Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes and alcohols and determination of lignin O-methyltransferase activities in dicot and monocot species.

To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes. The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols. Relative O-methyltransferase activities against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medicago sativa), and crude extracts from the model legume Medicago truncatula, tobacco, wheat and tall fescue. Extracts from all these species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxycinnamic acids.     

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.     

Optimization of process variables for minimization of byproduct formation during fermentation of blackstrap molasses to ethanol at industrial scale

Aims: To investigate the effect of molasses concentration, initial pH of molasses medium, and inoculum’s size to maximize ethanol and minimize methanol, fusel alcohols, acetic acid and aldehydes in the fermentation mash in industrial fermentors. Methods and Results: Initial studies to optimize temperature, nitrogen source, phosphorous source, sulfur supplement and minerals were performed. The essential nutrients were urea (2 kg in 60 m³), 0·5 l each of commercial phosphoric acid and sulfuric acid (for pH control) added at the inoculum preparation stage only. Yields of ethanol, methanol, fusel alcohols, total acids and aldehydes per 100‐l fermentation broth were monitored. Molasses at 29°Brix (degree of dissolved sugars in water), initial pH 4·5, inoculum size 30% (v/v) and anaerobic fermentation supported maximum ethanol (7·8%) with Y_P/S = 238 l ethanol per tonne molasses (96·5% yield) (8·2% increase in yield), and had significantly lower values of byproducts than those in control experiments. Conclusions: Optimization of process variables resulted in higher ethanol yield (8·2%) and reduced yield of methanol, fusel alcohols, acids and aldehydes. Significance and Impact of the Study: More than 5% substrate is converted into byproducts. Eliminating or reducing their formation can increase ethanol yield by Saccharomyces cerevisiae, decrease the overall cost of fermentation process and improve the quality of ethanol.     

Formation of volatile alcohols and esters from aldehydes in strawberries

The aldehydes acetaldehyde, propanal, 2-methylpropanal, butanal, 3-methylbutanal, pentanal and hexanal, were reduced to their corresponding alcohols by incubation with strawberry fruit. The alcohols formed were then converted to their acetate, propionate, n-butyrate, isovalerate and n-caproate esters during the incubation with strawberry fruit. Simultaneous reaction of isobutyric acid, n-valeric acid and isocaproic acid with aldehyde and strawberry fruit resulted in the formation of esters of these acids. In all seven alcohols and 54 esters were produced by means of incubation of aldehydes and volatile fatty acids with strawberry fruit.     

Detoxification of promutagenic aldehydes derived from methylpyrenes by human aldehyde dehydrogenases ALDH2 and ALDH3A1

Methylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulpho conjugation to reactive esters, which can induce mutations and tumours. Yet, further oxidation of the alcohol may compete with this toxification. We previously demonstrated that several human alcohol dehydrogenases (ADH1C, 2, 3 and 4) oxidise various benzylic alcohols (derived from alkylated pyrenes) to their aldehydes with high catalytic efficiency. However, all these ADHs also catalysed the reverse reaction, the reduction of the aldehydes to the alcohols, with comparable or higher efficiency. Thus, final detoxification requires elimination of the aldehydes by further biotransformation. We have expressed two human aldehyde dehydrogenases (ALDH2 and 3A1) in bacteria. All pyrene aldehydes studied (1-, 2- and 4-formylpyrene, 1-formyl-6-methylpyrene and 1-formyl-8-methylpyrene) were high-affinity substrates for ALDH2 (K_m = 0.027–0.9 μM) as well as ALDH3A1 (K_m = 0.78–11 μM). Catalytic efficiencies (k_cat/K_m) were higher for ALDH2 than ALDH3A1 by a moderate to a very large margin depending on the substrate. Most important, they were also substantially higher than the catalytic efficiencies of the various ADHs for the reduction the aldehydes to the alcohols. These kinetic properties ensure that ALDHs, and particularly ALDH2, can complete the ADH-mediated detoxification.     

Ripening‐induced acceleration of volatile aldehyde generation following tissue disruption in tomato fruit

