replaced by catalytic

processes [1]. Although catalytic tools are not only a

cornerstone of our present economy and society, but also

a key feature of basic life processes, most of the catalysts

used in the automotive, fuel refining, and chemical

industries consist either of inorganic, organometallic or

of organic catalysts in heterogeneous form, as for

example, catalysts involved in pollutant removal from

the exhaust leaving the car engines. The use of biocata-

lysts in chemical transformations has really taken off with

the focus on safe, healthy, resource efficient and econ-

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omical, energy saving, and environment-friendly pro-

duction procedures. The global needs for clean

manufacturing technologies, nonrenewable raw materials,

management of hazardous chemicals and waste present

new research challenges to both chemistry and biotech-

nology. These sciences are taking up these challenges

and the initiatives in Green/sustainable chemistry [2,3]

and white/industrial biotechnology [4] have emerged in

their disciplines independently. It is therefore of crucial

importance for the success of implementation and trans-

lation of science and technology into standard industrial

practice to develop a common chemistry–biotechnology

interface. One common opportunity for improvement and

invention is the current use of protecting groups for

overcoming nonselective and incompatible reactivities

in synthesis and biomimetic as well as enzyme-catalyzed

synthesis can provide the selectivities needed to over-

come barriers [5]. The manufacturing of molecular com-

plexity from simple starting materials with a minimum

number of steps, avoiding protection–deprotection loops

and orientation towards function of the product attract

much interest and biocatalytic process steps are well

positioned for contributing to the solutions of the

above-mentioned challenges [6].

The creation of sustainable value by viable industrial

processes and synthetic pathways requires not only

research progress in chemistry and biotechnology, but

in addition the integration of research from molecular and

engineering sciences, thereby enabling a large range of

industrial biotransformations [7–10]. As reaction devel-

opment serves different practical needs, progress in the

working areas single-step reactions, multi-step reactions,

and multi-component reactions (MCRs) will be discussed

in the following sections. Despite the enormous achieve-

ments in the chemical synthesis of organic compounds,

once believed to be accessible only by biological pro-

cesses and ‘vital forces’, over the past two centuries, many

present state-of-the-art processes are highly inefficient

[3]. This and additional boundary conditions like safety,

health and environment issues in industrial processes

have revitalized the interest in the discovery/invention

of novel biocatalytic reactions and reaction method-

ologies, which have been evolved by nature to achieve

highly efficient and selective transformations. Therefore

the section on the development of new biocatalytic

reaction methodology addresses this important industrial

innovation area.

Industrial biocatalytic single-step reactionsThe early success of single biocatalytic reaction steps

in classical organic synthesis schemes has led to an


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http://dx.doi.org/10.1016/j.copbio.2010.09.016

714 Chemical biotechnology

increasing number of established industrial processes and

continues to be a useful approach for the introduction of

biocatalysis into industrial practice. The discovery and

development of novel biocatalytic reaction steps can

thereby focus on overcoming synthetic bottleneck reac-

tions and improving the performance of existing chemical

reactions according to industrial requirements. Biocata-

lytic versions of reactions which are impossible or imprac-

tical by existing chemistry tools generate high interest

and stimulate further process research and development

work in industry.

Oxidation and reduction reactions

Oxidations and reductions catalyzed by oxidoreductases

have progressed towards the tools of choice (Figure 1) due

to their improved performance with respect to reaction

selectivity, safety, health, and environment aspects. Se-

lective introduction of one or two oxygen atoms by

biocatalysts has continued to attract a lot of industrial

interest. Among the reactions introducing one oxygen

atom, selective asymmetric hydroxylations, epoxidations,

and Baeyer–Villiger oxidations [11–13,14�] have made

significant progress and are of interest for the oxyfunc-

tionalization of inexpensive organic building blocks. Se-

lective biocatalytic oxidations of one out of several

hydroxygroups, as for example, in alcohols and sugars,

continue to be of industrial interest since the thirties of

the last century and have additional sustainability

benefits compared to the classical chemical oxidations

[7]. Since classical chemical oxidations often use stoichio-

metric oxidants in excess, the selective removal of

remaining oxidants is decisive for the product quality

and enzymatic methods have become standard practice in

production.

