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Alcohol dehydrogenases catalyse the conversion of a large variety of ketone substrates to the corresponding chiral products. Due to their high regio- and stereospecificity, they are key components in a wide range of industrial applications. A novel alcohol dehydrogenase from Comamonas testosteroni (CtADH) was identified in silico, recombinantly expressed and purified, enzymatically and biochemically investigated as well as structurally characterized. These studies revealed a broad pH profile and an extended substrate spectrum with the highest activity for compounds containing halogens as substituents and a moderate activity for bulky–bulky ketones. Biotransformations with selected ketones—performed with a coupled regeneration system for the co-substrate NADPH—resulted in conversions of more than 99% with all tested substrates and with excellent enantioselectivity for the corresponding S-alcohol products. CtADH/NADPH/substrate complexes modelled on the basis of crystal structures of CtADH and its closest homologue suggested preliminary hints to rationalize the enzyme’s substrate preferences

A suitable "process window" was identified for the combination of an asymmetric organocatalytic aldol reaction and subsequent biocatalytic reduction in aqueous medium, which thus enabled the enantio- and diastereoselective synthesis of 1,3-diols in a tandem-type, one-pot process. A key feature of this one-pot synthesis is the high 500 mM loading of the aldehyde substrate used as a starting material.

Whole-cell catalysts overexpressing two enzymes for a double reduction cascade in which aliphatic alpha-branched alpha,beta-unsaturated aldehydes are converted into Guerbet alcohols as a highly demanded class of lubricants were constructed and characterized. The reaction conditions were optimized to suppress by-product formation, achieving high conversion and selectivity. Application of methanol as a co-solvent on a 500 mL scale enabled a substrate concentration of 18.5 g L-1 obtaining 97% yield and 88% purity without further distillation. Addressing the needs of industrial processes, substrate loading was increased up to 94 g L-1 and the E-factor (environmental factor) was significantly improved compared to the original process with isolated enzymes.

For an alternative synthetic approach toward the heterocyclic industrial chemical e-caprolactone, which is based on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydrogenase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out identifying isooctane as a suitable co-solvent. Biotransformations in an aqueous-isooctane biphasic solvent system were found to proceed faster at both investigated substrate concentrations of 40 and 80 mm, respectively, compared with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high compatibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on the enzymes used. In contrast, so far, the use of recombinant whole-cells gave less satisfactory results, which might be due to limitations of the permeation of, for example, the substrate through the cell membrane.

The stereoselective synthesis of chiral 1,3-diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of beta-hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a beta-hydroxy-beta-trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2-trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the beta-hydroxy-beta-trifluoromethyl ketone was identified after purification and subsequent MALDI-TOF mass spectrometric analysis. As a result, a new NADP(+) -dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the beta-hydroxy-beta-trifluoromethyl ketone to its corresponding 1,3-diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N- or C-terminal His-tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N-terminal His-tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the beta-hydroxy-beta-trifluoromethyl ketone. 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel synthetic approach towards Guerbet alcohols, which are important intermediates in the production of plasticizers, lubricants, and surfactants, was developed. In contrast to the harsh reaction conditions of Guerbet alcohols produced today, which include high temperatures, the new developed process runs at room temperature. The key feature of this alternative process is the combination of organocatalytic and enzymatic steps towards a chemoenzymatic synthesis. In detail, the piperidinyloxyl-catalyzed oxidation of 1-hexanol by using hypochlorite and the lysine-catalyzed homoaldol condensation of the resulting aldehyde were combined with two subsequent enzymatic reductions of the C=C and C=O bonds of the in situ formed 2-branched α,β-unsaturated aldehyde by means of an ene reductase from Gluconobacter oxydans and an alcohol dehydrogenase from Rhodococcus sp. under in situ cofactor regeneration. The desired 2-branched aliphatic primary alcohol was obtained with high conversion and selectivity and without the need for intermediate purifications.

In this contribution, we report the chemoenzymatic preparation of a key building block for the active pharmaceutical ingredient rosuvastatin, one of the "top 5 blockbuster drugs" with a worldwide market value of 6.25 billion USD in 2012, via a seven-step synthesis without isolation of intermediates and with incorporation of two highly efficient biotransformations. This chemoenzymatic process reaches excellent space-time yields by using high substrate concentrations (several hundred grams per liter), emphasizing the potential of biocatalysis for industrial processes related to pharmaceutical drug synthesis and the compatibility of enzyme chemistry with classical organic synthesis.

