A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.     

Impact modification of lactic acid based poly(ester-urethanes) by blending

Branched biodegradable poly(ester-urethane)(PEU) was blended with two elastic biodegradable copolymers in proportions 5, 10, 15, and 20 wt % to investigate their effect on this hard and brittle polymer. Copolymer of L-lactide and ϵ-caprolactone, P(L-LA50/CL50), was synthesized by ring-opening polymerization and the other elastic poly(L-lactic acid-co-ϵ-caprolactone)urethane, P(LA50/CL50)U, was prepared by direct polycondensation of L-lactic acid and ϵ-caprolactone, followed with urethane bonding. In addition, four elastic biodegradable copolymers, three of them P(L-LA/CL) and one P(LA/CL)U, were blended with linear PEU to investigate their modifying effect on PEU. These compositions studied were 10, 15, and 20 wt % of P(L-LA40/CL60), P(L-LA60/CL40), P(L-LA80/CL20), and P(LA40/CL60)U in PEU. Blending was done in a batch mixer. PEU became more ductile when blended with P(L-LA/CL) and P(LA/CL)U, and its impact resistance improved markedly. In general, an addition of 15 wt % of copolymer appeared to give the most desirable mechanical properties. Moreover, the more L-lactide in the P(L-LA/CL) copolymer, the better was the miscibility of the blends, as shown by dynamic mechanical thermal analysis (DMTA) and scanning electron microscopy (SEM). One P(L-LA/CL) was also blended with poly(DL-lactide) (PDLLA) to see if the dispersion of rubbery copolymer particles was the same in PDLLA and PEU. A well-known commercial nonbiodegradable rubber [styrene/ethylene/butylene copolymer (SEBS)] was blended with linear PEU to compare its effect on impact strength. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 1335–1343, 1997     

Screening,Molecular Cloning,and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β‐Hydroxy‐β‐trifluoromethyl Ketone

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 β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐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 β‐hydroxy‐β‐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 β‐hydroxy‐β‐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 β‐hydroxy‐β‐trifluoromethyl ketone.     

Convergent Cascade Catalyzed by Monooxygenase–Alcohol Dehydrogenase Fusion Applied in Organic Media

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.     

High Regio- and Stereoselective Multi-enzymatic Synthesis of All Phenylpropanolamine Stereoisomers from β-Methylstyrene

We present a one-pot cascade for the synthesis of phenylpropanolamines (PPAs) in high optical purities (er and dr up to >99.5 %) and analytical yields (up to 95 %) by using 1-phenylpropane-1,2-diols as key intermediates. This bioamination entails the combination of an alcohol dehydrogenase (ADH), an ω-transaminase (ωTA) and an alanine dehydrogenase to create a redox-neutral network, which harnesses the exquisite and complementary regio- and stereo-selectivities of the selected ADHs and ωTAs. The requisite 1-phenylpropane-1,2-diol intermediates were obtained from trans- or cis-β-methylstyrene by combining a styrene monooxygenase with epoxide hydrolases. Furthermore, in selected cases, the envisioned cascade enabled to obtain the structural isomer (1S,2R)-1-amino-1-phenylpropan-2-ol in high optical purity (er and dr >99.5 %). This is the first report on an enzymatic method that enables to obtain all of the four possible PPA stereoisomers in great enantio- and diastereo-selectivity.     

Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO_Acineto); this used NADH 4200‐fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen‐bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.     

Artificial Fusions between P450 BM3 and an Alcohol Dehydrogenase for Efficient (+)-Nootkatone Production

Multi-enzyme cascades enable the production of valuable chemical compounds, and fusion of the enzymes that catalyze these reactions can improve the reaction outcome. In this work, P450 BM3 from Bacillus megaterium and an alcohol dehydrogenase from Sphingomonas yanoikuyae were fused to bifunctional constructs to enable cofactor regeneration and improve the in vitro two-step oxidation of (+)-valencene to (+)-nootkatone. An up to 1.5-fold increased activity of P450 BM3 was achieved with the fusion constructs compared to the individual enzyme. Conversion of (+)-valencene coupled to cofactor regeneration and performed in the presence of the solubilizing agent cyclodextrin resulted in up to 1080 mg L⁻¹ (+)-nootkatone produced by the fusion constructs as opposed to 620 mg L⁻¹ produced by a mixture of the separate enzymes. Thus, a two-step (+)-valencene oxidation was considerably improved through the simple method of enzyme fusion.     