Depending on the enzyme properties and the cofactor

recycling system, both the oxidative and the reductive

directions of an oxidoreductase application are of interest

[15,16]. Sustainable enzymatic reductions of aldehydes

and ketones are reliable, scalable and inexpensive routes

to optically active alcohols and have been extensively

employed in organic synthesis despite the vast number of

asymmetric reductions [17]. Even in the area of the

reduction of carbon–carbon double bonds, where catalytic

hydrogenation with hydrogen gas in autoclaves is per-

formed routinely, new asymmetric biocatalytic reductions

of activated alkenes bearing an electron-withdrawing

group have become interesting methods for preparing

the corresponding saturated products in up to >99% ee

and for side stepping the use of hydrogen gas [18]. High

enantioselectivity was also observed for the asymmetric

reduction of activated a,b-unsaturated enones catalyzed

by pentaerythritol tetranitrate reductase for reaction pro-

duct stereogenic centers at the beta-carbon atom [19].

Enoate reductases have also been used for the conversion

of a series of a,b-unsaturated nitriles to the optically


active nitrile products in high yields and excellent enan-

tioselectivities [20].

Amination reactions

As in nature, industrial biocatalytic aminations have been

performed by the two different routes of transamination

and reductive amination. The use of amino acid dehydro-

genases in reductive amination of prochiral precursors

continues to play an important role in the enzymatic

production of D-enantiomers and L-enantiomers of both

natural and non-natural amino acids. Transaminases have

obtained increased interest for the asymmetric synthesis

of amines from prochiral ketones [21–24] and amination is

becoming a key reaction (Figure 2) in industrial biotrans-

formations [9]. New routes to nonchiral amines are also of

interest and a new biocatalytic transamination of pyri-

doxal-50-phosphate has been achieved with complete

conversion [25]. The efficiency of the manufacturing

process for the antidiabetic compound sitagliptin has

been greatly improved by replacing the Rh(Jobiphos)-

catalyzed asymmetric hydrogenation of an enamine at

high pressure with a direct transaminase-catalyzed amin-

ation of prositagliptin ketone [26��]. The best engineered

enzyme could convert 200 g/l prositagliptin ketone to

sitagliptin with an excellent ee of >99.95%. A 53%

productivity increase, 19% waste reduction, elimination

of heavy metals, cost reductions and avoiding specialized

high-pressure hydrogenation equipment have been found

as specific advantages of the biocatalytic process [26��].

Glycosylation reactions

As selective chemical glycosylation reactions require a

substantial synthetic effort involving various protecting

group chemistries in organic solvents, the use of glyco-

syltransferases for coupling glycosyl donors to nonpro-

tected acceptors in aqueous media (Figure 3) continues to

attract a lot of interest [27–29]. Methods based on the

application of glycosyltransferases are currently recog-

nized as being the most effective for the preparation of

complex and highly pure oligosaccharides [30�]. The

trihexosylceramides Gb3 and iGb3 have been synthes-

ized by specific galactosyltransferases using lactosylcer-

amide as acceptor [31]. Sialyltransferases have been used

in chemoenzymatic or whole-cell approaches for the

synthesis of a large library of sialoside standards and

derivatives [32�]. Carbohydrate-based drug design makes

use of various glycosyltransferases for the production of

novel glycosylated compounds, as no single universal

glycosyltransferase has been found [33]. The final hexose

to be transferred from the NDP-hexose to the aglycon can

thereby be diversified by a variety of enzymes like

dehydratases, epimerases and aminotransferases.

Hydrolysis and reverse hydrolysis reactions

On the basis of the vast number of established enzymatic

reactions using hydrolases in aqueous and nonaqueous

systems, this area has become well established and new


Sustainable industrial chemistry Wohlgemuth 715

Figure 1

Selected biocatalytic oxidation and reduction reactions. The cyclohexanone-monooxygenase-catalyzed Baeyer–Villiger oxidation of bicycloheptenone

has been applied industrially by Sigma–Aldrich with the substrate-feed-product-recovery-technology (SFPR) using Optipore L-493 as adsorber for

high space-time yield [14�].

applications appearing in various fields of organic chem-

istry can build on this experience (Figure 4). The large-

scale availability of many hydrolases like acylases,

amidases, esterases, lipases, proteases and their ease of

use without any cofactors has been a key factor for the

rapid growth of this reaction class in industry [34]. The

robustness and scalability of these reactions with stan-

dard equipment have been useful for resolutions, dera-

cemizations, desymmetrizations in early steps or mild


deprotections in late steps of a synthesis. The complete

conversion of a substrate into one product of high enan-

tiomeric purity is particularly attractive, as for example,

in desymmetrizations of prochiral diols or diesters. Inex-

pensive acyl donors like acids or simple esters are pre-

ferred for cost-sensitive productions, but require tools to

drive reactions to completion. Lipase-catalyzed acyla-

tions with activated acyl donors like enol esters and acid

anhydrides are practically irreversible. Lipase-catalyzed



Figure 2

Selected biocatalytic amination reactions. The transaminase-catalyzed asymmetric amination of prositagliptin ketone to sitagliptin has been applied

industrially by Merck. Abbreviations: PLP = pyridoxal-50-phosphate; MBA = (S)-a-methylbenzylamine.

