Andrea Weckbecker

Cloning, expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir

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 corresponding alcohols with >99% ee and in the case of diketones >99% de.

Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent alcohol dehydrogenase and NAD+-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 α- and β-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 12 h, 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.

Glucose Dehydrogenase for the Regeneration of NADPH and NADH

Glucose dehydrogenases (GDHs) occur in several organisms such as Bacillus megaterium and Bacillus subtilis. They accept both NAD+ and NADP+ as cofactor and can be used for the regeneration of NADH and NADPH. In order to demonstrate their applicability we coupled an NADP+-dependent, (R)-specific alcohol dehydrogenase (ADH) from Lactobacillus kefir with the glucose dehydrogenase from B. subtilis. The ADH reduces prochiral ketones stereoselectively to chiral alcohols. The reduction requires NADPH, which was regenerated by the glucose dehydrogenase. Glucose dehydrogenase from B. subtilis (EC 1.1.1.47) is a tetramer with a molecular weight of 126,000. The enzyme shows a pH optimum at 8.0 and a broad temperature optimum at 45–50°C. We investigated the conversion of acetophenone in a cell-free system with purified ADH and GDH. Furthermore, we constructed two plasmids containing the genes encoding ADH and GDH by inserting them one after the other. These two plasmids differ from each other in the order of the genes. Because of the low solubility of the compounds, we examined the reaction in a water/organic solvent two-phase system.

Bioreductions Catalyzed by an Alcohol Dehydrogenase in Non‐aqueous Media

Highly productive biocatalytic reductions were established using an isolated alcohol dehydrogenase (ADH) under water‐deficient conditions. First, a solvent‐free system was evaluated for the reduction of 2‐butanone catalyzed by ADH evo‐1.1.200 promoted by the “smart cosubstrate” 1,4‐butanediol. ADH evo‐1.1.200 excelled by its activity and stability under high reagent concentrations and hence was the enzyme of choice. However, conversion of 2‐butanone was limited to <1 % in 10 days under the solvent‐free conditions. Therefore, water‐immiscible organic solvents were evaluated whereby the highest conversions were achieved in MTBE and toluene. MTBE was chosen as its different boiling point compared to other reaction components (e.g., 2‐butanone, 2‐butanol, diol cosubstrate, and lactone coproduct) would simplify the downstream processing. Further on, by tuning substrate loading, the productivity of the ADH evo‐1.1.200 was successfully increased to a turnover number (TON) of 64 000.

A High‐Throughput Screening Method for Chiral Alcohols and its Application to Determine Enantioselectivity of Lipases and Esterases

Chiral alcohols are valuable intermediates in the synthesis of pharmaceutical, agricultural, and fine chemicals, which can be produced either by hydrocarbon oxidation, ketone reduction, or ester hydrolysis. Nevertheless, these reactions usually produce non-enantiopure compounds. For this reason, several methods for the enantioselective synthesis of alcohols have been developed, which range from the synthesis of catalysts by combinatorial chemistry to the in vitro directed evolution of enzymes. In any case, high-throughput methods need to be applied to measure the enantiomeric excess (ee) or enantiopurity of the produced alcohols within a large number of samples. Several methods for high-throughput screening for enantioselectivity of catalysts have been reported, including electrospray ionization coupled to mass spectrometry, HPLC coupled to circular dichroism, FTIR spectroscopy, and enzymatic methods. Some of these sophisticated methods require, however, isotopically labeled pseudo-enantiomers for the assay and occasionally expensive equipment. Herein, we report a new colorimetric method for the evaluation of the ee values of alcohols based on enantioselective alcohol dehydrogenases (ADHs) coupled to a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (diaphorase) and its successful application in directed evolution for the screening of mutant libraries of lipases for enantioselective ester hydrolysis. The assay is based on the enantioselective oxidation of alcohols by two different ADHs assayed separately in parallel assays: the (R)-specific ADH from Lactobacillus kefir (LKADH) and the (S)-specific ADH from Rhodococcus erythropolis (READH), of which enantioselectivities and catalytic properties have been reported. The oxidation of either (R)-1 or (S)-1 produces NAD(P)H, which is again oxidized to NAD(P) by diaphorase from Clostridium kluyveri with the concomitant reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) 3 to its corresponding red formazan derivative 4 (Scheme 1). The formation of this dye can be easily followed at 492 nm. The reaction is carried out within five minutes, during which the slope of color development over time is linear. The regeneration of the oxidized form of the coenzyme also ensures high reaction rates of ADHs. As in any other coupled assay, the amount of diaphorase, the coupling enzyme, was kept in excess relative to the ADH enzymes in order to follow first-order kinetics. Previous studies using one or two enzymes to measure ee values have been reported and their accuracy was well demonstrated. Berkowitz et al. coupled (S)and (R)-ADHs with the Co-salen-catalyzed hydrolytic kinetic resolution of epoxides, whereas the group of Turner coupled Ror S-selective alcohol oxidases with the enzymatic reduction of ketones to determine the enantioselectivity of ketoreductases. The mathematical framework for the analysis of these data has been developed based on the Michaelis–Menten equation. Nevertheless, enzymes may display different kinetic behaviors in the presence of diverse compounds. In our study, neither LKADH nor READH display hyperbolic initial-rate curves. Instead, both enzymes displayed sigmoidally shaped kinetic curves (Figure 1). Scheme 1. Enantioselective reactions catalyzed by READH and LKADH and its coupling to the diaphorase redox system. Oxidation of either alcohol (S)-1 or (R)-1 correspondingly produces a molecule of coenzyme, NADH or NADPH, which in turn are oxidized by diaphorase through the reduction of 3 to produce the corresponding formazan red dye 4.