Selection of microbial biocatalysts for the reduction of cyclic and heterocyclic ketones

The reduction of carbonyl compounds plays an important role in the synthesis of complex chiral molecules. In particular, enantiopure substituted cyclic and heterocyclic compounds are useful intermediates for the synthesis of several antiviral, antitumor, and antibiotic agents, and recently, they have also been used as organocatalysts for C-C addition. Alcohol dehydrogenases (ADH) are enzymes involved in the transformation of prochiral ketones to chiral hydroxyl compounds. While significant scientific effort has been paid to the use of aliphatic and exocyclic ketones as ADH substrates, reports on (hetero)cyclic carbonyl compounds as substrates of these enzymes are scarce. In the present study, 109 bacteria and 36 fungi were screened, resulting in 10 organisms belonging to both kingdoms capable of transforming cyclic and heterocyclic ketones into the corresponding alcohols. Among them, Erwinia chrysanthemi could quantitatively reduce cyclododecanone and Geotrichum candidum could stereoselectively reduce N-Boc-3-piperidone and N-Boc-3-pyrrolidinone to their corresponding (S)-alcohols; however, the anti-Prelog isomer was obtained when acetophenone was the substrate.     

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4′-chloroacetophenone, the K_m value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4′-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The V_max value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD⁺ or NADP⁺ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity.     

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.     

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost‐effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co‐immobilized them on modified poly‐p‐hydroxybutyrate synthase (PhaC)‐inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled‐coil interaction. Enzyme‐loaded particles could be easily purified by centrifugation. Total conversion of 4'‐chloroacetophenone to (S)‐4‐chloro‐α‐methylbenzyl alcohol could be accomplished using enzyme‐loaded particles, catalytic amounts of NAD⁺ and formate as substrates for FDH. Chiral GC‐MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost‐effective alternative to coupled reactions using purified enzymes.     

Bioreduction of prochiral ketones by growing cells of Lasiodiplodia theobromae: Discovery of a versatile biocatalyst for asymmetric synthesis

Growing cells of the phytopathogen fungus Lasiodiplodia theobromae in potato dextrose broth have shown their potential for the stereoselective bioreduction of different prochiral aromatic and aliphatic ketones. Optically active alcohols were obtained under mild reaction conditions in high conversions (up to 90%) and moderate to excellent enantioselectivity (35–≥99% ee) depending on the ketone structure. Prelog alcohols were isolated, except in the bioreduction of cyclohexylmethylketone and octan-2-one where anti-Prelog alcohols were obtained.     

Improved synthesis of chiral alcohols with <Emphasis Type="Italic">Escherichia coli</Emphasis> cells co-expressing pyridine nucleotide transhydrogenase,NADP<Superscript>+</Superscript>-dependent alcohol dehydrogenase and NAD<Superscript>+</Superscript>-dependent formate dehydrogenase

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD⁺ and NADPH. The pnta and pntb genes encoding for the - and -subunits were cloned and co-expressed with NADP⁺-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.     

Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes

Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols.     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase–ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E·NAD⁺) and three ternary (E·NAD⁺·acetone, E·NAD⁺·3-pentanone and E·NAD⁺·cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.     

Production of chiral alcohols by enantioselective reduction with NADH-dependent phenylacetaldehyde reductase from Corynebacterium strain,ST-10

Phenylacetaldehyde reductase (PAR) (systematic name, 2-phenylethanol: NAD⁺ oxidoreductase) isolated from styrene-assimilating Corynebacterium strain ST-10 was used to produce chiral alcohols. This enzyme with a broad substrate range reduced various prochiral 2-alkanones and aromatic ketones to yield optically active secondary alcohols with an enantiomeric purity of 87–100% enantiomeric excess (e.e.). The stereochemistry of PAR revealed that the pro-R hydrogen of NADH was transferred to carbonyl moiety of acetophenone derivatives or alkanones through its re face. The combination with a NADH-regenerating system using formate dehydrogenase and formate was able to practically produce optically pure alcohols.     

Green and scalable synthesis of chiral aromatic alcohols through an efficient biocatalytic system

Chiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium-chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2-hydroxyacetophenone (2-HAP) to (R)-(-)-1-phenyl-1,2-ethanediol ((R)-PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg⁻¹ and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme-coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two-phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l⁻¹). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale.     