polymerization in an organic solvent or one bulk mono-

mer is advantageous in reducing energy consumption and

in polymerizing multifunctional monomers or monomers

which undergo side reactions or are degraded under

process conditions [35]. The enzymatic resolution of a

substrate with a remote stereogenic center has been

realized in the first enantioselective synthesis of (S)-

monastrol [36]. An interesting high yield synthesis of

12-aminolauric acid from v-laurolactam has been devel-

oped by enzymatic transcrystallization using v-laurolac-

tam hydrolase from Acidivorax sp. [37�]. This method has

been chosen because of low conversion ratios by the use

of organic solvents and biphasic systems. Enzymatic


transcrystallization starts with the addition of crystalline

substrate to the aqueous reaction medium, which dis-

solves the substrate up to its solubility limit, and the

enzymatic reaction can then give the soluble product,

which will crystallize, when the product concentration

from the enzymatic conversion exceeds the product

solubility. Overall, the process resembles a SFPR system

[11], where the crystalline substrate is converted into

crystalline product in a highly efficient and environment-

friendly process without organic solvent, acid or alkali. A

nitrilase-catalyzed kinetic resolution of 2-cyano-1,4-ben-

zodioxane and 2-cyano-6-formyl-1,4-benzodioxane to

optically active 1,4-benzodioxane-2-carboxylic acids



Figure 3

Selected biocatalytic glycosylation reactions representing the enormous potential of glycosyltransferases for future industrial applications.

enables mild and enantioselective nitrile hydrolysis

without damage to labile functional groups like the

formyl group [38].

Carbon–carbon formation reactions and carbon–carbon

bond cleaving reactions

The formation and cleavage of carbon–carbon bonds is of

prime importance for constructing the carbon skeleton

not only in synthetic organic chemistry, but also in the


metabolic pathways of living cells. Among the great

variety of enzymes, hydroxynitrile lyases, aldolases,

and transketolases have attracted much interest

(Figure 5). Hydroxynitrile lyases have been valuable

for manufacturing enantiopure target cyanohydrins from

aldehydes, as versatile bifunctional building blocks for

chemical synthesis [39]. Strategies for overcoming reac-

tion limitations and suppression of nonenzymatic side

reactions combine approaches from enzyme and reaction



Figure 4

Selected biocatalytic hydrolysis and reverse hydrolysis reactions. A recombinant novel isoform of pig liver esterase termed alternative pig liver esterase

(APLE) has been applied industrially by DSM.

engineering [40]. Crude hydroxynitrile lyase has also

been used for the enantioselective cyanohydrin synthesis

in a microreactor [41]. Biocatalysis by means of aldolases

offers a unique stereoselective and green tool to perform

carbon–carbon bond formation or cleavage. Recent

advances in aldolase-catalyzed stereoselective carbon–carbon bond formation reactions are valuable for gener-

ating molecular diversity and for synthetic improvements

from small chiral polyfunctional molecules to highly


complex oligosaccharide analogs [42]. Aldolase-cata-

lyzed carbon–carbon bond formation has been used for

the large-scale synthesis of a chloromethyl-substituted,

a,b-unsaturated d-lactone [43��]. The synthetic poten-

tial of thiamin diphosphate-dependent enzymes for

asymmetric carboligations, such as asymmetric cross-

benzoin condensations, has been extended appreciably

and a variety of enantiomerically pure 2-hydroxyketones

have been synthesized by enzymatic carbon–carbon



Figure 5

Selected biocatalytic carbon–carbon bond formation reactions. The double aldol condensation of acetaldehyde with chloroacetaldehyde catalyzed by

deoxyribose-5-phosphate aldolase (DERA-aldolase) has been applied industrially by DSM in the production of chiral lactones.

bond ligation of aldehydes [44]. The use of benzaldehyde

lyase and benzoylformate decarboxylase in recombinant

Escherichia coli resting cells in a MTBE/aqueous buffer

biphasic medium has improved substrate solubility and

extractive workup [45]. Another route to enantiomeri-

cally pure 2-hydroxy-ketones is the enzymatic chain

elongation of aldehydes by a two-carbon unit, which

can be catalyzed by transketolase and driven to com-

pletion by the use of the irreversible C2-ketol donor b-

hydroxypyruvate [46–48].


