Toward cell‐free biofuel production: Stable immobilization of oligomeric enzymes

To overcome the main challenges facing alcohol‐based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell‐free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β‐galactosidase, and an alcohol‐producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long‐term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto‐acid decarboxylase free in solution. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:324–331, 2014     

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.     

Corynebacterium glutamicum tailored for efficient isobutanol production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y_P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y_P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y_P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H⁺ to NADPH+H⁺. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y_P/S of about 0.48 mol per mol of glucose in the production phase.     

Use of the valine biosynthetic pathway to convert glucose into isobutanol

Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259–2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also—as has been demonstrated previously (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009)—used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.     

Dietary ethanol and lipid synthesis in Drosophila melanogaster

When cultured on a defined diet, ethanol was an efficient substrate for lipid synthesis in wild-type Drosophila melanogaster larvae. At certain dietary levels both ethanol and sucrose could displace the other as a lipid substrate. In wild-type larvae more than 90% of the flux from ethanol to lipid was metabolized via the alcohol dehydrogenase (ADH) system. The ADH and aldehyde dehydrogenase activities of ADH were modulated in tandem by dietary ethanol, suggesting that ADH provided substrate for lipogenesis by degrading ethanol to acetaldehyde and then to acetic acid. The tissue activity of catalase was suppressed by dietary ethanol, implying that catalase was not a major factor in ethanol metabolism in larvae. The activities of lipogenic enzymes, sn-glycerol-3-phosphate dehydrogenase, fatty acid synthetase (FAS), and ADH, together with the triacylglycerol (TG) content of wild-type larvae increased in proportion to the dietary ethanol concentration to 4.5% (v/v). Dietary ethanol inhibited FAS and repressed the accumulation of TG in ADH-deficient larvae, suggesting that the levels of these factors may be subject to a complex feedback control.This research was supported by National Institutes of Health Grant GM-28779 to B.W.G. and a Monash University Research Grant to S.W.M.     

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

Scale up of the production of xylitol dehydrogenase and alcohol dehydrogenase from Pachysolen tannophilus grown on xylose

Summary Coproduction of two enzymes, xylitol dehydrogenase (XDH) and alcohol dehydrogenase (ADH), was attempted using the yeast Pachysolen tannophilus. Production of both enzymes was scaled up to a volume of 500 l with the yeast growing on xylose a the sole carbon source. Maximal amount of XDH was obtained by harvesting the cells at the end of the logarithmic growth phase. Activity of XDH was induced by imposing anaerobic conditions, thereby yielding 10 times more enzyme than was obtained for aerobic growth conditions. In crude extracts, obtained by passage through a French press (20 000 p.s.i.), XDH activity was 0.057 u/mg protein and ADH was 0.15 u/mg for aerobic growth and 1.5 u/mg for microaerophilic growth. Extraction of intracellular enzymes on a large sale was performed with a combination of cell wall lytic enzymes and an industrial homogenizer (Manton-Gaulin, 3000 p.s.i.). This system enabled a continuous mode of operation (single passage through homogenizer) with a high cell density (100 g/l) and the extracts contained 0.033 u/mg of XDH and 0.45 u/mg protein of ADH.     

Characterization of Enzymes in the Oxidation of 1,2-Propanediol to <Emphasis Type="SmallCaps">d</Emphasis>-(−)-Lactic Acid by <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> DSM 2003

Although Gluconobacter oxydans can convert 1,2-propanediol to d-(−)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of d-(−)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in d-(−)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to d-(−)-lactic acid by G. oxydans DSM 2003.     

Refolding of a Thermostable Glyceraldehyde Dehydrogenase for Application in Synthetic Cascade Biomanufacturing

The production of chemicals from renewable resources is gaining importance in the light of limited fossil resources. One promising alternative to widespread fermentation based methods used here is Synthetic Cascade Biomanufacturing, the application of minimized biocatalytic reaction cascades in cell free processes. One recent example is the development of the phosphorylation independent conversion of glucose to ethanol and isobutanol using only 6 and 8 enzymes, respectively. A key enzyme for this pathway is aldehyde dehydrogenase from Thermoplasma acidophilum, which catalyzes the highly substrate specific oxidation of d-glyceraldehyde to d-glycerate. In this work the enzyme was recombinantly expressed in Escherichia coli. Using matrix-assisted refolding of inclusion bodies the yield of enzyme production was enhanced 43-fold and thus for the first time the enzyme was provided in substantial amounts. Characterization of structural stability verified correct refolding of the protein. The stability of the enzyme was determined by guanidinium chloride as well as isobutanol induced denaturation to be ca. −8 kJ/mol both at 25°C and 40°C. The aldehyde dehydrogenase is active at high temperatures and in the presence of small amounts of organic solvents. In contrast to previous publications, the enzyme was found to accept NAD⁺ as cofactor making it suitable for application in the artificial glycolysis.     