Tailor-made strategies for the stereo- and regioselective multi-step enzymatic synthesis of glycoconjugates require well characterized glycosyltransferases and carbohydrate modifying enzymes. We here report on a novel enzyme cascade for the synthesis of uridine 5'-diphospho-alpha-D-glucuronic acid (UDP-GlcA) and the non-sulfated human natural killer cell-1 (HNK-1) epitope including in situ regeneration of UDP-GlcA and the cofactor nicotinamide adenine dinucleotide NAD(+) by the combination of four enzymes in one-pot. In the first enzyme module sucrose synthase 1 (SuSy1) is used to produce uridine 5'-diphospho-alpha-D-glucose (UDP-Glc) from sucrose and uridine 5'-diphosphate (UDP). The combination with UDP-Glc dehydrogenase in the second enzyme module leads to the synthesis of UDP-GlcA with concomitant in situ regeneration of the cofactor NAD(+) by nicotinamide adenine dinucleotide hydride (NADH)-oxidase. In the third enzyme module the mammalian glucuronyltransferase GlcAT-P catalyzes the synthesis of the non-sulfated HNK-1 epitope by regioselective transfer of GlcA onto N-acetyllactosamine type 2 (LacNAc type 2). We present a comprehensive study on substrate kinetics, substrate specificities, variation and relation of enzyme activities as well as cross inhibition of intermediate products. With optimized reaction conditions we obtain superior product yields with streamlined synthesis costs for the expensive nucleotide sugar UDP-GlcA and cofactor NAD(+).

In this study an evaluation of the synthetic potential of a broad range of recombinant ene reductases was performed. In detail, a library of 23 ene reductases was used to screen the C=C reduction of 21 activated alkenes from different compound classes as substrates. The chosen set of substrates comprises nitroalkenes with an aryl substituent at the beta-position and a methyl substituent at the alpha- or beta-position, alpha,beta-unsaturated carboxylic acids and their esters with and without substituents at the beta-position, a range of cyclic alpha,beta-unsaturated ketones with different ring sizes and substitution patterns and one alpha,beta-unsaturated boronic acid. After we obtained insight into the substrate scope, several biotransformations were prioritised and further investigated in a screening of 41 reaction parameters (which included chaotropic and kosmotropic salts, polyols, buffer solutions, amino acids and organic solvents) towards their impact on the activity and enantioselectivity of the applied ene reductases. Under the optimised conditions, selected reduction processes were performed on an increased lab scale (up to 30 mL) with up to 10% substrate concentration, which led in general to both high conversion and (if chiral products were formed) enantioselectivity.

A Wacker oxidation using CuCl/PdCl2 as a catalyst system was successfully combined with an enzymatic ketone reduction to convert styrene enantioselectively into 1-phenylethanol in a one-pot process, although the two reactions conducted in aqueous media are not compatible due to enzyme deactivation by Cu ions. The one-pot feasibility was achieved via compartmentalization of the reactions. Conducting the Wacker oxidation in the interior of a polydimethylsiloxane thimble enables diffusion of only the organic substrate and product into the exterior where the biotransformation takes place. Thus, the Cu ions detrimental to the enzyme are withheld from the reaction media of the biotransformation. In this one-pot process, which formally corresponds to an asymmetric hydration of alkenes, a range of 1-arylethanols were formed with high conversions and 98-99 % ee. In addition, the catalyst system of the Wacker oxidation was recycled 15 times without significant decrease in conversion.

Eine Wacker-Oxidation unter Verwendung von CuCl/PdCl2 als Katalysatorsystem wurde erfolgreich mit einer enzymatischen Ketonreduktion zur enantioselektiven Umwandlung von Styrol in 1-Phenylethanol in einem Eintopf-Verfahren kombiniert, obwohl beide im wassrigen Medium verlaufende Reaktionen aufgrund der Enzymdeaktivierung durch Cu-Ionen nicht miteinander kompatibel sind. Die Machbarkeit als Eintopf-Verfahren wurde durch Kompartimentierung der beiden Reaktionen erreicht. Die Durchfuhrung der Wacker-Oxidation im inneren Bereich einer Polydimethylsiloxan-Hulse erlaubt nur die Diffusion des organischen Substrats und Produkts in den Ausenbereich, in dem die Biotransformation stattfindet. Somit werden die fur das Enzym nachteiligen Cu-Ionen vom Reaktionsmedium der Biotransformation ferngehalten. In diesem Eintopf-Verfahren, welches formal einer asymmetrischen Hydrierung von Alkenen entspricht, werden eine Reihe von 1-Arylethanolen mit hohen Umsatzen und 98–99 % ee gebildet. Zudem wurde das Katalysatorsystem fur die Wacker-Oxidation 15-mal wiederverwendet, ohne signifikanten Ruckgang an Umsatz.