A novel biocatalytic carbon–carbon bond formation reac-

tion equivalent to Friedel–Crafts alkylation has been

catalyzed by methyltransferases using S-adenosyl-L-

methionine and analogs [49��]. A very broad range of

acceptor substrates including cyclic and open-chain

ketones as well as diketones and a-ketoesters and

b-ketoesters have been found in the first enzymatic

asymmetric intermolecular aldehyde–ketone cross-

coupling reaction, using the thiamine-dependent enzyme

YerE [50��]. Changing the substrate specificity of the



Figure 6

Scheme of a biocatalytic multi-step and a biocatalytic multi-component reaction.

carbon–carbon bond forming enzyme tyrosine phenol

lyase has been key for replacing the cumbersome chemi-

cal multi-step synthesis of nonnatural 3-substituted tyro-

sine derivatives by a single-step biocatalytic synthesis

with complete conversion and excellent enantioselectiv-

ity, starting from the corresponding phenol precursor,

pyruvate and ammonia [51��].

Industrial biocatalytic multi-step reactionsMulti-step processes coupling two or more biocatalytic

reactions in one pot (Figure 6) are attractive because of

the reduction in the number of process steps, productivity

improvements and overcoming thermodynamic barriers

[66]. One-pot synthetic methods involving multiple bond

formation steps such as domino, tandem or cascade reac-

tions eliminate also time-consuming recovery and purifi-

cation steps. Biocatalytic reactions have thereby been

combined with other chemical or biocatalytic reactions.

A prominent two-step example is the conversion of cepha-


losporin C to 7-aminocephalosporanic acid by D-amino acid

oxidase and cephalosporin acylase. The synthesis of ator-

vastatin has been achieved by the biocatalytic reduction of

ethyl-4-chloroacetoacetate using a ketoreductase-cata-

lyzed reaction as the first step and a halohydrin-dehalo-

genase-catalyzed substitution reaction of the chloro-

substituent with the cyano-group [52]. Another two-step

reaction sequence has been used in the conversion of an

aromatic alkene to a chiral 2-hydroxy ketone. The carbon–carbon double bond in the olefin trans-anethole to

para-anisaldehyde has been cleaved biocatalytically with

a Trametes hirsuta extract and with molecular oxygen

as oxidant. The second reaction step catalyzed the

condensation of para-anisaldehyde to acetaldehyde by

the enantiocomplementary C–C bond forming enzymes

benzaldehydelyase and benzoylformatedecarboxylase,

respectively, to yield either (R)-2-hydroxy-1-(4-methoxy-

phenyl)-propanone or (S)-2-hydroxy-1-(4-methoxyphe-

nyl)-propanone [53].



Natural product synthesis and modification by biocataly-

tic multi-step reactions is of much interest because of the

challenges in large-scale production of bioactive small

molecules from natural sources or by total synthesis.

Many opportunities exist for preparing a wide range of

natural product variants due to the substrate flexibility of

the pathway enzymes. The bottom-up assembly of plant

biosynthetic pathways in microorganisms is of interest for

exploring the fascinating capabilities of the individual

enzymes as well as for facilitating scalable production

platforms for the synthesis of natural and unnatural alka-

loids [54]. The optimization of biocatalytic pathways to

macrotetrolides is highly attractive, because chemical

synthesis of compounds like nonactin has not been com-

petitive for large-scale production [55]. This is related to

the exquisite selectivity and orthogonality of biocatalytic

functional group transformations, which enables the

organization of multi-step reactions in defined reaction

spaces in an analogous way as in biological cells, cell

compartments or multi-enzyme machineries. The

achievements of biocatalytic multi-step reactions serve

as gold standard for the reaction development in organic

chemistry [4].

Industrial biocatalytic multi-componentreactionsWhile the tactics of step-by-step reactions is based on a

cascade of subsequent functional group transformations,

the goal for MCRs is to construct several bonds between

the components by a parallel operation of different reac-

tions with completely independent reactivity and selec-

tivity. MCRs are therefore step-efficient procedures

converging towards the product and avoiding protecting

group chemistry. MCRs enable building molecular com-

plexity directly from more than two components.

Although MCRs like the Strecker reaction are important

industrial reactions in organic chemistry, the develop-

ment of biocatalytic MCRs (Figure 6) has only recently

attracted interest.

