Trends and innovations in industrial biocatalysis for the production of fine chemicals

Sven Panke, Martin Held and Marcel WubboltsÃ
Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure a- and b-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.
Addresses DSM Research Campus Geleen, Advanced Synthesis & Catalysis, PO Box 18, 6160 MD Geleen, The Netherlands à e-mail: marcel.wubbolts@dsm.com

for this review, we performed a survey of patents in the area of biotransformations over the past few years with a priority date after 2000 that were issued by companies in the pharmaceutical, fine chemical, and flavour and fragrances area. In total, we judged 205 patents as relevant to this review (Table 1). In the following, we discuss a selection of these patents, based on the information provided therein and supporting publications in the open scientific literature.

The production of a- and b-amino acids
Aminotransferases that produce a-amino acids from a-keto acids using an amino donor such as L-aspartic or L-glutamic acid are — despite the 100% theoretical yield — typically hampered by moderate yields owing to the reaction equilibrium. In the case of L-aspartic acid, the oxaloacetate that is generated can undergo decarboxylation to pyruvate, thereby pulling the reaction in the desired direction. Similarly, Great Lakes (http:// www.greatlakesfine.com) has developed a biocatalyst that combines L-glutamate-preferring a-amino transferases, such as tyrosine aminotransferase (TAT) and branchedchain aminotransferase (BCAT), with L-ornithine damino transferase (Figure 1). The latter enzyme converts 2-ketoglutarate and L-ornithine into L-glutamic acid and L-glutamic acid semialdehyde, which spontaneously cyclizes to form D1-pyrroline-5-carboxylate. As a result the reaction equilibrium of the coupled a-amino transferase reaction can be shifted towards the synthesis direction. Thus, the synthesis of L-2-aminobutyrate from 2-ketobutyric acid (TAT, 92% yield) and L-tert-leucine from trimethylpyruvic acid (BCAT, 73% yield) was achieved [2]. Degussa (http://www.degussa.com) has described the use of engineered whole-cell biocatalysts producing balanced levels of the enzymes L-carbamoylase, L-hydantoinase and hydantoin racemase to synthesize unnatural L-amino acids [3,4]. The company has further improved this dynamic kinetic resolution technology for the production of D-amino acids as well, by including a D-specific carbamoylase and a D-hydantoinase obtained by directed evolution from the L-specific enzyme [3]. In another known dynamic kinetic resolution process of Degussa, racemic N-acetyl amino acids are resolved using an acylase in combination with an N-acetyl amino acid racemase. A novel N-acetyl amino acid racemase from Amycolatopsis orientalis, which is no longer prone to substrate inhibition, provides a significant improvement over the old process [3].
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Current Opinion in Biotechnology 2004, 15:272–279 This review comes from a themed issue on Protein technologies and commercial enzymes Edited by Karl-Erich Jaeger Available online 17th July 2004 0958-1669/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.06.011

Abbreviations DERA ee LNT NeuAc PDF PEP deoxyribose-5-phosphate aldolase enantiomeric excess lacto-N-tetraose N-acetyl-D-neuraminic acid peptide deformylase phosphoenolpyruvate

Introduction
Enzymes and whole-cell biocatalysts, as a result of their complex chiral constitution, are predominantly suited for the manufacture of optically pure stereoisomers. The production of optically active intermediates is an area of growing demand in the fine chemical industry and here biocatalysis has developed from a niche technology to a widely used manufacturing method [1]. In this review we discuss recent biocatalysis developments and innovations in industry grouped by product class and we have restricted ourselves to a- and b-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids, epoxides and complex multifunctional molecules. As the basis
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Innovations in industrial biocatalysis Panke, Held and Wubbolts 273

