Acetate formation from CO and CO2 by cell extracts of Peptostreptococcus productus (strain Marburg)

Cell extracts of Peptostreptococcus productus (strain Marburg) obtained from CO grown cells mediated the synthesis of acetate from CO plus CO₂ at rates of 50 nmol/min × mg of cell protein. ¹⁴CO was specifically incorporated into C₁ of acetate. No label exchange occurred between ¹⁴C₁ of acetyl-CoA and CO, indicating that ¹⁴CO incorporation into acetate was by net synthesis rather than by an exchange reaction. In acetate synthesis from CO plus CO₂ the latter substrate could be replaced to some extent by formate or methyl tetrahydrofolate as the methyl donor. The methyl group of methyl cobalamin was incorporated into acetate ony at very low activities. The cell extracts contained high levels of enzyme activities involved in acetate or cell carbon synthesis from CO₂. The following enzymic activities were detected: CO: methyl viologen oxidoreductase, formate dehydrogenase, formyl tetrahydrofolate synthetase, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate dehydrogenase, methylene tetrahydrofolate reductase, phosphate acetyltransferase, acetate kinase, hydrogenase, NADPH: benzyl viologen oxidoreductase, and pyruvate synthase. Some kinetic and other properties were studied.     

Substrate Specificity,Mode of Action,and of Inhibition by Organic Acids of Purified Acetylesterase from Sclerotinia Fungus

Acetylesterase (AcE) of Sclerotinia libertiana was purified approximately 1170-fold, and proved homogeneous by electrophoresis, ultracentrifugation and chromatography. The purified AcE hydrolyzed various acetyl esters in the following order; vinyl acetate, tri-acetin, n-butyl acetate, p-nitrophenylacetate, diacetin, ethylene glycol diacetate, monoacetin, ethyl acetate, acetylcholine, methyl acetate. It also had apparently a slight activity on tannic acid, benzoylcholine, methyl butyrate and acetic anhydride.

The mode of AcE reaction on these substrates could be divided into two types of group by Lineweaver-Burk plot, one forms the enzyme-substrate complex, ES, and the other, SES additionally combining substrate at a high substrate concentration.

From the inhibition experiment by organic acids, it was suggested that the neighbouring carboxyl groups of the di-, or tribasic acid such as citric, cis-aconitic, succinic, and maleic acid have a significance on inhibition of the AcE. Also, choline esterase inhibitor partially inhibited the activity on acetylcholine, and bivalent metal ion increased the activity on triacetin. Thus, the AcE was supposed to have a many adjacent sites of interaction with the substrate.     

Gibberellin A36, Isolation from Gibberella fujikuroi,Structure and Conversion to Gibberellin A37

A cDNA encoding a putative extracellular α-L-arabinofuranosidase was cloned from the basidiomycete Coprinopsis cinerea (CcAbf62A). CcAbf62A belongs to glycoside hydrolase family 62 (GH62), but is phylogenetically distinct from previously characterized GH62 enzymes. The recombinant CcAbf62A, expressed in Pichia pastoris, released L-arabinose from both wheat arabinoxylan and oat-spelt xylan. The enzyme activity for wheat arabinoxylan was increased by the addition of CcEst1, a carbohydrate esterase from C. cinerea.     

The incorporation of [1-14C]acetate into the methyl ketones that occur in steam-distillates of bovine milk fat

