The yeast alcohol dehydrogenase catalysed conversion of cinnamaldehyde to cinnamyl alcohol

The kinetics of the conversion of trans-cinnamaldehyde to trans-cinnamyl alcohol catalysed by yeast alcohol dehydrogenase (EC 1.1.1.1) have been characterised. Reaction with the free enzyme is curtailed after a short time as a result of inactivation by the substrate. It has been shown that immobilisation of the enzyme provides substantial protection against this inactivation. Use of the immobilised enzyme in a flow-through reactor further enhanced the enzyme lifetime, creating a viable synthetic process. The product cinnamyl alcohol may be recovered by continuous solvent extraction.     

KINETIC STUDIES ON OXIDATION OF ALLYL ALCOHOL BY YEAST ALCOHOL DEHYDROGENASE

The kinetics of the oxidation of allyl alcohol by yeast alcohol dehydrogenase (YADH) was studied with stopped-flow technique. The experimental results showed that the specific activity of YADH remained constant within theYADH concentration range ofO-16.4mg/dm₃ and YADH exhibited maximum activity at pH 7.9 and a phosphate buffer concentration of 0.25 mol/dm₃ The oxidation rate of allyl alcohol by YADH followed the Arrhenius equation within the temperature range of 10 to 40°C The YADH was denatured irreversibly at the temperatures above 40°C. The rate equation for the YADH-catalyzed oxidation of allyl alcohol was obtained. A mixed-type substrate inhibition occurred when the concentration of allyl alcohol was higher than 200 mmol/dm₃ The presence of LiCl, NaCl and KG could enhance the activity of YADH. The YADH lost activity significantly in the presence of organic solvents, particularly benzene and its derivatives.     

An Alcohol Dehydrogenase from the Short-Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane-1,6-diol

Biocatalytic production of lactones, and in particular ϵ-caprolactone (CL), have gained increasing interest as a greener route to polymer building blocks, especially through the use of Baeyer–Villiger monooxygenases (BVMOs). Despite several advances in the field, BVMOs, however, still suffer several practical limitations. Alcohol dehydrogenase (ADH)-mediated lactonization of diols in turn has received far less attention and very few enzymes have been identified for the conversion of diols to lactones, with horse-liver ADH (HLADH) remaining the catalyst of choice. Screening of a diverse panel of ADHs, AaSDR-1, a member of the short-chain dehydrogenase/reductase family, was found to produce ϵ-caprolactone from hexane-1,6-diol. Moreover, cofactor regeneration by an NADH oxidase eliminated the requirement of co-substrates, yielding water as the sole by-product. Despite lower turnover frequencies as compared to HLADH, higher selectivity was found for the production of CL, with HLADH forming significant amounts of 6-hydroxyhexanoic acid and adipic acid through aldehyde dehydrogenation/oxidation of the gem-diol intermediates. Also, CL yield were shown to be dependent on buffer choice, as structural elucidation of a Tris adduct confirmed the buffer amine to react with aliphatic aldehydes forming a Schiff-base intermediate which through further ADH oxidation, forms a tricyclic acetal product.     

Kinetic behavior of yeast alcohol dehydrogenase in AOT/isooctane reverse micelles

The kinetic behavior of yeast alcohol dehydrogenase (YADH) in sodium di-2-ethylhexylsulfosuccinate (AOT) isooctane reverse micelles has been studied using methyl ethyl ketone (MEK), NADH and Tris as the substrate, coenzyme and buffer, respectively. The solubility diagrams of aqueous buffer in the isooctane solution of AOT were established as a function of temperature and molar ratio of water of surfactant (ω₀) at various Tris and AOT concentrations. The dependence of enzyme activity on enzyme concentration, pH, ω₀ and Tris concentration was determined. The optimal ω₀ was at 10–15, increasing slightly with an increase in Tris concentration. The YADH entrapped in reverse micelles exhibited minimum activity at a Tris concentration of 0·1 mol dm^?3, while in aqueous buffer enzyme activity was not significantly affected by Tris concentration. Comparing the rate equation of the reduction of MEK by YADH in reverse micelles with that in aqueous buffer, the association of YADH and NADH could apparently have proceeded with an irreversible reaction before the substrate was bound, when performed in a reverse micellar system. Although the YADH entrapped in reverse micelles was less stable than when dissolved in aqueous buffer, the enzyme retained at least 20% activity after 21 h at 25°C and ω₀ = 20. This result was an improvement over previously reported data.     