Table 1 Indication of the industrial focus reflected by selecteda patents published in the area of biocatalysis and biotransformation. Compound class Alcohols Subdivision/identified technologies Ketone reduction (Dynamic) kinetic resolution Aldol condensation Oxygenases Other Hydantoin/carbamoyl hydrolysis Amide hydrolysis Transamination b-Amino acids Peptide synthesis by kinetic coupling Other Resolution of esters Nitrile/cyanohydrin hydrolysis Oxidation by oxygenases Acids from lyase reactions Resolution of amides Other Glycosyltransferases/oligosaccharides/glycoconjugates Glycoprotein remodeling Activated monosaccharides Monosaccharides Others Antibiotics Steroids Other modification of complex scaffolds Epoxides/peroxides/diols Amines Aldehydes Cyanohydrins Others Number of patents 35b 13 5 3 1 17 7 3 5 8 1 14 5 4 3 2 1 10 9 3 1 4 9 5 2 9 9 6 6 5

Amino acids/peptides

Acids

Sugars

Complex natural products

Others

a

Patents with priority date after 2000 were retrieved from Chemical Abstracts by SciFinder using enzym*, biocatal* and biotransform* search keys and were cross-checked for all companies known to be active in the field. Fermentation processes were excluded. bIncluding nine patents that primarily deal with aspects of cofactor regeneration.

Figure 1

O HO O 2-Ketoacid R α-Amino transferase HO

O R NH 2
L-Amino

acid

L-Glutamic

acid

2-Ketoglutarate

O HO N 3,4-Dihydro-2 H-pyrrole2-carboxylic acid

O HO NH 2
L-Glutamic

O O Ornithine δ-amino transferase (OAT) HO NH 2
L-Ornithine Current Opinion in Biotechnology

NH 2

acid semialdehyde

Great Lakes process for the combined utilization of a-amino tranferases with L-ornithine-d-amino transferase to produce a-amino acids [2]. www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:272–279

274 Protein technologies and commercial enzymes

Figure 2

HOOC HOOC NH2

NH2

HOOC

NH2 HOOC NH2

HOOC N S

NH2

S HOOC NH2 1 N N COOH 13 HOOC N N N N N 2 OH

N

N 3 4 HOOC SO3 H NH2 5

HOOC

NH2

NH2 S 6 SH N S 7 S

HOOC HOOC NH2

NH2 HOOC S NH2 HOOC HOOC NH2 NH2

N

N S N N+ 12 11 10 9 N–
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Se N O

N

O O N H

8

The Wacker Chemie process for L-cysteine (shown in box) production, which provides access to a wide range of unnatural (S)-a-amino acids [5]. Compounds that have been made include the (S)-enantiomers of tetrazole-2-yl alanine 1, hydroxyethylcysteine 2, pyrazole-1-yl-alanine 3, sulfocysteine 4, cyanoalanine 5, phenylcysteine 6, thiazole-2-yl-cysteine 7, quisqualic acid 8, azidoalanine 9, phenylselenocysteine 10, thien-2-ylcysteine 11, triazole-1-yl-alanine 12 and 5-carboxybenzo-triazole-2-yl-alanine 13.

A production method for the manufacture of cysteine by fermentation — to replace cysteine produced by extraction from human hair — has been developed by Wacker Chemie (http://www.wacker.com/). Building on this technology in an engineered Escherichia coli strain, the enzyme O-acetylserine sulfhydrylase, which accepts a broad range of substrates, was used to produce a stunning number of unnatural amino acids, such as triazole-1-yl-alanine, (S)hydroxyethylcysteine and phenylselenocysteine, by feeding a fermentation with unnatural precursor molecules (Figure 2) [5]. These novel unnatural amino acids may prove useful for the synthesis of combinatorial (peptide) libraries and pharmaceutical intermediates. Peptide deformylase (PDF) catalyzes the hydrolysis of the N-formyl group from the N-terminal N-formylmethionine group from nascent polypeptides. Interestingly, the enzyme is also active with an almost complete enantioselectivity (E ratio >500) on several N-formylated a- and b-amino acids, a-amino acid amides and a-amino nitriles
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[6]. PDF was used for the enantioselective formylation of a-amino nitriles, such as phenylalanine nitrile, to yield (S)-N-formyl-phenylalanine nitrile with an enantiomeric excess (ee) of over 99.5%. In addition, the enzyme can be used for the mild and selective deprotection of N-formylprotected peptides [6]. In addition to the above-mentioned use of PDF [6] and more established resolution methods using N-acylases and lipases, a novel route for the production of b-amino acids from b-amino nitriles has been described that uses nitrilases from Rhodococcus sp. R312 and Rhodococcus erythropolis NCIMB 11540 [7].