1. The ¹⁴C-labelling of the fatty acids and the methyl ketones in steam-distillates of milk fat from a lactating cow that had been injected intravenously with [1-¹⁴C]acetate was determined. 2. The labelling patterns of the C₆–C₁₆ fatty acids and the corresponding methyl ketones with one fewer carbon atoms were similar, particularly so for the C₅–C₁₀ compounds at 9 and 22hr. after the injection of [1-¹⁴C]acetate. The isolation of ¹⁴C-labelled methyl ketones in the range C₃–C₁₅ is evidence that the β-oxo acid precursors, which are glyceride-bound in the milk fat, are synthesized in the mammary gland from acetate. The absence of heptadecan-2-one in steam-distillates and the extremely low specific radioactivity of stearic acid are further evidence for this biosynthetic pathway. 3. The specific radioactivities of the C₅–C₁₅ methyl ketones were higher (with the exception of C₉ methyl ketone in the second milking) than the specific activities of the corresponding fatty acids with one more carbon atom. This is consistent with the methyl ketone precursors' being formed during the biosynthesis of fatty acids rather than being products of β-oxidation of fatty acids.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium

A new enzymatic route for biodiesel production from soybean oil was developed using methyl acetate as a novel acyl acceptor. Novozym 435 (immobilized Candida antarctica lipase) gave the highest methyl ester (ME) yield of 92%. The optimum conditions of the transesterification were 30% enzyme based on oil weight; a molar ratio of methyl acetate/oil of 12:1; temperature 40 °C and reaction time 10 h. Since no glycerol was produced in the process, this method is very convenient for recycling the catalyst and by-product triacetylglycerol showed no negative effect on the fuel property.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

Fatty acid synthesis by slices from developing leaves

Fatty acid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivium L.) leaves. The basal regions of the leaves had the lowest rates of fatty acid synthesis and accumulated small amounts of very long chain fatty acids. Fatty acid synthesis was highest in the middle leaf sections in all four plants. Linolenic acid synthesis from [1-¹⁴C]acetate was highest in the distal leaf sections of rye grass. The labelling of the fatty acids of individual lipids of rye grass was examined and it was found that [¹⁴C]linolenic acid was highest in the galactolipids. Synthesis of this acid in the galactolipids was most active in leaf segment C. Only traces of [¹⁴C]linolenic acid were ever found in phosphatidylcholine and it is concluded that this phospholipid cannot serve as a substrate for linoleic acid desaturation in rye grass. The synthesis of fatty acids was sensitive to arsenite, fluoride and the herbicide EPTC. The latter was only inhibitory towards those leaf segments which made very long chain fatty acids. Formation of fatty acids from [1-¹⁴C]acetate was also studied in chloroplasts prepared from successive leaf sections of rye grass. Chloroplasts isolated from the middle leaf sections had the highest activity. Palmitic and oleic acids were the main fatty acid products in all chloroplast preparations. Linolenic acid synthesis was highest in chlorplasts isolated from the distal leaf sections of rye grass.     

Synthesis of symmetric and asymmetric diamides of citric acid and amino acids

Summary A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.Abbreviations AcOtBu tert-butyl acetate - i-Bu iso-butyl - tBu tert-butyl - Bzl benzyl - p-OH-Bzl p-hydroxybenzyl - m,p-(OH)₂-Bzl m,p-dihydroxybenzyl - DCCI dicyclohexylcarbodiimide - Et ethyl - Me methyl - Su succinimidyl - SuOH N-hydroxysuccinimide - Ph phenyl     

Formation of Volatile Esters in Strawberries

Aliphatic alcohols including methyl, ethyl, isopropyl, npropyl, isobutyl, nbutyl, isoamyl, namyl and hexyl alcohol were converted to their acetate, propionate, nbutyrate, isovalerate and caproate esters during the incubation with strawberry fruit tissue. Formate, isobutyrate and n-valerate esters were formed when alcohols were incubated together with these fatty acids and strawberry.

Seventy esters were formed from various combinations of alcohols and acids by means of incubation with strawberry.

No ester formation was observed when strawberry was homogenized.     

Optical resolution of two isomeric naphthylalanines by immobilized enyzmes

Optical resolution of β-(1-naphthyl)alanine and β-(2-naphthyl)alanine have been efficiently carried out through enzymatic hydrolysis of their methyl ester and/or N-acetyl ester derivatives by immobilized enzymes. Difficulties related to the lipophilic character of these amino acids were overcome by using emulsions of n-butyl acetate–water as reaction medium. The use of an automatic recirculating apparatus allowed reproducible and repetitive use of the immobilized biocatalysts.     