In industriellem Masstab wird Poly-e-caprolacton (PCL) gegenwartig nur chemisch produziert, wobei mit Peressigsaure ein gefahrliches Reagens als Oxidationsmittel genutzt wird. Baeyer-Villiger-Monooxygenasen (BVMOs) ermoglichen im Prinzip die enzymatische Synthese von e-Caprolacton (e-CL) direkt ausgehend von Cyclohexanon mit molekularem Sauerstoff, doch gegenwartige Systeme leiden unter niedriger Produktivitat sowie Substrat- und Produktinhibierung. Wir uberwanden wesentliche Limitationen eines solchen biokatalytischen Wegs durch die Kombination einer Alkoholdehydrogenase mit einer BVMO fur die effiziente Oxidation von Cyclohexanol zu e-CL. Entscheidend war die direkte Ringoffnungs-Oligomerisierung des in situ gebildeten e-CL in wassriger Phase unter Nutzung der Lipase A aus Candida antarctica. So wurde das Problem der Produktinhibierung gelost, und Oligo-e-CL wurde mit >20 g L−1 ausgehend von 200 mM Cyclohexanol erhalten. Dieses Oligomer konnte chemisch leicht zu PCL polymerisiert werden.

In this contribution the use of an artificial in situ-cofactor regeneration with [Fe(III)TSPPICI for enzymatic amino acid oxidation, exemplified for the L-glutamate dehydrogenase-catalyzed synthesis of a-ketoglutarate from sodium L-glutamate, is reported. In comparison of two L-glutamate dehydrogenases, the one isolated from Clostridium difficile turned out to be the preferred enzyme. At a substrate concentration of 15 mM of L-glutamate in situ-cofactor regeneration using [Fe(III)TSPP]CI as an "artificial NADH-oxidase" proceeded smoothly, leading to up to >99% overall conversion and 88% conversion related to the formation of alpha-ketoglutarate after 24h. At an increased concentration of 50 mM of L-glutamate, a somewhat decreased conversion of 43% was observed (which, however, corresponds to a nearly doubled volumetric productivity of 3.95 g/(Ld) compared to the experiments at 15 mM). Thus, the iron complex [Fe(III)TSPP]CI turned out to be capable to be used for cofactor regeneration of the cofactor NAD(+) for enzymatic amino acid oxidation. (C) 2014 Elsevier B.V. All rights reserved.

A novel type of organic synthesis enabling a direct one-pot transformation of α,β-unsaturated aldehydes into saturated carboxylic acids is described. As sole reagent water is required, which is integrated completely in the product. This tandem process proceeds under perfect atom economy, and consists of two coupled redox biotransformations without the need of external co-substrates for cofactor regeneration. The initial reduction of the CC double bond of an α,β-unsaturated aldehyde is catalyzed by an NADPH-dependent ene reductase, leading to the formation of the saturated aldehyde and NADP+. The aldehyde intermediate is then oxidized to the corresponding carboxylic acid, thus regenerating NADPH for the next catalytic cycle. When using prochiral α,β-unsaturated aldehydes as substrates the corresponding carboxylic acids are formed enantioselectively with up to >99 % ee as demonstrated, e.g., for the transformation of citral to (S)-citronellic acid.

A gene encoding a novel 7 alpha-specific NADP(+)-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60 degrees C, but the enzyme is inactivated at this temperature with a half-life time of 5 min. Contrary to other known 7 alpha-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP(+) using an NADPH oxidase that resulted in a complete conversion (

It doesn't take much to oxidize cycloalkanes directly to the corresponding cyclic ketones: molecular oxygen as the oxidant, water as the solvent, the cofactor NADP(+) (and a little 2-propanol to reduce it), as well as two catalytic enzymes-a hydroxylating P450 monooxygenase and an alcohol dehydrogenase. (Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-β-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.