A novel lipase-catalyzed direct Mannich reaction in water

has been discovered, involving aniline, a nonenolizable

substituted benzaldehyde as electrophile and the enoliz-

able acetone as a source of nucleophile [56]. A sequence

of a biocatalytic desymmetrization of a 3,4-substituted

meso-pyrrolidin with monoamine oxidase N from Asper-gillus niger and the use of the resulting enantiopure

1-pyrrolin as component in an Ugi-type 3-component

reaction has been performed in two separate operations

in order to achieve the best yields, diastereomeric ratio

and ee values [57��]. An interesting approach towards a

biocatalytic asymmetric Strecker reaction has combined

transimination with imine-cyanation in a double dynamic

covalent system under thermodynamic control and sub-

sequently coupled in a one-pot process with lipase-

catalyzed transacylation under kinetic control [58]. A

biocatalytic Biginelli 3-component reaction has been


developed for the high-yield synthesis of 3,4-dihydropyr-

imidine-2-(1H)-ones, consisting of the condensation of

urea or thiourea with a substituted benzaldehyde and a

1,3-ketoester at room temperature in aqueous phosphate

buffer pH 7.0 and using Saccharomyces cerevisiae as bioca-

talyst [59].

Development of new biocatalytic reactionmethodologyIndustrial applications aim at stable processes with

robust, simple and sustainable operation and product

recovery as well as high molecular economy [60–63].

The search for new biocatalytic reaction methodology

is experiencing a boost by progress in a number of

relevant areas like the connection between the broad

set of natural product biosynthetic reactions and the

genes that encode them [64] or the tremendous progress

in engineering enzymes by directed evolution [65��].

Whether the reaction is performed on a small or large

scale, the confinement or localization of enzymes in a

certain reaction space, while retaining their catalytic

activities under process conditions, is key. Expanding

the organic chemistry of enzyme-catalyzed reactions

and interfacing the enzyme reactions with classical chemi-

cal reactions in this reaction space, with no need for using

protecting groups, is promising [66]. A concise approach to

the synthesis of all 24 hexoses and 5-deoxy-hexoses, still

ongoing, is based on a range of biocatalysts which inter-

convert polyols and ketoses, aldose isomerases for the

equilibration of ketoses and aldoses, and D-tagatose-3-

epimerase for the C-3 equilibration of a wide range of

substrates like ketoses, deoxysugars, and C-branched

sugars [67�]. An interesting biocatalytic domino reaction

between phenol and various cyclic 1,3-dicarbonyl com-

pounds yielded annulated benzofurans, using the

enzymes tyrosinase and laccase from Agaricus bisporus [68].

The capturing and activation of carbon dioxide by

enzymes has obtained increased interest [69], as on the

one hand the chemistry of direct carboxylation reactions

is underdeveloped and on the other hand many carbox-

ylating and decarboxylating enzymes are occurring widely

in nature. The novel continuous flow enzymatic carbox-

ylation of pyrrole to pyrrole-2-carboxylate by immobilized

Bacillus megaterum represents an interesting green engin-

eering approach [70]. Salicylic acid decarboxylase from

Trichosporon monilliforme has been discovered to catalyze

the enzymatic Kolbe–Schmitt reaction from phenol to

salicylic acid [71]. 3,4-Dihydroxybenzoate decarboxylase

from Enterobacter cloacae enabled the mild regioselective

carboxylation of catechol to 3,4-dihydroxy-benzoic acid

with 3 M potassium hydrogencarbonate at 308C [72].

ConclusionsBiocatalytic single-step reaction platforms developed over

the last years have progressed rapidly in the industrial



production environment and many more methodologies

developed at the research scale are waiting to be applied

and to be scaled up. Discovery and development of novel

biocatalytic single-step reactions continues to be import-

ant, especially in areas where no direct functional group

transformation is known or where the known chemical

transformation is lacking safety, selectivity or sustainabil-

ity. The innate selectivity and orthogonality advantage of

biocatalytic reactions bears a lot of potential for major

improvements in multi-step reactions. Attention needs

to be paid to both the molecular and the engineering

aspects of the architecture of such biocatalytic multi-step

systems. Whatever route is selected, key to further

advances in sustainable chemical reactions is the devel-

opment of novel biocatalytic reaction methodologies,

which are modular, scalable, and compatible with the

development of chemical reactions. The science, technol-

ogy and industry of chemical synthesis and catalysis on the

other hand is accepting established biocatalytic reaction

platforms, because of the need for method and route

simplification, molecular economy, safety, health, and

environment improvements. Therefore the knowledge

building in industrial biocatalysis and its practical imple-

mentation is key for value creation in a future bioeconomy.