Amines
Enantiomerically pure amines are important chiral intermediates with both pharmaceutical and agricultural applications. BASF (http://www.basf.com/) has developed a very versatile Burkholderia plantarii lipase platform for the resolution of amines using methoxy-acetic acid ethyl ester as acylating agent [8]. The B. plantarii lipase has been improved by mutagenesis [9] and other lipases
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Innovations in industrial biocatalysis Panke, Held and Wubbolts 275

Figure 3

OH R

OH

O

OR 15 OC(O)OMe O (a) EtO O O (b) Cl OC(CH3)3 O OEt O rec. L. brevis ADH Cl α-Chymotrypsin HO O OEt OC(O)OMe O 16 98.1% ee Steps 15

OH

O

O

17 > 99.5% ee OC(CH3)3

Steps 15

O (c) Cl +

O

DERA Cl

OH

OH

O Cl

O

OH Steps 15

2 equivalents OH KCN Cl NC

18, 96.6% de OH Nitrilase CN NC

OH Steps 15

O (d)

COOH

19 >95% ee

Current Opinion in Biotechnology

Various biocatalytic routes to (R,R)-3,5-dihydroxy-hexanoate ester derivatives (shown in box; 15), that serve as precursors for the synthesis of statins. (a) Prochiral 3-substituted glutarates undergo desymmetrization by a-chymotrypsin to (R)-3-methoxyacetylglutaryl monoethyl ester 16 [12]. (b) The reduction of 3,5-dioxo-hexanoic acid tert-butyl ester to (S)-6-chloro-5-hydroxy-3-oxo-hexanoic acid tert-butyl ester 17 [13]. The reaction is catalysed by L. brevis ADH. (c) The condensation of chloroacetaldehyde with two molecules of acetaldehyde to form (3R,5S)-6-chloro-3,5-dihydroxy-hexanal (18) is catalyzed by DERA [16]. The hexanal spontaneously cyclizes with retention of the stereochemistry to the hemi-acetal, which drives the equilibrium in the desired direction. (d) Nitrilase-catalyzed desymmetrization of 3-hydroxy-glutarodinitrile, which is easily accessible via epichlorohydrin, yields (R)-4-cyano-3-hydroxy-butyric acid 19 [19]. The enantiomeric excess (ee) values are given as percentages.

and acylating agents have also been successfully used for amine resolutions by BASF, as reviewed in Breuer et al. [1]. Diversa (http://www.diversa.com/) has explored the potential of nitroreductases for the production of (chiral) amines and has performed high-throughput screening to obtain suitable enzymes for this purpose [10]. Alternative methods for amine resolutions, based on penicillin acylases that act by either hydrolyzing N-acyl amines (hydrolysis mode) or by N-acylation of the amine starting compounds (synthesis mode), have been described by DSM (http://www.dsm.com/) [11].

Alcohols and acids
The manufacture of optically active alcohols is an important activity in biotransformation. For example, optically
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pure alcohols are a key intermediate in the manufacture of the sidechains of synthetic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)-inhibiting (cholesterol lowering) drugs such as atorvastatin, cerivastatin, fluvastatin or rosuvastatin. Several approaches have recently been discussed in the academic and patent literature (Figure 3). One approach involves the desymmetrization of a methoxyacetyl-ester of glutaric acid diethyl ester with a-chymotrypsin (Figure 3a) [12]. Alternatively, a 3,5dioxocarboxylate can be reduced with excellent enantioselectivity employing a recombinant NADPH-dependent Lactobacillus brevis alcohol dehydrogenase (Figure 3b) [13]. Another promising route was reported in patent and open literature by both DSM and Diversa [14,15]. This route employs a deoxyribose-5-phosphate aldolase (DERA) that catalyzes a tandem aldol condensation in which two equivalents of acetaldehyde are added to chloroacetaldehyde to produce a lactol derivative that
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276 Protein technologies and commercial enzymes