Ceriopsins A–D, diterpenoids from Ceriops decandra

Chemical examination of the ethyl acetate solubles of the CH₃OH:CH₂Cl₂ (1:1) extract of the roots of Ceriops decandra collected from Kauvery estuary resulted in the isolation of four new diterpenoids, ceriopsins A–D (1–4). The structures of the new diterpenoids were elucidated by a study of their physical and spectral data as methyl 17-hydroxy-16-oxobeyeran-18-oate (1), methyl 16(R)-16,17-dihydroxybeyeran-18-oate (2), 1β,15(S)-isopimar-7-ene-1,15,16-triol (3), and 8,15(R)-epoxypimarane-1β,16-diol (4).     

Phospholipids from the hepatopancreas of Indian horseshoe crab Carcinoscorpius rotundicauda

Total phospholipids were extracted from the heart, hepatopancreas, and hemolymph of the Indian horseshoe crab Carcinoscorpius rotundicauda by the conventional method. Characteristic group reaction and 2-dimensional thin-layer chromatography on silica gel were used for identification of different phospholipids. The phospholipid profile obtained from hemolymph and 2 major organs are comparable and show phosphatidyl choline (PC) and phosphatidyl ethanolamine to be the major phospholipids. A phospholipid has been consistently detected migrating immediately below the PC in the thin-layer chromatogram of lipids extracted from the hepatopancreas. When mixed methyl esters of this slower moving PC are resolved on a silica gel plate ran in hexane ether:acetic acid 80:20:1, with appropriate controls, an additional spot is seen just below the normal methyl ester, indicating a difference between the fatty acid compositions of 2 PC (e.g., regular and slower). The slower mixed methyl esters were found to comprise mainly the 4 saturated fatty acids: lauric, myristic, palmitic, and stearic. The slow moving PC seems to consist mainly of molecular species with the above-mentioned saturated fatty acids at both Sn 1 and Sn 2 positions.     

Rhythms of volatile release from female and male sea buckthorn plants and electrophysiological response of sea buckthorn carpenter moths

The sea buckthorn, Hippophae rhamnoides L., is a thorny, nitrogen-fixing, dioecious, and deciduous shrub which has been attacked by a catastrophic outbreak of Holcocerus hippophaecolus in the ‘Three North Areas’ of China recently. The behavioral responses of female individuals to their dioecious host sea buckthorn, H. rhamnoides ssp. sinensis, were tested by Y-tube bioassay, and intraspecific emission variations and the circadian rhythm of male and female sea buckthorn plants were compared, together with the electrophysiological responses of sea buckthorn carpenter moths to these parameters. Y-tube olfactometry indicated that mated female H. hippophaecolus individuals did not display a significant preference for either sex of sea buckthorns. Additionally, no unique chemical compound was found. Female antennae significantly responded to 1-octene, methyl salicylate, and (Z)-3-Hexen-1-ol acetate, among which methyl salicylate was more abundant in females than in males. In addition, the circadian variation of (Z)-3-Hexen-1-ol acetate suggested that it was an effective compound for host location.     

Antidiabetic activity of phenolic compounds from Pecan bark in streptozotocin-induced diabetic rats

The n-butanol fraction (BF) of bark of Pecan tree, Carya illinoinensis (Wangenh) K. Koch (Juglandaceae) afforded two new flavonol methyl ether: caryatin-3′ sulfate (6) and caryatin-3′ methyl ether-7-O-β-d-glucoside (7) while five known phenolics (1–5) were isolated from its ethyl acetate fraction (EAF). The structures of isolated compounds were established based on 1D and 2D NMR spectroscopy. The isolated compounds were investigated for their hypoglycaemic, antioxidant as well as the aldose reductase (AR) inhibitory effect in lenses of streptozocin diabetic rats. All the isolated compounds showed significant hypoglycaemic and antioxidant activities, except 5 and 6. A marked AR-inhibitory effect was identified for compounds 2, 3 and 7.     