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)–water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K _m and V _max values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA–water medium.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

Purification and characterization of a novel carbonyl reductase involved in oxidoreduction of aromatic β-amino ketones/alcohols

Aromatic β-amino ketones/alcohols such as adrenalone play an important role in some stereoselective synthesis of pharmaceuticals. Unfortunately, the transformation of aromatic β-amino ketones to their chiral alcohols has been carried out chemically as no corresponding biocatalyst has been available. Here, a novel carbonyl reductase responsible for the reduction of adrenalone to (R)-(−)-epinephrine was identified and characterized from Kocuria rhizophila. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by ion-exchange column chromatography, hydrophobic chromatography and gel chromatography. The purified enzyme yielded pure (R)-enantiomer product with high activity and utilized NADH as the cofactor. The enzyme had special significance by showing selectivity for many aromatic β-amino ketones/alcohols such as 2-amino-acetophenone, 2-amino-4′-hydroxyacetophenone, isoproterenol and ephedrine. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for adrenalone and NADH were 14.62 μmol/(min mg) protein and 0.189 mM, 11.66 μmol/(min mg) protein and 0.204 mM respectively. These properties ensure the enzyme a promising future for industrial application as a replacement of chemical synthesis of aromatic β-amino chiral alcohols.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Enzymatic preparation of optically pure t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca

Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe³⁺ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals.     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.     

Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell-free system

Alcohol dehydrogenase (ADH) and amine dehydrogenase (AmDH)-catalyzed one-pot cascade conversion of an alcohol to an amine provides a simple preparation of chiral amines. To enhance the cofactor recycling in this reaction, we report a new concept of coupling whole-cells with the cell-free system to enable separated intracellular and extracellular cofactor regeneration and recycling. This was demonstrated by the respective biotransformation of racemic 4-phenyl-2-butanol 1a and 1-phenyl-2-propanol 1b to (R)-4-phenylbutan-2-amine 3a and (R)-1-phenylpropan-2-amine 3b . Escherichia coli cells expressing S-enantioselective CpsADH, R-enantioselective PfODH, and NADH oxidase (NOX) was developed to oxidize racemic alcohols 1a–b to ketones 2a–b with full conversion via intracellular NAD⁺ recycling. AmDH and glucose dehydrogenase (GDH) were used to convert ketones 2a–b to amines (R)- 3a–b with 89–94% conversion and 891–943 times recycling of NADH. Combining the cells and enzymes for the cascade transformation of racemic alcohols 1a–b gave 70% and 48% conversion to the amines (R)- 3a and (R)-3 b in 99% ee, with a total turnover number (TTN) of 350 and 240 for NADH recycling, respectively. Improved results were obtained by using the E. coli cells with immobilized AmDH and GDH: (R)- 3a was produced in 99% ee with 71–84% conversion and a TTN of 1410-1260 for NADH recycling, the highest value so far for the ADH–AmDH-catalyzed cascade conversion of alcohols to amines. The concept might be generally applicable to this type of reactions.     

Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP(+). Metal analyses revealed that this NADP(+)-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K(m) values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP(+), respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.     

Superabsorbed alcohol dehydrogenase—a new catalyst for asymmetric reductions

A new immobilisate of alcohol dehydrogenase (ADH) is described in which all components for the reaction, i.e. enzyme, the coenzyme NADP⁺, the buffer and other cofactors (trace elements), are immobilized together. It is an all-inclusive catalyst. The support is a cheap, commercially-available, superabsorbent polymer. The immobilisation is easy to achieve. The superabsorbed ADH is, even when dried, a stable and storable catalyst for at least five weeks at −18°C. Asymmetric reductions of the prochiral ketones, acetophenone, 4-acetylpyridine and ethyl acetoacetate, with a superabsorbed ADH from Lactobacillus brevis (ADH 002) and a superabsorbed ADH from Thermoanaerobicum sp. (ADH 005) in 2-propanol as both the organic solvent and the cofactor-regenerating substrate are given. Yields of chiral (R) and (S)-alcohols from 97–100% were achieved within 18 to 48 h with enantiomeric excesses of >99%. The superabsorbed ADH was easily separated by filtration and could be reused at least four times.