Hexanal and cis-3-hexenal are principal flavor volatiles in ripe tomato fruit, but whether they accumulate during ripening or are formed upon maceration of the tissue has not been clarified. This has been addressed by measuring levels of these aldehydes in green and ripe fruit with discrimination between intrinsic aldehyde content and aldehyde generation following tissue disruption. Volatile sampling of tomato fruit homogenates was accomplished by purge/trapping, followed by thermal desorption on a gas chromatograph equipped with a mass selective detector. Incubation of some samples with alcohol dehydrogenase to convert the aldehydes to their respective alcohols permitted positive identification of the isomeric form of hexenal as cis-3-hexenal. Red and green tomato fruit homogenized in buffer with saturated CaCl₂ contained low (0.1-0.8 µg g^?1 fresh weight) levels of hexanal and cis-3-hexenal; thus there is minimal endogenous volatile content in intact fruit. Volatile levels increased rapidly, up to 10-fold, following homogenization of ripe tomato fruit in the absence of CaCl₂, and more modestly in corresponding green tomato fruit homogenates. Incubation with the appropriate lipoxygenase/hydroperoxide lyase substrate (linoleic acid for hexanal, linolenic acid for cis-3-hexenal) doubled the amount of volatile compound produced. Hexanal generation was suppressed in the presence of linolenic acid, suggesting that the enzyme complex has greater affinity for this substrate. As well, levels of cis-3-hexenal, but not hexanal, tended to decline within 30 min of homogenization, possibly reflecting a specific degradative process. The results collectively indicate that the contribution of six-carbon aldehydes to tomato fruit flavor is attributable to metabolism invoked following tissue disruption rather than within the intact fruit.     

Screening for reduction of aldehydes and ketones by solventogenic cultures of the strict anaerobe,Clostridium acetobutylicum

Summary A simple procedure for screening Clostridium acetobutylicum for biotransformations to produce alcohols was developed by supplementing rich medium with glucose and butyric acid to obtain butanol-producing cultures. No special apparatus was required to maintain anaerobic conditions. The validity of the method was established by demonstrating that the cultures could reduce alicyclic and aromatic aldehydes and ketones to the corresponding alcohols.     

The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components

Primary alcohols varying in chain length from C₁₃ to C₁₆, and in number, position, and geometric configuration of double bonds, were applied in dimethyl sulfoxide to the surface of the female sex pheromone glands of Heliothis subflexa (Gn.) and Hydraecia micacea (Esper). Capillary gas chromatographic analysis of extracts of the treated glands indicated that the alcohols were converted to the corresponding aldehydes by H. subflexa females and to the acetates by H. micacea females. Conversions of the alcohols showed no preferences for molecular weight, number, position, or geometry of the double bonds in either species. Application of the acetates of the primary alcohols to the gland surface of H. subflexa females resulted in the production of both the corresponding alcohols and aldehydes, while neither alcohols nor aldheydes were produced when acetates were applied to the glands of H. micacea. In addition, application of the acetates to the gland surface of Heliothis virescens (F.) resulted in the production of both the corresponding alcohols and aldehydes. However, no evidence was found to indicate that acetates are ever produced by the pheromone gland of females of H. virescens.     

Accumulation of l-Glutamic Acid from Dibasic Carboxylic Acids by a Hydrocarbon Utilizing Bacterium

In order to know the substrate specificity in a hydrocarbon utilizing bacterium, the following materials were examined: n-alkanes, n-alkenes, monohydric alcohols, aldehydes, monobasic carboxylic acids, dihydric alcohols and dibasic carboxylic acids.

It was found that dibasic carboxylic acids were well utilized, and a great deal of l-glutamic acid was accumulated from them. Then suberic acid, which is C₈ dibasic carboxylic acid, was compared with n-dodecane in the effects of thiamine, penicillin, C/N ratio and substrate concentration on l-glutamic acid accumulation and cell growth.     

Characterization of oxidase(s) associated with the sex pheromone gland in Manduca sexta (L.) females

An oxidase that converts primary aliphatic alcohols into aldehydes was discovered in the cuticle of the sex pheromone gland and in the papillae anales on the tip of the abdomen of Manduca sexta females. Oxidase activity was not found in the epidermal cells of the pheromone gland where fatty acid precursors of the pheromonal aldehydes are found. This oxidase requires oxygen and water to function and appears to have a rather broad substrate specificity. The activity of the oxidase is reduced by the application of piperonyl butoxide, which also interferes with the PBAN induced production of the natural pheromone aldehydes. However, endogenous alcohols cannot be found in the pheromone gland. Thus, it is not yet clear whether or not the oxidase is involved in the terminal step of biosynthesis of the pheromone aldehydes in M. sexta females. © Wiley-Liss, Inc.

1 This article is a U.S. Government work and, a such, is in the public domain in the United States of America.

     

Stereochemical analysis of chiral amines,diamines, and amino alcohols: Practical chiroptical sensing based on dynamic covalent chemistry

Practical chiroptical sensing with a small group of commercially available aromatic aldehydes is demonstrated. Schiff base formation between the electron-deficient 2,4-dinitrobenzaldehyde probe and either primary amines, diamines, or amino alcohols proceeds smoothly in chloroform at room temperature and is completed in the presence of molecular sieves within 2.5 hours. The substrate binding coincides with a distinct circular dichroism signal induction at approximately 330 nm, which can be correlated to the absolute configuration and enantiomeric composition of the analyte. The usefulness of this sensing method is highlighted with the successful sensing of 18 aliphatic and aromatic amines and amino alcohols and five examples showing quantitative %ee determination with good accuracy.     