Promotive action of ceramics on yeast ethanol production,and its relationship to pH,glycerol and alcohol dehydrogenase activity

Summary A yeast strain, Saccharomyces cerevisiae KPY32 isolated from pito, a traditional West-African alcoholic beverage, was immobilized in porous ceramic beads as a means of improving its ethanol production. Stationary fermentation cultures at different temperatures were made using semi-synthetic medium and fermentation parameters including ethanol production, sugar consumption, cell growth and pH were monitored. Glycerol production, and the activity of alcohol dehydrogenase (ADH) of the various systems were monitored. It was found that immobilization of the yeast resulted in improved ethanol production, at conversion rates above 93% of the theoretical value. The pH of the immobilized systems was also stabilized at around 4.0, glycerol production was higher, and the ADH activities were higher than those of free-cell systems. Ethanol production at the high temperature of 37° C was also improved by immobilization. The promotive action was found to be related to the pH, presence of glycerol and the enhancement of ADH activity.Offprint requests to: B. Demuyakor     

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.     

Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels]

The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner–Doudoroff pathway

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvC^TM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD⁺ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.     

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.     

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.     

Non-enzymatic PLP-dependent oxidative deamination of amino acids induces higher alcohol synthesis

Pyridoxal phosphate (PLP) is an organic cofactor found in all transaminase enzymes. In this study PLP was used to replace the enzymatic deamination step in the Ehrlich pathway, for the oxidative conversion of amino acids into 2-keto acids. PLP functions in an enzymeindependent manner. It was further used in the synthesis of higher alcohols through a sequential enzymatic reduction in vitro and in vivo. PLP-dependent oxidation was investigated against five representative amino acids: valine, leucine, isoleucine, norvaline, and phenylalanine. In vitro amino acid oxidation resulted in approximately 45 ~ 75% [mole/mole] of each 2-keto acid conversion and in vitro ammonia formation was less than 2-keto acid formation, with 20% of conversion yields. Whole cell E. coli expressing reduction enzymes KivD/ADH with both single amino acid and amino acid mixture (4% yeast extract) gave the highest yield (30 ~ 55%) in the presence of the PLP-Cu complex and following enzymatic reactions.     

Isozymic patterns and functional states of cell lines ofDrosophila melanogaster cultured in vitro. II

Summary Our previous isoenzyme investigation ofDrosophila melanogaster cell lines in vitro has been completed with twelve further enzyme systems. The enzyme profiles seem to be in good agreement with a previous hypothesis concerning the precise origin of these cell lines (probably from imaginal discs or nervous tissues). Our results have been summarized with reference to the biochemical genetic map ofDrosophila melanogaster in order to consider a possible functional organization of the genome.Abbreviations NAD nicotine adenine nucleotide - NADP nicotine adenine nucleotide phosphate - NBT nitroblue tetrazolium - PMS phenazine methosulfate - EDTA ethylene diamine tetraacetic acid - GOT Glutamate-oxaloacetate transaminase - PGK Phosphoglycerate kinase - GPDH -glycerophosphate dehydrogenase - MDH Malate dehydrogenase - PGM Phosphoglucomutase - Aph Alkaline phosphatase - MDH-NADP Malic enzyme - Lap Leucine Amino-Peptidase - LDH Lactate dehydrogenase - -1-OHDH L-3-hydroxyacid dehydrogenase - ADH Alcohol dehydrogenase - Aldox Aldehyde oxydase - 6PGD 6 Phosphogluconate dehydrogenase - G6PD Glucose-6-Phosphate dehydrogenase - Hex3 Fructokinase - IDH Isocitrate dehydrogenase - Est 6 Esterase 6 - Est C Esterase C - ODH Octanol dehydrogenase - XDH Xanthine dehydrogenase - AcPh Acid Phosphatase 1     

N-acylethanolamines as novel alcohol dehydrogenase 3 substrates

N-Acylethanolamines (NAEs) are members of the fatty acid amide family. The NAEs have been proposed to serve as metabolic precursors to N-acylglycines (NAGs). The sequential oxidation of the NAEs by an alcohol dehydrogenase and an aldehyde dehydrogenase would yield the N-acylglycinals and/or the NAGs. Alcohol dehydrogenase 3 (ADH3) is one enzyme that might catalyze this reaction. To define a potential role for ADH3 in NAE catabolism, we synthesized a set of NAEs and evaluated these as ADH3 substrates. NAEs were oxidized by ADH3, yielding the N-acylglycinals as the product. The (V/K)_app values for the NAEs included here were low relative to cinnamyl alcohol. Our data show that the NAEs can serve as alcohol dehydrogenase substrates.     

Mode of Action of α, β Unsaturated Carbonyl Compounds

The toxicity of the α, β unsaturated carbonyl compounds (α, β UCC_s) (patulin, penicillic acid, parasorbic acid, tulipalin and plumbagin) towards Pythium sp. group F (Van Der Plaat -Niterinks 1981) was neutralized by the addition of an excess of cysteine. This suggests that the mode of action of these compounds could be due to a binding of the α, βi UCC_s to sulphydryl groups in enzymes or other macromolecules. Alcohol dehydrogenase (ADH), an enzyme with a sulphydryl group at the active site, was assayed spectrophotometrically and all the α, β UCC_s inhibited ADH.