is similar to the 3,5-dihydoxy sidechain of synthetic statins (Figure 3c). Diversa screened for novel DERAs that were both tolerant to high substrate concentrations and that were more active than DERA from E. coli [14]. A DERA variant with less than 30% identity to the E. coli enzyme led to a significant improvement over the E. coli wild-type DERA based process originally described by Wong et al. [16]. The application of rational mutagenesis, based on the available crystal structure of the E. coli enzyme, expanded the range of suitable acceptor substrates for DERA to azido-substituted aldehydes, further facilitating access to, for example, the atorvastatin sidechain [17]. An interesting variation on the desymmetrisation approach discussed above is presented by Diversa’s nitrilase technology, applied again for the manufacture of the key intermediate for atorvastatin [18]. Here, a prochiral dinitrile is hydrolyzed selectively to (R)-3-hydroxy-4cyanobutyrate (Figure 3d), which in several subsequent steps can be converted into 6-cyano-3R,5R-dihydroxyhexanoic acid ethyl ester as an advanced intermediate to atorvastatin [19]. The most prominent method to produce optically active alcohols by biocatalysis is the enantioselective reduction of ketones. Novel industrially relevant alcohol dehydrogenases continue to become available and novel process engineering concepts have also been developed. For example, an NAD+-dependent (S)-alcohol dehydrogenase from R. erythropolis DSM43297 has recently been expressed in E. coli [20]. This enzyme displays excellent enantioselectivity and accepts aliphatic and aromatic ketones as well as b-keto esters [21]. Such reactions typically suffer from the low solubility of the organic substrate in aqueous buffer. The addition of a second liquid — organic — phase as a substrate reservoir is an attractive approach, but has been hampered by the sensitivity of the cofactor recycling enzyme formate dehydrogenase from Candida boidinii to such systems. However, conditions have been identified where an engineered variant of formate dehydrogenase [22] and R. erythropolis dehydrogenase remained stable — a twophase system consisting of aqueous buffer and 20% hexane or heptane [23] — thus enabling simple batch processes with substrate concentrations between 100 mM and 200 mM. Another industrially very suitable approach is a monophasic system where the polarity is controlled by the addition of water-miscible organic solvents. Ideally, such solvents should also function as a hydrogen source for cofactor regeneration, preferably employing the same enzyme that is used for the enantioselective reduction. In fact, biocatalysts that function in such systems have recently become available [24]. Whole cells of Rhodococcus ruber DSM 44541 reduce aliphatic and aromatic
Current Opinion in Biotechnology 2004, 15:272–279

ketones in excellent enantioselectvity [25], and the cells retain activity in the presence of 20% acetone or 50% 2propanol. An additional benefit is that the excess of reducing agent drives the reduction to completion. The responsible alcohol dehydrogenase has recently been isolated and tolerates even larger amounts of organic solvent in the water phase than lyophilized R. ruber cells [26]. Employing the latter, enantioselective ketone reductions with only one enzyme and substrate concentrations of up to 400 mM could be achieved [27].