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Savile CK, Janey JM, Mundorff EC, Morre JC, Tam S, Jarvis WR,Colbeck JC, Krebber A, Fleitz FJ, Brands J et al.: Biocatalyticasymmetric synthesis of chiral amines from ketones appliedto sitagliptin manufacture. Science 2010, 329:305-309.

A variety of enzyme engineering techniques have been applied to thecreation of a transaminase biocatalyst with the required properties andactivity toward the prositagliptin ketone. This has resulted in an efficientbiocatalytic trans-amination process to replace a rhodium-catalyzedasymmetric hydrogenation for the large-scale manufacturing of the anti-diabetic compound sitagliptin.

27. Chokhawala HA, Huang S, Lau K, Yu H, Cheng J, Thon V, Hurtado-Ziola N, Guerrero JA, Varki A, Chen X: Combinatorialchemoenzymatic synthesis and high-throughput screening ofsialosides. ACS Chem Biol 2008, 3:567-576.

28. Wohlgemuth R: Tools and ingredients for the biocatalyticsynthesis of carbohydrates and glycoconjugates. BiocatalBiotransformation 2008, 26:42-48.

29. Wanga Z, Gilbert M, Eguchi H, Yu H, Cheng J, Muthanad S, Zhou L,WangPG, Chen X, Huang X: Chemoenzymatic syntheses oftumor-associated carbohydrate antigen Globo-H and



stage-specific embryonic antigen 4. Adv Synth Catal 2008,350:1717-1728.

30.�

Weijers CAGM, Franssen MCR, Visser GM: Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides. BiotechnolAdv 2008, 26:436-456.

Review on the effective application of glycosyltransferases for the pre-paration of complex and highly pure oligosaccharides.

31. Adlercreutz D, Weadge JT, Petersen BO, Duus JØ, Dovichi NJ,Palcic MM: Enzymatic synthesis of Gb3 and iGb3 ceramides.Carbohydr Res 2010, 345:384-388.

32.�

Chen X, Varki A: Advances in the biology and chemistry of sialicacids. ACS Chem Biol 2010, 5:163-176.

A sialic acid review including recent advances in chemoenzymatic synth-esis as well as large-scale E. coli.

33. Luzhetskyy A, Mendez C, Salas JA, Bechthold A:Glycosyltransferases, important tools for drug design. CurrTop Med Chem 2008, 8:680-709.

34. Wohlgemuth R: Large-scale applications of hydrolasesin biocatalytic asymmetric synthesis. In Large-scaleAsymmetric Catalysis. Edited by Blaser HU, Federsel HJ.Weinheim: Wiley-VCH; 2010.

35. Gross RA, Ganesh M, Lu W: Enzyme-catalysis breathes new lifeinto polyester condensation polymerizations. TrendsBiotechnol 2010, 28:435-443.

36. Blasco MA, Thumann S, Wittmann J, Giannis: A, Groger H:Enantioselective biocatalytic synthesis of (S)-monastrol.Bioorg Med Chem Lett 2010, 20:4679-4682.

37.�

Fukuta Y, Komeda H, Yoshida Y, Asano Y: High yield synthesis of12-aminolauric acid by ‘enzymatic transcrystallization’ of v-laurolactam using v-laurolactam hydrolase from Acidivoraxsp. T31. Biosci Biotechnol Biochem 2009, 73:980-986.

An interesting high-yield enzymatic hydrolysis of v-laurolactam by v-laurolactam hydrolase from Acidivorax sp. has been developed. Crystal-line v-laurolactam, added to the enzyme solution, has been converted tocrystalline 12-aminolauric acid with the high volume yield of >200 g/l,high purity and >97% conversion.

38. Benz P, Muntwyler R, Wohlgemuth R: Chemoenzymaticsynthesis of chiral carboxylic acids via nitriles. J Chem TechnolBiotechnol 2007, 82:1087-1098.

39. Purkarthofer T, Skranc W, Schuster C, Griengl H: Potential andcapabilities of hydroxynitrile lyases as biocatalysts in thechemical industry. Appl Microbiol Biotechnol 2007, 76:309-320.

40. Andexer JN, Langermann JV, Kragl U, Pohl M: How to overcomelimitations in biotechnological processes—examples fromhydroxynitrile lyase applications. Trends Biotechnol 2009,27:599-607.

41. Koch K, van den Berg RJF, Nieuwland PJ, Wijtmans R,Schoemaker HE, van Hest JCM, Rutjes FPJT: Enzymaticenantioselective C–C-bond formation in microreactors.Biotechnol Bioeng 2008, 99:1028-1033.