Direct Access to Medium‐Chain α,ω‐Dicarboxylic Acids by Using a Baeyer–Villiger Monooxygenase of Abnormal Regioselectivity

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium‐chain α,ω‐dicarboxylic acids through a Baeyer–Villiger oxidation–hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long‐chain aliphatic keto acids (C₁₆–C₂₀) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω‐dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω‐dicarboxylic acids from renewable oils and fats.     

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor

(R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called “focused rational iterative site-specific mutagenesis” (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.     

Cascade Synthesis from Cyclohexane to ϵ-Caprolactone by Visible-Light-Driven Photocatalysis Combined with Whole-Cell Biological Oxidation

Cyclohexane was directly oxy-functionalised to ϵ-caprolactone through a cascade reaction sequence combining visible-light-driven photocatalysis with cyclohexanone monooxygenase (CHMO) whole-cell biocatalysis. Two available photocatalysts, Au-doped TiO₂ (Au-TiO₂) and graphitic carbonitride (g-C₃N₄), were evaluated in the experiment and some optimisations to the cascade reaction were applied. In stepwise mode, the highest degree of conversion from cyclohexanol to ϵ-caprolactone can be up to 41 %, with use of g-C₃N₄. The cascade reaction from cyclohexane to ϵ-caprolactone is achievable under a light intensity of 149 μW cm⁻².     

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.     

Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products (“normal” and “abnormal”) when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k_cat/K_M) 8.9-fold to 484 s⁻¹ mM⁻¹ for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO_C308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L⁻¹ d⁻¹. This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.     

Inhibition of horse liver and yeast alcohol dehydrogenase by aromatic and aliphatic aldehydes

The reactivity of yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) towards 12 different aldehydes was tested. YADH was inhibited by pre-incubation with citral, citronellal, p-cuminaldehyde, p-anisaldehyde, m-tolualdehyde, trans-cinnamaldehyde, salicylaldehyde, p-hydroxybenzaldehyde and benzaldehyde, although of these aldehydes only trans-cinnamaldehyde acted as a substrate for the enzyme. HLADH was inhibited, to a much smaller extent, by pre-incubation with citral, salicylaldehyde, citronellal, p-anisaldehyde, piperonaldehyde, trans-cinnamaldehyde, p-hydroxybenzaldehyde and p-cuminaldehyde and all of these aldehydes acted as substrates for the HLADH. Immobilisation of the enzymes on CNBr-activated Sepharose 4B gave protection against inhibition by the aldehydes, suggesting a means of significantly extending the useful lifetime of the enzymes when they are used in industrial processes.     

Prevention of Swelling Phenomenon of Alginate Beads To Improve the Stability and Recyclability of Encapsulated Horse Liver Alcohol Dehydrogenase

Horse Liver Alcohol Dehydrogenase (HLADH) has been immobilized on calcium-alginate beads and used for both oxidation and reduction reactions. To avoid swelling of the beads and their subsequent breakage, calcium ions were added to both reaction and storage solutions, allowing the beads to maintain the initial structural features. The techniques used for this purpose revealed that 2 mM Ca²⁺ is the optimal concentration, which does not significantly change the weight of the beads, the amount of water in them, and their external and internal structure. The optimized experimental procedure has been used to verify the properties of the enzyme in terms of reusability, storage, and thermal stability. The addition of calcium ions allows the enzyme to retain more than 80 % of its initial activity for fourteen cycles and approximately 50 % at the twentieth cycle. Moreover, when the biocatalyst has been stored in a buffer solution containing 2 mM Ca²⁺, the retention of enzyme activity after 30 days was 100 %, compared to that measured before incubation. The encapsulated enzyme exhibits greater thermal stability than free HLADH up to at least 60 °C, preventing dimer dissociation into the two subunits.     

Chemo‐ and Stereodivergent Preparation of Terminal Epoxides and Bromohydrins through One‐Pot Biocatalysed Reactions: Access to Enantiopure Five‐ and Six‐Membered N‐Heterocycles

Different enantiopure terminal epoxides or bromohydrins have chemoselectively been synthesised in one‐pot starting from the corresponding α‐bromo ketones through alcohol dehydrogenase (ADH)‐catalysed processes adding an organic co‐solvent and tuning appropriately the medium pH and the temperature. Thus, at neutral pH enantiopure bromohydrins were obtained while using basic conditions (pH 9.5–10) epoxides were isolated as the main product. Furthermore, by simple selection of the biocatalyst, chemo‐ and stereodivergent transformations were achieved to obtain, e.g., enantiopure prolinol or piperidin‐3‐ol.     