Immobilization of yeast alcohol dehydrogenase by entrapment and covalent binding to polymeric supports

Yeast alcohol dehydrogenase (YADH), which catalyzes oxidoreductions of a broad spectrum of substrates, was immobilized by entrapping it into a network of a poly(acrylamide‐co‐hydroxyethyl methacrylate) copolymer and was also covalently bound onto porous chitosan beads activated through glutaraldehyde. Maximum retention of YADH activity achieved was 90 and 24% for entrapment and covalent binding, respectively. The results obtained for thermal, storage, and operational stability of entrapped and covalently bound YADH were compared with free YADH. The immobilized enzyme showed improved thermal and storage stability. The immobilized enzymes also retained 50% activity after six and eight cycles. Enzyme‐catalyzed oxidation of ethanol was observed to be diffusion‐controlled through Lineweaver–Burk plots. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1299–1305, 2001     

Antignawing Factor Derived from Cinnamomum cassia Bark Against Mice

The antignawing activity of cinnamomi cortex (the dried bark of Cinnamomum cassia) derived materials against laboratory-reared mice was evaluated using a wire-dipping method. The biologically active component of C. cassia bark was characterized by spectral analysis as cinnamaldehyde. The antignawing activity of the compound was compared with that of four commercially available compounds: cinnamyl alcohol, trans-cinnamic acid, eugenol, and salicylaldehyde. Cinnamaldehyde and cinnamyl alcohol exhibited potent repellent activity, whereas little or no activity was produced from salicylaldehyde. Moderate activity was observed in trans-cinnamic acid and eugenol. Cinnamaldehyde and cinnamyl alcohol exhibited potent and moderate repellent activity at 5 and 2.5% solutions, respectively, of these compounds in ethanol. Little or no activity was observed in 1% solution. As a naturally occurring repellent, cinnamaldehyde and cinnamyl alcohol could be useful as a new preventive agent against various kinds of damage caused by rodents.     

Density‐Dependent Formation of the Pure trans‐Isomer of the Unsaturated Alcohol by Selective Hydrogenation of Citral in Supercritical Carbon Dioxide

Selective hydrogenation of citral to unsaturated alcohol [geraniol (trans) + nerol (cis)] was carried out in supercritical carbon dioxide (scCO₂) using an MCM‐41 supported plantinum catalyst (∼1 wt% Pt). A remarkable rate of isomerization of the unsaturated alcohol [nerol (cis) to geraniol (trans)] during the hydrogenation of citral was achieved simply by tuning the density of CO₂. Optimum reaction conditions were developed to obtain only geraniol (trans) with a selectivity of 98.8% and citral conversion of 99.8%. A significant change in the cis:trans ratio of the product (1:82.3) from the substrate (1:1.3) was observed depending on the various reaction parameters like carbon dioxide and hydrogen pressure, reactant concentration, reaction time and, particularly, the total selectivity for unsaturated alcohol [geraniol (trans) +nerol (cis)]. It has been observed that the presence of hydrogen is necessary for isomerization. Our results were explained in terms of a density‐dependent, two‐step model. The kinetic behaviour shows that the rate of isomerization was higher in scCO₂ compared to other organic solvents and the pure form of geraniol (trans) was obtained exclusively. A probable reaction pathway was proposed in order to explain the isomerization during hydrogenation of citral in scCO₂ medium.     

亲和反胶团选择性萃取分离酵母乙醇脱氢酶

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH>pI with electrostatic attraction between YADH and CTAB. Th     

Antimicrobial Effect of α- or β-Cyclodextrin Complexes with Trans-Cinnamaldehyde Against Staphylococcus aureus and Escherichia coli

Molecular inclusion or encapsulation based on cyclodextrin (CD) is the most widely used method in encapsulation technology. α- and β-CD complexes consisting of antibacterial trans-cinnamaldehyde were prepared by the molecular inclusion method. The characteristics of the complex were determined, including examination of their antimicrobial effects after the drying process. The particle sizes of the trans-cinnamaldehyde–CD complexes were observed in the range of 300 to 500 nm after production. Particle sizes of both trans-cinnamaldehyde–CD complexes were slightly increased increasing in a dose-dependent manner of trans-cinnamaldehyde. Although β-CD complexes were larger than α-CD complexes, they possessed a lower polydispersity index with a narrow size distribution. The encapsulation efficiency of trans-cinnamaldehyde–CD complexes was >90% in all formulations. Trans-cinnamaldehyde affected reduction of Staphylococcus aureus and Escherichia coli, with antibacterial activity increasing in a dose-dependent manner of trans-cinnamaldehyde concentration. In addition, β-CD complexes showed more effective antimicrobial effects compared to α-CD complexes.     