42. Clapes P, Fessner WD, Sprenger GA, Samland AK: Recentprogress in stereoselective synthesis with aldolases. Curr OpinChem Biol 2010, 14:154-167.

43.��

Wolberg M, Dassen BHN, Schurmann M, Jennewein S,Wubbolts MG, Schoemaker HE, Mink D: Large-scale synthesisof new pyranoid building blocks based on aldolase-catalysedcarbon–carbon bond formation. Adv Synth Catal 2008,350:1751-1759.

Large-scale aldolase-catalyzed carbon–carbon bond formation, basedon a reaction discovered by CH Wong and coworkers, has permitted thehighly stereoselective synthesis of substituted d-lactones.

44. Muller M, Gocke D, Pohl M: Thiamin diphosphate in biologicalchemistry: exploitation of diverse thiamin diphosphate-dependent enzymes for asymmetric chemoenzymaticsynthesis. FEBS J 2009, 276:2894-2904.

45. Dominguez e Maria P, Stillger T, Pohl M, Kiesel M, Liese A,Groger H, Trauthwein H: Enantioselective C–C bond ligationusing recombinant Escherichia coli-whole-cell biocatalysts.Adv Synth Catal 2008, 350:165-173.


46. Wohlgemuth R: C2-ketol elongation by transketolase-catalyzedasymmetric synthesis. J Mol Catal B: Enzym 2009,61:23-29.

47. Shaeri J, Wright I, Rathbone EB, Wohlgemuth R, Woodley JM:Characterization of enzymatic D-xylulose 5-phosphatesynthesis. Biotechnol Bioeng 2008, 101:761-767.

48. Wohlgemuth R, Smith MEB, Dalby PA, Woodley JM:Transketolases. In Encyclopedia of Industrial Biotechnology.Edited by Flickinger MC. Hoboken, NJ: Wiley; 2010.

49.��

Stecher H, Tengg M, Ueberbacher BJ, Remler P, Schwab H,Griengl H, Gruber-Khadjawi M: Biocatalytic Friedel–Craftsalkylation using non-natural cofactors. Angew Chem Int Ed2009, 48:9546-9548.

The SAM-dependent methyltransferases NovO from Streptomyces spher-oides and CouO from Streptomyces rishiriensis, cloned and expressed inE. coli, have been shown to accept modified cofactors and to catalyze thesynthesis of a range of monosubstituted methylated, allylated, propargy-lated and benzylated arenes with excellent regioselectivity.

50.��

Lehwald P, Richter M, Rohr C, Liu HW, Muller M: Enantioselectiveintermolecular aldehyde–ketone cross-coupling through anenzymatic carboligation reaction. Angew Chem Int Ed 2010,49:1-5.

The thiamindiphosphate-dependent enzyme YerE has been shown tocatalyze the asymmetric cross-coupling of aldehydes and ketones tochiral teriary alcohols.

51.��

Seisser B, Zinkl R, Gruber K, Kaufmann F, Hafner A, Kroutil W:Cutting long syntheses short: access to non-natural tyrosinederivatives employing an engineered tyrosine phenol lyase.Adv Synth Catal 2010, 352:731-736.

The laborious and time-consuming multi-step synthesis of 3-substitutedtyrosine derivatives has been replaced by a single biocatalytic one-stepreaction using an engineered tyrosine phenol lyase.

52. Ma SM, Gruber J, Davis C, Newman L, Gray D, Wang A, Grate J,Huisman GW, Sheldon RA: A green-by-design biocatalyticprocess for atorvastatin intermediate. Green Chem 2010,12:81-86.

53. Kurlemann N, Lara M, Pohl M, Kroutil W, Liese A: Asymmetricsynthesis of chiral 2-hydroxy ketones by coupled biocatalyticalkene oxidation and C–C bond formation. J Mol Catal B:Enzymatic 2009, 61:111-116.

54. Leonard E, Runguphan W, O’Connor S, Jones Prather K:Opportunities in metabolic engineering to facilitate scalablealkaloid production. Nat Chem Biol 2009, 5:292-300.

55. Jani P, Emmert J, Wohlgemuth R: Process analysis ofmacrotetrolide biosynthesis during fermentation by means ofdirect infusion LC–MS. Biotechnol J 2008, 3:1-7.

56. Li K, He T, Li C, Feng XW, Wang N, Yu XQ: Lipase-catalyzeddirect Mannich reaction in water: utilization of biocatalyticpromiscuity for C–C bond formation in a ‘one-pot’ synthesis.Green Chem 2009, 11:777-779.