Evolution of a Transition State: Role of Lys100 in the Active Site of Isocitrate Dehydrogenase

An active site lysine essential to catalysis in isocitrate dehydrogenase (IDH) is absent from related enzymes. As all family members catalyze the same oxidative β‐decarboxylation at the (2R)‐malate core common to their substrates, it seems odd that an amino acid essential to one is not found in all. Ordinarily, hydride transfer to a nicotinamide C4 neutralizes the positive charge at N1 directly. In IDH, the negatively charged C4‐carboxylate of isocitrate stabilizes the ground state positive charge on the adjacent nicotinamide N1, opposing hydride transfer. The critical lysine is poised to stabilize—and perhaps even protonate—an oxyanion formed on the nicotinamide 3‐carboxamide, thereby enabling the hydride to be transferred while the positive charge at N1 is maintained. IDH might catalyze the same overall reaction as other family members, but dehydrogenation proceeds through a distinct, though related, transition state. Partial activation of lysine mutants by K⁺ and NH₄⁺ represents a throwback to the primordial state of the first promiscuous substrate family member.     

Modeling Alcohol Dehydrogenase Catalysis in Deep Eutectic Solvent/Water Mixtures

The use of oxidoreductases (EC1) in non-conventional reaction media has been increasingly explored. In particular, deep eutectic solvents (DESs) have emerged as a novel class of solvents. Herein, an in-depth study of bioreduction with an alcohol dehydrogenase (ADH) in the DES glyceline is presented. The activity and stability of ADH in mixtures of glyceline/water with varying water contents were measured. Furthermore, the thermodynamic water activity and viscosity of mixtures of glyceline/water have been determined. For a better understanding of the observations, molecular dynamics simulations were performed to quantify the molecular flexibility, hydration layer, and intraprotein hydrogen bonds of ADH. The behavior of the enzyme in DESs follows the classic dependence of water activity (a_W) in non-conventional media. At low a_W values (<0.2), ADH does not show any activity; at higher a_W values, the activity was still lower than that in pure water due to the high viscosities of the DES. These findings could be further explained by increased enzyme flexibility with increasing water content.     

Characterization of four diol dehydrogenases for enantioselective synthesis of chiral vicinal diols

Enantiopure vicinal diols are important building blocks used in the synthesis of fine chemicals and pharmaceutical compounds. Diol dehydrogenase (DDH) mediated stereoselective oxidation of racemic vicinal is an efficient way to prepare enantiopure vicinal diols. In this study, four new bacterial DDHs (AnDDH from Anoxybacillus sp. P3H1B, HcDDH from Hazenella coriacea, GzDDH from Geobacillus zalihae and LwDDH from Leptotrichia wadei) were mined from the GenBank database and expressed in E. coli T7. The four DDHs were purified and biochemically characterized for oxidation activity toward (R)-1-phenyl-1,2-ethanediol, with the optimal reaction condition of pH9.0 (AnDDH), 10.0 (HcDDH) and 11.0 (GzDDH and LwDDH) and the temperatures at 40℃ (AnDDH), 50℃ (HcDDH) and 60℃ (GzDDH and LwDDH), respectively. The four enzymes were stable at the pH from 7.0 to 9.0 and below 40℃. Kinetic parameters of four DDHs showed that the HcDDH from Hazenella coriacea had high activity toward a broad range of vicinal diols. A series of racemic vicinal diols were successfully resolved by recombinant E. coli (HcDDH-NOX) resting cells co-expression of an NADH oxidase (NOX), affording (S)-diols and (1S, 2S)-trans-diols in ≥ 99% ee. The synthetic potential of HcDDH was proved by E. coli (HcDDH-NOX) via kinetic resolution of racemic trans-1,2-indandiol on a 100 ml scale reaction, (S, S)-trans-1,2-indandiol was prepared in 46.7% yield and >99% ee. In addition, asymmetric reduction of four α-hydroxy ketones (10-300 mmol·L^-1) by E. coli (HcDDH-GDH) resting cells resulted in >99% ee and 69-98% yields of (R)-vicinal diols. The current research expands the toolbox of DDHs to synthesize chiral vicinal diols and demonstrated that the mined HcDDH is a potential enzyme in the synthesis of a broad range of chiral vicinal diols.     

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5–5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.     

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

Gluconacetobacter diazotrophicus is a N₂-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.