New Steroids from the South China Sea Soft Coral Lobophytum sp.

Two new steroids, (22R,23S)‐3β‐hydroxy‐23‐methyl‐17,20‐epoxyergost‐5‐en‐22‐yl acetate and (22R,23S)‐5‐hydroperoxy‐23‐methyl‐5α‐17,20‐epoxyergost‐6‐ene‐3β,22‐diol, were isolated from the South China Sea soft coral Lobophytum sp., together with two related known ones. The structures of all compounds were elucidated by extensive spectroscopic analysis and by comparing their spectral data with those previously reported. The structure of (22R,23S)‐3β‐hydroxy‐23‐methyl‐17,20‐epoxyergost‐5‐en‐22‐yl acetate was further confirmed through chemical correlation. All the isolates were evaluated for the in vitro inhibitory activity against NF‐κB, a potential target for the treatment of cancer, and (22R,23S)‐5‐hydroperoxy‐23‐methyl‐5α‐17,20‐epoxyergost‐6‐ene‐3β,22‐diol exhibited moderate inhibition activity with IC₅₀ value of 8.96 μg/mL.     

Methane emission from feather moss stands

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH₄. However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH₄ turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH₄ cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH₄ emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH₄ h^?1 gdw^?1. Temperature and water content positively influenced CH₄ emission. Methanogenic potentials determined under N₂ atmosphere in darkness ranged between 22 and 157 pmol CH₄ h^?1 gdw^?1. Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH₄ was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 10⁴ and 10⁵ copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH₄ was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH₄ produced was depleted in ¹³C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3–62 mM). The δ¹³C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH₄ by hydrogenotrophic methanogenesis and causing net emission of CH₄ under ambient conditions, albeit at low rates.     

Chromatographic analysis of simple phenols in some species from the genus Salix

Introduction – Salicis Cortex, made from willow bark is a herbal remedy, which is standardised based on the content of salicin, a compound with analgesic and antiphlogistic properties. However, clinical trials suggest that other compounds also present in Salicis Cortex can contribute to the pharmacological effects. Objective – To characterise the composition of phenolic acids in the barks of different species and clones from the genus Salix by use of chromatographic methods—HPTLC and HPLC. Methodology – The phenolic acid composition was analysed by MGD (multiple gradient development)–HPTLC technique. The separation was performed on HPTLC Diol plates with gradient elution using a mixture of chloroform:hexane:ethyl acetate with increasing concentration of ethyl acetate from 10 to 25%. Derivatisation with thymol reagent was employed for the first time for specific detection of phenolic acids containing methoxyl groups. Results – The presence of all phenolic acids previously reported in the genus Salix was confirmed, namely p‐hydroxybenzoic, vanillic, cinnamic, p‐coumaric, ferulic and caffeic acids. Furthermore, pyrocatechol as a constituent of willow bark was revealed. The highest concentration of this compound was observed in the S. purpurea bark (2.25 mg/g). Conclusion – The presence of a relatively high content of pyrocatechol in Salix species may raise doubts about the safe application of this herbal medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

A new irreversible enzyme-aided esterification method in organic solvents

A new irreversible esterification method for carboxylic acids catalyzed by a lipase from Candida antarctica (Novozyme 435) in organic solvents has been developed. The water produced during the process is chemically destroyed by a corresponding ester of acetoacetate, which acts as a sacrificial substrate in this reaction. The flavour esters isobutyl acetate, methyl butyrate, ethyl butyrate and benzyl butyrate were synthesized either in small scale (0.05 mol) or large scale (1 mol). The yields range from 82 to 92% within 24 h at 52°C. Optimal molar ratios of reactants were 1:1:1 (carboxylic acid:alcohol:acetoacetate).