Characterization and application of xylene monooxygenase for multistep biocatalysis

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.     

Antimicrobial Browning-Inhibitory Effect of Flavor Compounds in Seaweeds

Since ancient times, the antimicrobial properties of seaweeds have been recognized. However, antimicrobial activities of volatile compounds in seaweeds have not been explored so far. Here, essential oils from seaweeds including green, brown and red algae such as Laminaria japonica, Kjellmaniella crassifolia, Gracilaria verrucosa and Ulva pertusa were prepared by using SDE (simultaneous distillation and extraction) apparatus. Volatile compounds in the essential oils were identified as aldehydes, ketones, carboxylic acids, alcohols and hydrocarbons by comparison of GC-retention times and MS data with those of authentic specimens. Flavor compounds such as (3Z)-hexenal, (2E)-hexenal and (2E)-nonenal in some essential oils showed strong antimicrobial activities against Escherichia coli TG-1, and Erwinia carotovora. Inhibition of browning can be achieved during either of two stages, namely, oxidation reaction by tyrosinase or subsequent non-enzymatic polymerization. Tyrosinase activity was measured by monitoring absorbance at 475 nm originating from dopachrome formed from L-DOPA. Many kinds of aliphatic carboxylic acids, aldehydes and alcohols were used as inhibitors for PPO activity. The results indicated that the α,β-unsaturated carbonyl compounds strongly inhibit tyrosinase activity. When seaweeds are damaged or macerated, the α,β-unsaturated aldehydes such as (2E)-hexenal and (2E)-nonenal are biosynthesized via the corresponding (3Z)-unsaturated aldehydes from linolenic acid and arachidonic acid. The flavor compounds that are formed could be valuable as safe antimicrobial browning-inhibitory agents of edible seaweed origin.     

Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with K _ms 10–20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit. Daniel Manríquez and Islam El-Sharkawy contributed equally to the work. Accession numbers for Cm-ADH1 (ABC02081), and Cm-ADH2 (ABC02082).     

The kinetics and mechanism of liver alcohol dehydrogenase with primary and secondary alcohols as substrates

1. The activity of liver alcohol dehydrogenase with propan-2-ol and butan-2-ol has been confirmed. The activity with the corresponding ketones is small. Initial-rate parameters are reported for the oxidation of these secondary alcohols, and of propan-1-ol and 2-methylpropan-1-ol, and for the reduction of propionaldehyde and 2-methylpropionaldehyde. Substrate inhibition with primary alcohols is also described. 2. The requirements of the Theorell-Chance mechanism are satisfied by the data for all the primary alcohols and aldehydes, but not by the data for the secondary alcohols. A mechanism that provides for dissociation of either coenzyme or substrate from the reactive ternary complex is described, and shown to account for the initial-rate data for both primary and secondary alcohols, and for isotope-exchange results for the former. With primary alcohols, the rapid rate of reaction of the ternary complex, and its small steady-state concentration, result in conformity of initial-rate data to the requirements of the Theorell-Chance mechanisms. With secondary alcohols, the ternary complex reacts more slowly, its steady-state concentration is greater, and therefore dissociation of coenzyme from it is rate-limiting with non-saturating coenzyme concentrations. 3. Substrate inhibition with large concentrations of primary alcohols is attributed to the formation of an abortive complex of enzyme, NADH and alcohol from which NADH dissociates more slowly than from the enzyme-NADH complex. The initial-rate equation is derived for the complete mechanism, which includes a binary enzyme-alcohol complex and alternative pathways for formation of the reactive ternary complex. This mechanism would also provide, under suitable conditions, for substrate activation or substrate inhibition in a two-substrate reaction, according to the relative rates of reaction through the two pathways.     

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. Received: 22 March 1999 / Received revision: 12 July 1999 / Accepted: 1 August 1999     

Preparation of (1S)-verbenone, aromatic and alicyclic carboxylic acids by oxidation of aldehydes, primary and secondary alcohols with Nocardia corallina

(lS)-Verbenone, (S)-perillyl acid, cinnamic acid, meta-nitrocinnamic acid, veratric acid and 2-naphthoic acid were prepared, at 1 mM scale, from the corresponding alcohols or aldehydes with whole cells of Nocardia corallina B-276, in yields from 19 to 71% (w/w). Similar microbiological oxidations gave poor yields with the heterocyclic alcohols: 3-pyridylmethanol, 4-flavanol and 4-chromanol.     

Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.     

Mammalian alcohol dehydrogenase — Functional and structural implications

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1–ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.