A gene encoding a novel 7α-specific NADP

Spontaneous electron transport to molecular oxygen led to regeneration of oxidised nicotinamide cofactor in cell lysates that contain an alcohol dehydrogenase, a quinone reductase and a quinone mediator. This concept allows the efficient oxidation of alcohols in the presence of alcohol dehydrogenase-containing E. coli lysates and catalytic amounts of the quinone lawsone.

We report the combination of a palladium-catalyzed Suzuki cross-coupling reaction with an enzymatic reduction in a one-pot process in aqueous medium, in which both reaction steps are conducted at room temperature. By using a water-soluble palladium catalyst system, which is prepared from commercially available palladium chloride and TPPTS (tris(3-sulfonatophenyl)phosphine hydrate, sodium salt), the palladium-catalyzed step runs at room temperature in a mixture of isopropanol and water. After adjusting the pH value to pH 7, the resulting biaryl ketone is then directly reduced in situ via an enzymatic asymmetric reduction in the presence of an alcohol dehydrogenase (ADH), leading to the desired alcohol with up to >95% conversion (over two steps) and excellent enantioselectivities of >99% ee. Notably, both enantiomers of the desired alcohol are accessible depending on the source of applied ADH.

12-Keto-UDCA is an important optically active component for the drug ursodeoxycholic acid (UDCA). Starting from the three-keto compound dehydrocholic acid, the carbonyl groups at position 3 and 7 have to be reduced stereo- and regioselectively. In this case we applied two hydroxysteroid dehydrogenases for this purpose, the NAD-dependent 3α-HSDH from Pseudomonas testosteroni and the NADP-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Both enzymes can be produced in high yields by an Escherichia coli strain as recombinant proteins. In order to avoid impurities by the 7a-hydroxysteroid dehydrogenase of Escherichia coli, a mutant strain with an inactivated 7a-enzyme was applied for producing the three enzymes. For bioconversion, the dehydrogenases can be used as crude enzyme samples and are applied simultaneously. A 1.8 L batch of 100mM DHCA incubated at pH = 8.0 and 25°C resulted in 80 g crude product with a quite high purity of ≥ 99:5% as judged by HPLC analysis.

A direct one-pot conversion of (substituted) styrene(s) into the corresponding (substituted) (R)-phenylethan-1-ol(s) in a highly enantioselective manner has been achieved by using a "chemoenzymatic catalytic system", comprising a palladium-catalyzed Wacker-Tsuji oxidation and subsequent enantioselective enzymatic reduction of the in situ formed (substituted) acetophenone(s). Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A gene encoding an NADPH-dependent carbonyl reductase from Neurospora crassa ( nccr ) was cloned and heterologously expressed in Escherichia coli . The enzyme (NcCR) was purified and biochemically characterised. NcCR exhibited a restricted substrate spectrum towards various ketones, and the highest activity (468 U/mg) was observed with dihydroxyacetone. However, NcCR proved to be very selective in the reduction of different α- and β-keto esters. Several compounds were converted to the corresponding hydroxy ester in high enantiomeric excess ( ee ) at high conversion rates. The enantioselectivity of NcCR for the reduction of ethyl 4-chloro-3-oxobutanoate showed a strong dependence on temperature. This effect was studied in detail, revealing that the ee could be substantially increased by decreasing the temperature from 40 °C (78.8%) to −3 °C (98.0%). When the experimental conditions were optimised to improve the optical purity of the product, ( S )-4-chloro-3-hydroxybutanoate ( ee 98.0%) was successfully produced on a 300 mg (1.8 mmol) scale using NcCR at −3 °C.

The development of aldol reactions of glycine with substituted benzaldehydes in the presence of recombinant L-threonine aldolases from Escherichia coil or Saccharomyces cerevisiae, which were obtained with excellent overexpression data, has been carried out. When using glycine and ortho-chlorobenzaldehyde, a high conversion of >95%, an enantioselectivity of >99% ee, and a diastereoselectivity with d.r.(syn/anti) = 80:20 was obtained for the resulting beta-hydroxy alpha-amino acid in such a biotransformation. It should be noted that this enzymatic process can be conducted at an elevated substrate concentration of 250 mM. (C) 2011 Elsevier Ltd. All rights reserved.