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.     

Cinnamyl derivatives and monoterpenoids as nonspecific ovipositional deterrents of the onion fly

Laboratory dose-response choice tests and discriminate-dosage bioassays revealed wide variation in the effectiveness of cinnamyl, cinnamoyl, monoterpene, and phenethyl alcohol derivatives as ovipositional deterrents toDelia antiqua (Meigen), the onion fly. (E)-Cinnamic acids were not detectably deterrent. When formulated in particles of polyethylene glycol, (E)-cinnamaldehyde had a BR₉₀ (concentration eliciting 90% deterrency) of 1.0% and (E)-4-methoxycinnamaldehyde had a BR₉₀ of 0.38%. Among nine monoterpenoids tested,p-cymene was inactive, citronellal had a BR₉₀ of 3.7%, and terpinene-4-ol had a BR₉₀ of 0.46%. Para-substituted phenethyl alcohols gave increasing deterrence in the order: –NO₂, CH₃O–, –Cl, –CH₃, –H. Wide varieties of structures were deterrent: C-8 to C-13, intermediate in polarity, and possessing either oxygen-containing or nitrile functional groups. The air concentration of (E)-cinnamaldehyde at its BR₉₀ was 1.7 ng/ml. This relatively high concentration, the diversity in deterrent structures, and the lack of differences in deterrency among positional and optical isomers suggest that ovipositional deterrency in onion flies is mediated by receptors broadly tuned for detecting phenylpropenoid, phenolic, monoterpenoid, and perhaps other classes of allelochemicals.     

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.     

Influence of Ru precursor,support and solvent in the hydrogenation of citral over ruthenium catalysts

A series of ruthenium supported catalysts were prepared and investigated in the liquid-phase hydrogenation of citral. The mechanism of the reaction was found to be dependent on the Ru precursor, support and solvent used. On the sample prepared from RuCl₃ the acetals of citronellal were formed with the subsequent hydrogenation of the isolated C=C double bond. On the samples obtained from precursors which do not contain chloride ions, citronellal was the main reaction product. Hydrogenation of the isolated C=C double bond was negligible. Using cyclohexane as solvent, a large amount of isopulegol was also obtained. The results of the hydrogenation of citral have been correlated to the presence of acid sites and incompletely reduced ruthenium which favour cyclization and acetals formation from citronellal. On the basis of the reported results, a reaction scheme for hydrogenation of citral over ruthenium catalysts is proposed.     

Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Directed evolution of stereo‐ or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building “small but smart” mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple‐code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino‐acid alphabets. By using the synthetically difficult‐to‐reduce prochiral ketone tetrahydrofuran‐3‐one as a substrate, highly R‐ and S‐selective variants were obtained (92–99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi–Dunitz trajectory.     

Highly Efficient Hydrogenation of Cinnamaldehyde Catalyzed by Pt-MCM-48 in Supercritical Carbon Dioxide

In supercritical carbon dioxide medium, a platinum-containing cubic mesoporous molecular sieve was shown to promote markedly the selective hydrogenation of trans-cinnamaldehyde, giving an excellent selectivity for cinnamyl alcohol. In contrast to the other platinum-containing conventional catalysts, Pt-MCM-48 can be recycled a number of times without showing any deactivation in the studied reaction condition. The selectivity mainly depends on the pressure of carbon dioxide, while the conversion depends on the pressure of carbon dioxide as well as hydrogen pressure.     

Enantioselective Reduction of Citral Isomers in NCR Ene Reductase: Analysis of an Active‐Site Mutant Library

A deeper understanding of the >99 % S‐selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active‐site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)‐citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)‐citral it remained ≥88 % (S) for all single‐residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A‐based double‐mutant library that enhanced the (E)‐citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)‐citral to 44 % (S). Formation of (R)‐citronellal from an (E/Z)‐citral mixture is a desire in industrial (?)‐menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.     