Carbohydrates
N-Acetyl-D-neuraminic acid (NeuAc) and related compounds are potential pharmaceuticals and have been launched as neuraminidase inhibitors (e.g. Relenza from GSK; http://www.gsk.com/) for the treatment of influenza. Access to NeuAc is difficult and several biocatalytic approaches have been developed to produce this compound. All start from the easily available N-acetyl-Dglucosamine that is epimerized to N-actyl-D-mannosamine (ManNAc). There are two possible routes from ManNAc: an aldol condensation with pyruvate, catalyzed by NeuAc aldolase, or a NeuAc-synthetase-catalyzed reaction with phosphoenolpyruvate (PEP). The first route has been implemented [28], but the equilibrium of the epimerization as well as the aldol-condensation is unfavourable. The equilibrium problem can be solved by the degradation of pyruvate after synthesis to facilitate workup [29], making use of a novel process for cheap access to pyruvate by engineered E. coli cells [30]. By substituting the alkaline epimerization with an enzymatic step, using an epimerase from hog kidney, the reaction sequence could be carried out with high productivity [31]. Researchers from Kyowa Hakko (http://www.kyowa.co.jp/eng/)_have established a very elegant alternative approach by functionally expressing the epimerase gene in E. coli. In addition, they substituted the pyruvaterequiring aldolase for a PEP-requiring synthase. The latter reaction is thermodynamically more favourable [32]. The provision of PEP has been addressed by the use of perforated Corynebacterium ammoniagenes that converts glucose into PEP. Given the importance of complex carbohydrate structures for biological function [33], oligosaccharides have become desirable drug ingredients. An attractive route for their production is the assembly of oligosaccharides from their monosaccharide constituents, in analogy to the Leloirpathway. This method depends on the availability of both the bacterial glycosyltransferases and the activated forms of the monosaccharides that are used by the glycosyltransferases to sequentially build up the oligosaccharide structure. For example, Galb1-3GlcNAcb1-3Galb1-4Glc (lacto-N-tetraose; LNT) is of use for fighting Pseudomonas aeruginosa infections [34]. A suitable starting material for LNT is lactose to which GlcNAc can be added to obtain
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Innovations in industrial biocatalysis Panke, Held and Wubbolts 277

Figure 4

O

Improved Prunus amygdalus hydroxynitrile lyase

OH H+, ∆T CN

OH CO2H Cl (R)-2-Chloromandelic acid
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Cl

Cl (R)-2-Chloromandelonitrile ee 96.5%

Hydroxynitrile lyase is used for the production of (R)-2-chloromandelonitrile. The enzyme has been engineered for several improvements: the inclusion of a secretion signal, active-site modification and recombinant expression of only one of the almond tree (Prunus amygdalus) isoenzymes [45]. The recombinant enzyme is also stable at acidic pH.

GlcNAcb1-3Galb1-4Glc (lacto-N-triose II; LNTII) using b1-3GlcNAc transferase. In a second step, galactose (Gal) is added with b1-3Gal transferase to obtain LNT. The latter enzyme has recently been cloned from Streptococcus agalactiae [35], while the former is available from Pasteurella multocida [36]. Combined with Kyowa Hakko’s technology, which provides access to UDP-Gal [37] and UDPGlcNAc [38], a route from lactose to LNT and then to Galb1-3GlcNAc is now available.

Manipulations of complex natural products
Biocatalysis is widely used for the manipulation of complex natural products, such as antibiotics and steroids. Over the past couple of years, this approach has been extended to a broader variety of natural products. The most prominent example is the manufacturing process for the anticancer drug Taxol (Paclitaxel), which in nature is a low yield secondary plant metabolite (<0.1% in T. brevifolia bark) [39]). To improve the supply of the key intermediate 10-deacylbaccatin, different taxanes were used as precursors together with a set of hydrolytic enzymes [40]. A similar approach has also been used with epothilones, complex macrolide structures that were isolated from the myxobacterium Sorangium cellulosum and which also have a role as anti-tumor agents [41]. Microorganisms have been isolated that selectively hydroxylate particular positions of the complex molecule, which is particularly important as unselective modification at carbon atoms C1 to C8 would lead to rapid inactivation [42]. The hydroxylation of epothilone at C21 has attracted commercial interest and a novel epothilone B C21-amino derivative has been synthesized [43]. This compound has entered phase I clinical trials for the treatment of advanced solid malignancies. Other selective biohydroxylations involve the methylenes at C9 and C14 [43] as well as the terminal C26 [44].

insecticides and 2-hydroxy acids such as mandelic acid derivatives. The production of ortho-substituted aromatic cyanohydrins, such as (R)-2-chloromandelic acid, is typically hampered by low turnover frequencies and/or low ee values. Furthermore, cyanohydrin formation from aldehydes typically requires a low pH to address the limited stability of cyanohydrins at physiological pH. These problems were approached in a step-by-step manner (Figure 4). First, a promising (R)-hydroxynitrile lyase isoenzyme from Prunus amygdalus (PaHNL) was efficiently overexpressed in Pichia pastoris and was engineered to contain a secretion signal. The limited activity of the enzyme was addressed by rational mutagenesis, based on the three-dimensional structure, resulting in a variant that was sixfold improved for the reaction in question relative to the wild-type enzyme. In this way, the production of (R)-2-chloromandelonitrile could be carried out in excellent yield and with significantly improved ee values (96.5%) [45].