The NADP-dependent glycerol dehydrogenase (EC 1.1.1.72) from Gluconobacter oxydans is a member of family 11 of the aldo-keto reductase (AKR) enzyme superfamily; according to the systematic nomenclature within the AKR superfamily, the term AKR11B4 has been assigned to the enzyme. AKR11B4 is a biotechnologically attractive enzyme because of its broad substrate spectrum, combined with its distinctive regioselectivity and stereoselectivity. These features can be partially rationalized based on a 2-Å crystal structure of apo-AKR11B4, which we describe and interpret here against the functional complex structures of other members of family 11 of the AKR superfamily. The structure of AKR11B4 shows the AKR-typical (β/α)(8) TIM-barrel fold, with three loops and the C-terminal tail determining the particular enzymatic properties. In comparison to AKR11B1 (its closest AKR relative), AKR11B4 has a relatively broad binding cleft for the cosubstrate NADP/NADPH. In the crystalline environment, it is completely blocked by the C-terminal segment of a neighboring protomer. The structure reveals a conspicuous tryptophan residue (Trp23) that has to adopt an unconventional and strained side-chain conformation to permit cosubstrate binding. We predict and confirm by site-directed mutagenesis that Trp23 is an accelerator of (co)substrate turnover. Furthermore, we show that, simultaneously, this tryptophan residue is a critical determinant for substrate binding by the enzyme, while enantioselectivity is probably governed by a methionine residue within the C-terminal tail. We present structural reasons for these notions based on ternary complex models of AKR11B4, NADP, and either octanal, d-glyceraldehyde, or l-glyceraldehyde.

A recombinant enoate reductase from Gluconobacter oxydans was heterologously expressed, purified, characterised and applied in the asymmetric reduction of activated alkenes. In addition to the determination of the kinetic properties, the major focus of this work was to utilise the enzyme in the biotransformation of different interesting compounds such as 3,5,5-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) and (E/Z)-3,7-dimethyl-2,6-octadienal (citral). The reaction proceeded with excellent stereoselectivities (>99% ee) as well as absolute chemo- and regioselectivity, only the activated C=C bond of citral was reduced by the enoate reductase, while non-activated C=C bond and carbonyl moiety remained untouched. The described strategy can be used for the production of enantiomerically pure building blocks, which are difficult to prepare by chemical means. In general, the results show that the investigated enoate reductase is a promising catalyst for the use in asymmetric C=C bond reductions.

Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon-carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of beta-hydroxy-alpha-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral beta-hydroxy-alpha-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a L: -threo-phenylserine derivative or L: -threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of L: -threonine or L: -allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of beta-hydroxy-alpha-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, co-expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6-fold increase in reaction rate caused by the addition of 1 mM NADP(+). Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole-cell catalyst was studied as well. Comparative studies between the whole-cell catalyst and the cell-free system were investigated. Furthermore, the successful application of the whole-cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole-cell system, which facilitated the access to L-glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole-cell catalyst shows several advantages over the cell-free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss-of-activity. The results obtained making the described whole-cell catalyst an improved catalyst for a more efficient production of enantiopure L-glyceraldehyde.

Direct enantioselective reduction of 2´-, 3´- and 4´ -hydroxyacetophenone without protection of the hydroxy moiety was carried out in the presence of (R)- and (S)-alcohol dehydrogenases as biocatalysts. Whereas reduction of 2´-hydroxyacetophenone gave only low to medium conversions, reduction of 3´- and 4´ -hydroxyacetophenone proceeded efficiently leading to the resulting 1-(3- hydroxyphenyl)ethan-1-ol and 1-(4-hydroxyphenyl)ethan-1-ol with high conversion (up to > 95 %) and excellent enantioselectivity (up to > 99% ee).

It was found that Delta(4,5)-steroid 5 beta-reductases are capable of reducing also small molecules bearing an activated C=C double bond such as monocyclic enones and acyclic enoate esters. As preferred Delta(4,5)-steroid 5 beta-reductase (5 beta-StR) for this purpose, 5 beta-StR from Arabidopsis thaliana was used. In part. enzyme activities are even higher than that for progesterone. Successful preliminary biotransformations with enzymatic in situ cofactor recycling were also carried out. When using the prochiral compound isophorone as a substrate, a high enantioselective reaction course (>99% ee) was observed.