From Sophorose Lipids to Natural Product Synthesis

Sophorose lipids (SL) are a readily available, scalable source for the disaccharide sophorose: In a four‐step sequence the crude product from the fermentation broth was converted to key disaccharide donors 7 that were further utilized in the synthesis of natural products Ebracteatoside C, Zizybeoside I and phenethyl glycoside. While the aglycones for Zizybeoside I and the phenethyl glycoside are commercially available simple primary alcohols, the synthesis of Ebrateatoside C required a secondary allylic alcohol (R)‐6‐hydroxyoct‐7‐enyl acetate. The enantiomerically pure secondary alcohol was synthesized in two steps using an alcohol dehydrogenase‐catalysed reduction as the key step.     

Preparation and catalysis of amorphous CoNiB and polymer-stabilized CoNiB catalysts for hydrogenation of unsaturated aldehydes

CoNiB and polymer polyvinylpyrrolidone (PVP)-stabilized CoNiB (PVP-CoNiB) catalysts were prepared by chemical reduction with NaBH₄. They were characterized and examined for their catalysis in the hydrogenation of unsaturated aldehydes. CoNiB and PVP-CoNiB catalysts were characterized by X-ray diffraction as amorphous structures and by transmission electron microscopy as having particle sizes in the range 3.5–7 nm – smaller than those of NiB (7–15 nm) and CoB (5–10 nm). CoNiB catalysts were significantly more active than NiB and CoB in the hydrogenations of furfural, crotonaldehyde and citral, and the PVP-CoNiB catalysts were significantly more active than CoNiB. The catalytic properties of CoNiB and PVP-CoNiB catalysts during the selective hydrogenations of crotonaldehyde and citral were similar to those of NiB, but different from those of CoB. The conjugated CC bonds in crotonaldehyde and citral were preferentially reduced to form butyraldehyde and citronellal. PVP-CoNiB could hydrogenate citral to citronellal at a low reaction temperature of 30 °C.     

Platinum/3,3´-thiodipropionic acid nanoparticles as recyclable catalysts for the selective hydrogenation of trans-cinnamaldehyde

Platinum nanoparticles as spherical aggregates were prepared by the reduction of a Pt(II) salt with hydrazine using 3,3´-thiodipropionic acid as a protective agent, and characterized by IR, XRF, XRD, and SEM where agglomerates have been visualized. The average crystallite size was 6 nm. For the first time such nanoparticles were evaluated as catalysts in the hydrogenation of unsaturated aldehydes. Hydrogenation of trans-cinnamaldehyde yielded cinnamyl alcohol with a selectivity of up to 83% at complete substrate conversion. At 30 °C, the catalyst could be recycled and reused for three runs with only slight losses in activity and selectivity.     

Negative effect of [bmim][PF6] on the catalytic activity of alcohol dehydrogenase: mechanism and prevention

BACKGROUND: [bmim][PF₆] is a hydrophobic ionic liquid which could be considered as an environmentally friendly solvent for biocatalysis. In pure [bmim][PF₆], however, alcohol dehydrogenase from yeast (YADH) has no catalytic activity. The aim of the present work was (1) to quantitatively study the negative effect of [bmim][PF₆] on the catalytic activity of YADH and the related mechanism and (2) to made an attempt to lessen the negative effect of [bmim][PF₆] on YADH by microemulsifying [bmim][PF₆]. RESULTS: The activity of YADH in the homogeneous solution formed by H₂O, CH₃CH₂OH and [bmim][PF₆] decreased rapidly with the increase of the molar fraction of [bmim][PF₆]. The inhibitory effect of [bmim][PF₆] on YADH was probably caused by the competition of the imidazole group of [bmim][PF₆] with the coenzyme NAD⁺ for the binding sites on YADH. In a water‐in‐[bmim][PF₆] microemulsion, YADH was catalytically active due to the formation of the interfacial membrane of the nonionic surfactant TritonX‐100, which separated YADH from [bmim][PF₆] and avoided the direct inactivation of [bmim][PF₆] on YADH. Under optimal conditions, the activity of YADH was as high as 51 µmol L^?1 min^?1. CONCLUSION: [bmim][PF₆] was an inhibitor of YADH and its negative effect on YADH could be lessened by its microemulsification. Copyright © 2008 Society of Chemical Industry