Conclusions
Biocatalysis and other forms of biotransformation provide access to (predominantly chiral) fine chemicals and are increasingly being applied in industry. Trends that can be observed are the continued use of hydrolases, where novel enzymes are now accessible as a result of highthroughput screening methods and genomics. In addition, there is an increase in the industrial application of oxidoreductases, especially for ketone reductions, amino transferases and carbon–carbon bond forming enzymes.

References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.  Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Stuermer R, Zelinski T: Industrial methods for the production of optically active intermediates. Angew Chem Int Ed Engl 2004, 43:788-824. Excellent overview of current methods — both chemical and biocatalysis — applied in industry for the synthesis of enantiomerically pure compounds. Current Opinion in Biotechnology 2004, 15:272–279

Cyanohydrins
Hydroxynitrile lyases are versatile enzymes for the synthesis of both (R)- and (S)-cyanohydrins, which are important starting compounds for the production of pyrethroid
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278 Protein technologies and commercial enzymes

2. 

Li T, Kootstra AB, Fotheringham IG: Nonproteinogenic a-amino acid preparation using equilibrium shifted transamination. Org Process Res Dev 2002, 6:533-538. Elegant combination of an a- and d-aminotransferase to produce L-a-amino acids in high yield. 3. May O, Verseck S, Bommarius A, Drauz K: Development of dynamic kinetic resolution processes for biocatalytic production of natural and nonnatural L-amino acids. Org Process Res Dev 2002, 6:452-457. Trauthwein H, May O, Dingerdissen U, Buchholz S, Drauz K: A new biocatalytic route to enantiopure N-carbamoyl amino acids by fast enzyme screening. Tetrahedron Lett 2003, 44:3737-3739.