57.��

Znabet A, Ruijter E, Decanter FJJ, Kohler V, Helliwell M, Turner NJ,Orru RVA: Highly stereoselective synthesis of substitutedprolyl peptides using a combination of biocatalyticdesymmetrization and multicomponent reactions. AngewChem Int Ed 2010, 49:5289-5292.

A highly diastereoselective Ugi-multicomponent reaction of opticallyactive 3,4-disubstituted 1-pyrrolines, obtained by monoamineoxidaseN-catalyzed desymmetrization of the corresponding meso-pyrrolidines,with isocyanides and carboxylic acids has been developed for thesynthesis of substituted prolylpeptides.

58. Pornrapee V, Ramstrom O: Dynamic asymmetricmulticomponent resolution: lipase-mediated amidation of adouble dynamic covalent system. J Am Chem Soc 2009,131:14419-14425.

59. Kumar A, Maurya RA: An efficient baker’s yeast catalyzedsynthesis of 3,4-dihydropyrimidin-2-(1H)-ones. TetrahedronLett 2007, 48:4569-4571.

60. Wohlgemuth R: Modular and scalable biocatalytic tools forpractical safety, health and environmental improvements inthe production of speciality chemicals. BiocatalBiotransformation 2007, 25:178-185.



61. Wohlgemuth R: Tools and ingredients for the biocatalyticsynthesis of metabolites. Biotechnol J 2009, 9:1253-1265.

62. Tao J, Xu JH: Biocatalysis in development of greenpharmaceutical processes. Curr Opin Chem Biol 2009, 13:43-50.

63. Wohlgemuth R: Green production of fine chemicals by isolatedenzymes. In Biocatalysis for Green Chemistry and ChemicalProcess Development. Edited by Tao JA, Kazlauskas RJ.Hoboken, NJ: Wiley; 2010.

64. Walsh CT, Fischbach MA: Natural products version 2.0:connecting genes to molecules. J Am Chem Soc 2010,132:2469-2493.

65.��

Reetz MT: Directed evolution of enantioselective enzymes: anunconventional approach to asymmetric catalysis in organicchemistry. J Org Chem 2009, 74:5767-5778.

An excellent perspective on the principles, strategies and methods of thedirected evolution of enantioselective enzymes and their successes andfuture challenges in their applications as asymmetric catalysts in organicchemistry.

66. Wohlgemuth R: Interfacing biocatalysis and organic synthesis.J Chem Technol Biotechnol 2007, 82:1055-1062.

67.�

Rao D, Best D, Yoshihara A, Gullapalli P, Morimoto K,Woemaid MR, Wilson FX, Izumori K, Fleet GWJ: A conciseapproach to the synthesis of all twelve 5-deoxyhexoses:D-tagatose-3-epimerase — a reagent that is both specific andgeneral. Tetrahedron Lett 2009, 50:3559-3563.


An interesting equilibration of 5-deoxy-D-fructose to 5-deoxy-D-psicoseand of 5-deoxy-L-psicose to 5-deoxy-L-fructose, providing substrates forthe preparation of all D-5-deoxy-aldohexoses and L-5-deoxy-aldo-hexoses.

68. Leutbecher H, Hajdok S, Braunberger C, Neumann M, Mika S,Conrad J, Beifuss U: Combined action of enzymes: the firstdomino reaction catalyzed by Agaricus bisporus. Green Chem2009, 11:676-679.

69. Glueck SM, Gumus S, Fabian WMF, Faber K: Biocatalyticcarboxylation. Chem Soc Rev 2010, 39:313-328.

70. Matsuda T, Marukado R, Koguchi S, Nagasawa T, Mukouyama M,Harada T, Nakamura K: Novel continuous carboxylation usingpressurized carbon dioxide by immobilized decarboxylase.Tetrahedron Lett 2008, 49:6019-6020.

71. Yoshida T, Inami Y, Matsui T, Nagasawa T: Regioselectivecarboxylation of catechol by 3,4-dihydroxybenzoatedecarboxylase of Enterobacter cloacae P. Biotechnol Lett 2010,32:701-705.

72. Kirimura K, Gunji H, Wakayama R, Hattori T, Ishii Y: EnzymaticKolbe–Schmitt reaction to form salicylic acid from phenol:enzymatic characterization and gene identification of a novelenzyme, trichosporon moniliiforme salicylic aciddecarboxylase. Biochem Biophys Res Commun 2010, 394:279-284.


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