The acetic acid bacterium Gluconobacter oxydans has a high potential for oxidoreductases with a variety of different catalytic abilities. One putative oxidoreductase gene codes for an enzyme with a high similarity to the NADP+-dependent glycerol dehydrogenase (GlyDH) from Hypocrea jecorina. Due to this homology, the GlyDH (Gox1615) has been cloned, over-expressed in Escherichia coli, purified and characterised. Gox1615 shows an apparent native molecular mass of 39 kDa, which corresponds well to the mass of 37.213 kDa calculated from the primary structure. From HPLC measurements, a monomeric structure can be deduced. Kinetic parameters and the dependence of the activity on temperature and pH were determined. The enzyme shows a broad substrate spectrum in the reduction of different aliphatic, branched and aromatic aldehydes. Additionally, the enzyme has been shown to oxidize a variety of different alcohols. The highest activities were observed for the conversion of D-glyceraldehyde in the reductive and L-arabitol in the oxidative direction. Since high enantioselectivities were observed for the reduction of glyceraldehyde, the kinetic resolution of glyceraldehyde was investigated and found to yield enantiopure L-glyceraldehyde on preparative scale.

The R-specific alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure-based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb-ADH. Mutations were introduced into Lb-ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb-ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.

The reduction of methyl acetoacetate was carried out in continuously operated biotransformation processes catalyzed by recombinant Escherichia coli cells expressing an alcohol dehydrogenase from Lactobacillus brevis . Three different cell types were applied as biocatalysts in three different cofactor regeneration approaches. Both processes with enzyme-coupled cofactor regeneration catalyzed by formate dehydrogenase or glucose dehydrogenase are characterized by a rapid deactivation of the biocatalyst. By contrast the processes with substrate-coupled cofactor regeneration by alcohol dehydrogenase catalyzed oxidation of 2-propanol could be run over a period of 7 weeks with exceedingly high substrate and cosubstrate concentrations of up to 2.5 and 2.8 mol L −1 , respectively. Even under these extreme conditions, the applied biocatalyst showed a good stability with only marginal leakage of intracellular cofactors.

A one-pot two-step process for the enantioselective synthesis of hydrophobic allylic alcohols was developed, which comprises ketone formation by the Wittig reaction and their enzymatic in situ biotransformation into the desired target products. By means of this combined Wittig reaction and bioreduction, the allylic alcohols were prepared with conversions of up to 90 %, and with excellent enantio selectivities of >99 % ee. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007).

A Wacker oxidation using CuCl/PdCl2 as the catalyst system is successfully combined with enzymatic ketone reduction to convert styrene enantioselectively into 1-phenylethanol in a one-pot process, although the two reactions conducted in aqueous media are not compatible due to enzyme deactivation by Cu ions.

White Biotechnology uses microorganisms and enzymes to manufacture a large variety of chemical products. Therefore, the demand for new and useful biocatalysts is steadily and rapidly increasing. We have developed methods for the isolation of new enzyme genes, constructed novel expression systems, and optimized existing enzymes for biotechnological applications by methods of directed evolution. Furthermore, we have isolated and characterized biocatalysts relevant for the preparation of enantiopure compounds.

Die Weise Biotechnologie verwendet Mikroorganismen und Enzyme, um chemische Produkte herzustellen. Daher nimmt der Bedarf an neuen Enzymen als Biokatalysatoren fur die Biotechnologie-Industrie stetig und schnell zu. Vorgestellt wird die Isolierung neuer Enzymgene, die Konstruktion von Expressionssystemen und Bakterienstammen, aber auch die Optimierung bereits vorhandener Enzyme mit Methoden der gerichteten Evolution. Daneben werden biotechnologisch relevante Enzymklassen untersucht, die zur Darstellung enantiomerenreiner Verbindungen verwendet werden konnen.

Lactobacillus kefir DSM 20587 produces an (R)-specific NADP-dependent alcohol dehydrogenase (ADH) with a broad substrate specificity. The gene of this ADH was isolated and the complete nucleotide sequence determined. The adh gene comprises 759 bp and encodes a protein of 252 amino acids with a calculated molecular weight of 26 781 Da. The deduced amino acid sequence indicated a high degree of similarity to short-chain dehydrogenases. After cloning and expression in Escherichia coli the enzyme was purified and characterized. For the reduction of acetophenone the specific activity of the homogeneous recombinant ADH was 558 U mg−1. The enzyme shows its maximum activity at 50°C while the pH optimum was at pH 7.0. In order to demonstrate its preparative application, purified ADH was used for the stereoselective reduction of several aliphatic and aromatic ketones as well as β-keto esters. Glucose dehydrogenase was added for the regeneration of NADPH. All prochiral ketones were stereoselectively reduced to the c...