19. Burk M, Desantis G, Morgan B, Zhu Z: Manufacture of (R)-ethyl 4-cyano-3-hydroxybutyric acid from 3-hydroxyglutaronitrile using nitrilases. 2002, Patent WO2003106415A2. 20. Abokitse K, Hummel W: Cloning, sequence analysis, and heterologous expression of the gene encoding a (S)-specific alcohol dehydrogenase from Rhodococcus erythropolis DSM 43297. Appl Microbiol Biotechnol 2003, 62:380-386. 21. Hummel W, Abokitse K, Drauz K, Rollmann C, Groger H: Towards a large-scale asymmetric reduction process with isolated enzymes: expression of an (S)-alcohol dehydrogenase in E. coli and studies on the synthetic potential of this biocatalyst. Adv Synthesis Catalysis 2003, 345:153-159. 22. Slusarczyk H, Felber S, Kula MR, Pohl M: Stabilization of NADdependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues. Eur J Biochem 2000, 267:1280-1289. ¨ 23. Groger H, Hummel W, Rollmann C, Chamouleau F, Husken H, ¨  Werner H, Groger H, Wunderlich C, Abokitse K, Drauz K, Buchholz S: Preparative asymmetric reduction of ketones in a biphasic medium with an (S)-alcohol dehydrogenase under in situ cofactor-recycling with a formate dehydrogenase. Tetrahedron 2004, 60:633-640. Reports the use of a two-liquid phase process for enantioselecive ketone reduction to reduce substrate mass-transfer limitations. 24. Stampfer W, Kosjek B, Moitzi C, Kroutil W, Faber K: Biocatalytic  asymmetric hydrogen transfer. Angew Chem Int Ed Engl 2002, 41:1014-1017. An elegant system for manufacturing (S)-alcohols. One enzyme is responsible for both reduction and cofactor regeneration. The hydrogen donor can be added in large quantitites to the aqueous phase, driving the reaction to completion and modifying the polarity of the medium at the same time. 25. Stampfer W, Kosjek B, Faber K, Kroutil W: Biocatalytic asymmetric hydrogen transfer employing Rhodococcus ruber DSM 44541. J Org Chem 2003, 68:402-406. 26. Kosjek B, Stampfer W, Pogorevc M, Goessler W, Faber K, Kroutil W: Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions. Biotechnol Bioeng 2004, 86:55-62. 27. Stampfer W, Edegger K, Kosjek B, Faber K, Kroutil W: Simple biocatalytic access to enantiopure (S)-1-heteroarylethanols employing a microbial hydrogen transfer reaction. Adv Synthesis Catalysis 2004, 346:57-62. 28. Mahmoudian M, Noble D, Drake CS, Middleton RF, Montgomery DS, Piercey JE, Ramlakhan D, Todd M, Dawson MJ: An efficient process for the production of N-acetylneuraminic acid using N-acetylneuraminic aldolase. Enzyme Microb Technol 1997, 20:393-400. 29. Wong C-H: Method for synthesizing 2-ketoaldonic acids. 1998, Patent US 5.759.825. 30. Zelic B, Gerharz T, Bott MC, Vasic-Racki D, Wandrey C, Takors R: Fed-batch process for pyruvate production by recombinant Escherichia coli YYC202 strain. Eng Life Sci 2003, 3:299-305. 31. Kragl U, Gygax D, Ghisalba O, Wandrey C: Enzymatic two-step synthesis of N-acetylneuraminic acid in the enzyme membrane reactor. Angew Chem Int Ed Engl 1991, 30:827-828. 32. Tabata K, Koizumi S, Endo T, Ozaki A: Production of N-acetyl-Dneuraminic acid by coupling bacteria expressing N-acetyl-Dglucosamine 2-epimerase and N-acetyl-D-neuraminic acid synthetase. Enzyme Microb Technol 2002, 30:327-333. 33. Koeller KM, Wong CH: Emerging themes in medicinal glycoscience. Nat Biotechnol 2000, 18:835-841. 34. Ramphal R, Carnoy C, Fievre S, Michalski JC, Houdret N, Lamblin G, Strecker G, Roussel P: Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Galb1-3GlcNAc) or type 2 (Galb1-4GlcNAc) disaccharide units. Infect Immun 1991, 59:700-704. 35. Watanabe M, Miyake K, Yanae K, Kataoka Y, Koizumi S, Endo T, Ozaki A, Iijima S: Molecular characterization of a novel b1,3galactosyltransferase for capsular polysaccharide synthesis www.sciencedirect.com

4.

5. 

Maier THP: Semisynthetic production of unnatural L-a-amino acids by metabolic engineering of the cysteine-biosynthetic pathway. Nat Biotechnol 2003, 21:422-427. Breakthrough methodology for the manufacture of a variety of unnatural (S)-a-amino acids by fermentation/biotransformation based on the cysteine biosynthesis pathway. 6. Sonke T, Kaptein B, Wagner AFV, Quaedflieg PJLM, Schultz S, Ernste S, Schepers A, Mommers JHM, Broxterman QB: Peptide deformylase as biocatalyst for the synthesis of enantiomerically pure amino acid derivatives. J Mol Catal, B Enzym 2004, 29:265-277. Preiml M, Hillmayer K, Klempier N: A new approach to b-amino acids: biotransformation of N-protected b-amino nitriles. Tetrahedron Lett 2003, 44:5057-5059. Balkenhohl F, Ditrich K, Hauer B, Ladner W: Optically active amines via lipase-catalyzed methoxyacetylation. Chemiker-Zeitung 1997, 339:381-384. Matuschek M, Stuermer R, Hauer B, Klebe G, Bocola M: Lipase variants, their production with recombinant cells, and their use in alcohol or amine acylation and ester hydrolysis. 2002, Patent EP11620[2003035878].

7.

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Current Opinion in Biotechnology 2004, 15:272–279

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